25-50 Micro Litre Ems
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Studies on the production of alpha amylase by Aspergillus oryzae using submerged fermentation ROHEENA ABDULLAH INSTITUTE OF INDUSTRIAL BIOTECHNOLOGY GC UNIVERSITY LAHORE 10 Bot - P h D - 2 0 0 5
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Transcript of 25-50 Micro Litre Ems
Studies on the production of alpha amylase by Aspergillus oryzae
using submerged fermentation
ROHEENA ABDULLAH
INSTITUTE OF INDUSTRIAL BIOTECHNOLOGY
GC UNIVERSITY LAHORE
10 Bot - P h D - 2 0 0 5
i
A THESIS TITLED
Studies on the production of alpha amylase by Aspergillus oryzae
using submerged fermentation
Submitted to GC University Lahore in fulfillment of the
requirements for the award of degree of
Doctor of Philosophy
IN BOTANY
By
ROHEENA ABDULLAH
INSTITUTE OF INDUSTRIAL BIOTECHNOLOGY
GC UNIVERSITY LAHORE
10 Bot - P h D - 2 0 0 5
ii
DECLARATION
I Miss Roheena Abdullah Roll No.10-Bot-PhD-2005 student of PhD in the
subject of botany, hereby declared that the matter printed in this thesis titled
“Studies on the production of alpha amylase by Aspergillus oryzae using submerged
fermentation” is my own work and has not been printed, published and submitted as
research work, thesis or publication in any form in any university, research
institution etc. in Pakistan or abroad.
Date:_______________ ______________________ Signature of Deponent
iii
RESEARCH COMPLETION CERTIFICATE
Certified that the research work contained in this thesis titled “Studies on the
production of alpha amylase by Aspergillus oryzae using submerged fermentation”
has been carried out and completed by Miss Roheena Abdullah Roll No 10-Bot -
phD-05 under my supervision during her Ph.D studies in the subject of Botany.
_________________ _______________________
Date Prof. Dr Ikram-ul-Haq (S.I) Supervisor
Submitted through ___________________ _____________________
Prof. Dr Ikram-ul-Haq (S.I) Controller of Examinations
Director, GC University, Lahore
Institute of Industrial Biotechnology
GC University, Lahore
iv
ACKNOWLEDGEMENTS
All praise for the, “ALMIGHTY ALLAH” who is the only supreme Authority and
whose presence has been figured on the two words i.e. “KUN FAYAKUN”. Every
tiny or massive entity moves with His permission. Countless thanks to Him for
accrediting me to accomplish this important task with in this specified time. All my
respect and regards to the Holy Prophet Hazrat Muhammad (peace be upon him)
who is forever a torch of guidance and knowledge for humanity. In view of his
saying:
“He who does not thank to people is not thankful to Allah”
I am highly obliged in paying deepest gratitude to my respected teacher
and research supervisor Prof. Dr. Ikram-ul-Haq, SI (Director, Institute of Industrial
Biotechnology, GCU, Lahore for his valuable guidance, encouragement, cooperation
and discussion. His enthusiastic inspiration and fatherly affection enabled me to
attain the objectives without any difficulty.
I most great fully acknowledge my indebtedness to Dr. M. A. Qadeer and Dr.
Muhammad Yaqub, Dr. Sikander Ali, Dr. Hamid Mukhtar, Dr. Mohsin Javed,
and Dr. Numan Aftab for their scholarly, scientific discussions and generous
advices when needed, during the entire period of my research work.
I am thankful to highly esteemed Dr. Zaheer-ud-Din Khan, (Chairperson
Department of Botany, GCU, Lahore) and Dr. Amin-ul-Haq Khan, Dean, Faculty
of Science and Technology, GCU, Lahore for providing all the necessary facilities
through out my research duration. I am grateful to Dr. Khalid Aftab, Vice
v
Chancellor, GC University, Lahore for providing me this opportunity to work in this
great Institute
The words are inadequate to express my heartfelt thanks to my friends and
fellows Aafia Aslam, Zahid Butt, Shazia Malik and Tehreema Iftikhar, for their
moral support in the research work.
I feel pleasure to acknowledge Dr. Shakeel (Assistant Professor Department of
Pathology Punjab University) for helping in the identification of strain.
I am also thankful to laboratory staff especially Mr. Fasial, Mr. Usman , Mr
Ramez and all others for their full cooperation during the whole period of my research.
Although feelings are deep but unfortunately words are too shallow, that cannot
follow the depths of my deep gratitude to my loving mother and father Mr. and Mrs.
Abdullah. My fortune is due, to their prayers.
ROHEENA ABDULLAH
vi
CONTENTS
Minor contents Page No Title i Declaration ii Research Completion Certificate iii Acknowledgements iv Contents vi List of Tables ix List of Figures x Abstract xiii Major contents
Chapter# 1 : INTRODUCTION 1 Objective 8 Chapter# 2: REVIEW OF LITRATURE 9 Uses of alpha amylase 44 Chapter# 3 : MATERIALS AND METHODS 49 3.1. Materials 49 3.2. Methods 49 3.2.1. Isolation of organism 49 3.3. Fermentation 51 3.3.1. Inoculum preparation 51 3.3.1.1. Conidial inoculum 51 3.3.1.2 Conidial count 51 3.3.1.3. Vegetative inoculum 51 3.3.2. Fermentation media 52 3.4. Shake flasks studies 52 3.5. Fermenter studies 53 3.6. Nutritional and cultural requirement of Aspergillus oryzae 53 3.6.1. Fermentation media 53 3.6.2. Incubation period 53 3.6.3. Effect of initial pH 54 3.6.4. Effect of temperature 54 3.6.5. Effect of volume 54 3.6.6. Effect of inoculum size 54 3.6.7. Effect of agitation 55 3.6.8. Evaluation of carbon sources 55 3.6.9. Evaluation of nitrogen sources 55 3.7. Induction of mutation 55 3.7.1. Minimal inhibitory concentration of 2-deoxy-D-glucose 55 3.7.2. Ultraviolet (UV) irradiation 56
vii
3.7.3. Nitrosoguanidine treatment 56 3.7.4. Nitrous acid treatment 57 3.7.5. EMS treatment 57 3.7.6. Selection of mutants 57 3.8. Analytical techniques 58 3.8.1. Estimation of alpha amylase 58 3.8.2. Estimation of total protein contents 58 3.8.3. Determination of mycelial morphology 59 3.8.4. Estimation of dry cell mass (DCM) 59 3.9. Statistical analysis 59 3.10. Kinetic study 59 3.11. Purification of alpha amylase 60 3.11.1. Separation of fungus from fermented broth 60 3.11.2. Ammonium sulfate precipitation 61 3.11.2.1. Anion exchange chromatography 61 3.11.2.2. Gel filtration 61 3.11.3. Dialysis 62 3.11.4. Electrophoresis 62 3.11.5. Protein Marker 62 3.12. Gel Preparation 62 3.12.1. Separating gel 63 3.12.2. Stacking gel 63 3.13. Characterization of enzyme 64 3.14. Standard curves 64 3.14.1. Maltose 64 3.14.2. Bovine serum albumin 64 3.15. Preparation of reagents/ buffers 65 3.15.1. DNS reagent 65 3.15.2. Brad ford reagent 65 3.15.3. Starch Solution 65 3.15.4. Acetate Buffer (pH 5.0) 66 3.15.5. Phosphate Citrate Buffer (pH 7.5) 66 3.15.6. Tris HCl buffer (pH 7.5) 66 3.15.7. Acrylamide bis acrylamide (30%) 66 3.15.8. Separating buffer (1.5 M Tris HCl, pH 8.8) 67 3.15.9. Stacking buffer (1M Tris HCl, pH 6.8) 67 3.15.10.Tank Buffer 67 3.15.11.Gel loading buffer 67 3.15.12. SDS Solution (10%) 67 3.15.13. Ammonium per sulfate 68 3.15.14. Staining and Destaining solution 68 Chapter # 4: RESULTS AND DISCUSSION 71 4.1. Identification, isolation and screening of organism 71 4.2.Strain improvement 75 4.2.1. Physical mutagenesis 75
viii
4.2.1.1. Screening of UV treated isolates 75 4.3. Chemical Mutagenesis 78 4.3.1. Screening of NG treated isolates 78 4.3.2. Screening of nitrous acid treated isolates 78 4.3.3.Screenning of EMS treated isolates 79 4.4. Optimization of cultural conditions in shake flasks 86 4.4.1. Screening of Culture media 86 4.4.2. Rate of alpha amylase production 86 4.4.3. Effect of incubation temperature 87 4.4.4. Effect of different initial pH 87 4.4.5. Effect of different volume of medium 87 4.4.6. Effect of inoculum size 88 4.5. Optimization of nutritional requirements of A. oryzae in shake flasks 95 4.5.1. Effect of starch from different sources 95 4.5.2. Effect of different concentrations of corn starch 95 4.5.3. Evaluation of additional carbon sources 96 4.5.4. Evaluation of inorganic nitrogen sources 96 4.5.5. Evaluation of organic nitrogen sources 97 4.5.6. Effect of surfactants 97 4.6. Optimization of cultural conditions in stirred fermenter 109 4.6.1. Rate of alpha amylase production 109 4.6.2. Effect of pH 110 4.6.3. Effect of aeration levels 110 4.6.4. Effect of dissolved oxygen 111 4.6.5. Effect of inoculum size 111 4.6.6. Effect of agitation intensity 112 4.7. Purification of alpha amylase 125 4.7.1. Ammonium sulfate precipitation 125 4.7.2. Step wise purification 125 4.7.2.1. Ammonium sulfate precipitation 125 4.7.2.2. Anion exchange chromatography 125 4.7.2.3. Gel filtration 126 4.8. Characterization 132 4.8.1. Temperature optima of purified alpha amylase 132 4.8.2.Effect of time of incubation on the activity of purified alpha amylase 132 4.8.3. Effect of distilled water and buffer on the activity of purified alpha amylase
132
4.8.4. Effect of pH on the activity of purified alpha amylase 133 4.8.5. Effect of metal ion on the activity of purified alpha amylase 133 Discussion 139 Conclusion 148 Chapter # 5 : References 149
ix
LIST OF TABLES Table Title of Table Page
4.1 Isolation and screening of A. oryzae for the alpha amylase production
72
4.1.1 Sub grouping of alpha amylase producing isolates of A. oryzae 74 4.2 Screening of UV isolates of A. oryzae IIB-30 for alpha amylase
production 76
4.2.1 UV treated survivors at different exposure time 77 4.2.2 Range of alpha amylase activity of UV isolates 77 4.3 Screening of NG treated A. oryzae UV-23 isolates for alpha
amylase production 80
4.3.1 NG treated survivors of A. oryzae 81 4.3.2 Range of alpha amylase activity of NG isolates 81 4.4 Screening of nitrous acid treated strains of A. oryzae NG-18 for
the alpha amylase production 82
4.4.1 Nitrous acid treated survivors of A. oryzae 83 4.4.2 Range of alpha amylase activity of nitrous acid treated isolates 83 4.5 Screening of EMS treated A. oryzae NA17 for the alpha
amylase production 84
4.5.1 EMS treated survivors of A. oryzae 85 4.5.2 Range of alpha amylase activity of EMS treated isolates 85 4.6 Kinetic evaluation of rate of fermentation for the alpha amylase
production by A. oryzae IIB-30 and its mutant derivative in stirred fermenter
114
4.7 Kinetic evaluation of different pH values of media for the alpha amylase production by A. oryzae IIB-30 and its mutant derivative in stirred fermenter
116
4.8 Kinetic evaluation of different aeration for the alpha amylase production by A. oryzae IIB-30 and its mutant derivative in stirred fermenter
118
4.9 Kinetic evaluation of different levels of dissolved oxygen for the alpha amylase production by A. oryzae IIB-30 and its mutant derivative in stirred fermenter
120
4.10 Kinetic evaluation of different inoculum sizes for the alpha amylase production by A. oryzae IIB-30 and its mutant derivative in stirred fermenter
122
4.11 Kinetic evaluation of different agitation speeds for the alpha amylase production by A. oryzae IIB-30 and its mutant derivative in stirred fermenter
124
4.12 Purification summary of alpha amylase produced by mutant strain of A. oryzae EMS-18 by using ammonium sulfate
127
4.13 Step wise purification profile of alpha amylase produced by mutant strain of A. oryzae EMS-18.
128
x
LIST OF FIGURES Figure Title of Figure Page
3.1 Standard curve of maltose 69
3.2 Standard curve of bovine serum albumin (BSA) 70
4.1 Screening of fermentation media for the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18
89
4.2 Rate of fermentation for the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18
90
4.3 Effect of incubation temperature on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18
91
4.4 Effect of different initial pH of fermentation medium on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18
92
4.5 Effect of different volume of media on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18
93
4.6 Effect of different inoculum sizes on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18
94
4.7 Effect of raw starch from different sources on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18.
99
4.8 Effect of different concentrations of starch on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18.
100
4.9 Effect of additional carbon sources on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18
101
4.10 Effect of different concentrations of lactose on the alpha amylase production by A. oryzae IIB-30 and its
102
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mutant derivative A. oryzae EMS-18
4.11 Effect of inorganic nitrogen sources on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18
103
4.12 Effect of different concentrations of ammonium sulfate on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18
104
4.13 Effect of organic nitrogen sources on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18
105
4.14 Effect of different concentrations of peptone on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18
106
4.15 Effect of different surfactants on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18.
107
4.16 Effect of different concentrations of Tween 80 on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18.
108
4.17 Comparison of rate on the alpha amylase production by wild (IIB-30) and mutant strain of A. oryzae (EMS-18) in stirred fermenter
113
4.18 Effect of initial pH of media on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18
115
4.19 Effect of different aeration levels on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18
117
4.20 Effect of different level of dissolved oxygen on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18
119
4.21 Effect of different inoculum size on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18
121
4.22 Effect of different agitation intensity on the alpha 123
xii
amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18
4.23 Elution pattern on Sephadex – DEAE 129
4.24 The elution profile on Sephadex G-100 130
4.25 SDS-PAGE analysis of pooled fractions of ion exchange chromatography and ammonium sulfate fractionation.
131
4.26 Effect of temperature on the activity of purified alpha amylase by mutant strain of A. oryzae EMS-18
134
4.27 Effect of time of incubation on the activity of purified alpha amylase by mutant strain of A. oryzae EMS-18
135
4.28 Effect of different buffers and distilled water on the activity of purified alpha amylase by mutant strain of A. oryzae EMS-18
136
4.29 Effect of different pH on the activity of purified alpha amylase by mutant strain of A. oryzae EMS-18
137
4.30 Effect of metal ions on the activity of purified alpha amylase by mutant strain of A. oryzae EMS-18.
138
xiii
Abstract
The present study, deals with the isolation, screening and selection of Aspergillus
oryzae for the alpha amylase production. Seventy eight isolates of A. oryzae were
isolated from different soil samples. The strains were initially selected qualitatively on
starch agar medium and screened quantitatively for enzyme production in shake flasks
and a strain producing 130 ±0.1U/ml of enzyme was selected which was assigned the
code IIB-30. The selected strain was subjected to physical and chemical mutagenic
treatments in order to improve its amylolytic potential. During the treatments, isolates
were qualitatively and quantitatively screened. Among these, EMS-18 exhibited the
highest enzyme activity (347±1.2 U/ml). This mutant showed 2.6 fold increased
activity over the parental strain in terms of enzyme production. The cultural conditions
and nutritional requirements of the selected strains (both wild and mutant) were
optimized in 250 ml Erlenmeyer flasks prior to scale up studies in a fermenter.
Six different fermentation media were evaluated for the alpha amylase
production by both wild and mutant strains of A. oryzae in shake flasks fermentation.
Of all the media, M4 containing (g/l); starch 20, yeast extract 8.5, NH4Cl 1.3,
MgSO4.7H2O 0.12, CaCl2 0.06 gave maximal enzyme production i.e., 168±2 (wild)
and 385±2 (mutant) which was highly significant (p≤0.05). The effect of incubation
temperature, initial pH, volume of media and inoculum size was investigated on the
enzyme production. The optimal enzyme production was obtained at 30°C, pH 5,
volume, 10 % and inoculum size 4 %, by both wild and mutant strains. The rate of
fermentation was also studied and the highest yield of enzyme was obtained 72 h after
inoculation.
xiv
Corn starch (2 %) and lactose (1.5 %) as carbon sources while, ammonium sulfate
(0.3 %) and peptone (0.2 %) as nitrogen sources were also optimized. Different
surfactants were added to the fermentation media and Tween 80 at the level of 0.1%
was found to be the best for enzyme production.
The scale up studies for alpha amylase production was carried out in a 7.5 L
stirred fermenter. The rate of fermentation for enzyme production by both wild and
mutant strains was investigated.) It was found that the enzyme production increased
gradually and reached maximum (335 U/ml and 608 U/ml) after 64 h (wild) and 48 h
(mutant). The kinetic depiction of results showed optimal fermentation period for
enzyme production to be 64 h and 48 h, respectively. The other cultural conditions
such as initial pH (5), aeration level (1.5 vvm), dissolved oxygen (15 %), inoculum
size (10 %) and agitation intensity (200 rpm) were optimized for enzyme production.
The fermented broth was subjected to ammonium sulfate precipitation at
different saturation levels (20-90 %). The optimum level of ammonium sulfate
saturation was found to be 70 % that gave 1.3 fold purification. By using Sephadex-
DEAE column, the active fractions were eluted using 0.05 M Tris-HCl buffer
containing 0.30 M NaCl at pH 7.5. The molecular weight of alpha amylase was found
to be 48 kDa on SDS-PAGE after gel filtration. A total of 9.5 fold enzyme purification
was accomplished. The effect of time, temperature, pH and metal ions on purified
enzyme was also investigated and maximum activity was achieved after 30 min at
40ºC and pH 5 in the presence of Ca+2 ion.
1
INTRODUCTION
The starch degrading enzyme alpha amylase (α-1,4 glucan-glucanohydrolase EC 3. 2.
1. 1) is widely distributed in nature. This extracellular enzyme hydrolyses α-1,4
glucosidic linkages randomly throughout the starch molecule in an endo-fashion
producing oligosaccharides and monosaccharides including maltose, glucose and
alpha limit dextrin (Omemu et al., 2005; Bhanja et al., 2007; Leman et al., 2009).
Alpha amylases are one of most important and widely used enzymes whose spectrum
of application has widen in many sectors such as clinical, medicinal and analytical
chemistry. Beside their use in starch saccharification they also find applications in
food, baking, brewing, detergent, textile and paper industries. These are important
enzymes used in starch processing industries for hydrolysis of polysaccharides such as
starch into simple sugar constituents. Increasing utility and consumption of alpha
amylase in different industries has placed a greater stress on increasing indigenous
enzyme production and search of more rapid processes (Carlsen et al., 1996;
Ramachandran et al., 2004; Kathiresan and Manivanan, 2006; Gupta et al., 2008).
Alpha amylase can be derived from several sources such as plants, animals and
microorganisms, but production from first two groups is limited for several reasons.
The concentration of enzymes in the plant material is generally low so the processing
of large amount of plant material is necessary; on the other hand enzyme of animal
origin is by- product of meat industry. In contrast, microbial source of alpha amylase
can be produced in amount meeting the demands of market. Different fungal and
bacterial strains have been extensively used for the enzyme production (Pandey et al.,
2
2000). Filamentous fungi have been well known for the starch and cellulose degrading
enzymes they naturally secrete. The ability of filamentous fungi to secrete large
amounts of extracellular protein has made them well suited for the industrial enzyme
production. The commonly used fungi included Trichoderma sp. Themomyces
lanuginosus, Penicillium griseoreseum, Fusarium moniliformis, Actinomycetes sp. and
Alternaria sp. (Arnesen et al., 1998; Ray 2001; Poornima et al., 2008). Many species
of Aspergillus such as A. niger, A. tamarii, A. awamori and A. oryzae have received
most attention to obtain many kinds of hydrolytic enzymes like alpha amylase, lipase
and protease. However, A. oryzae is the organism of choice because of its ubiquitous
nature, non fastidious nutritional requirements and high productivity of alpha amylase
(Abe et al., 1988; Archer and Wood, 1995; Agger et al., 2001; Zangirolami et al.,
2002).
Strain selection is a critical step in the development of a biotechnological
process, and it is based on a number of factors such as physiological stability, yield
consistency, incubation time required for maximum production as well as the
tolerance to temperature, aeration and shear stress etc. (Laluce et al., 1991).
Production of enzyme is greatly effected by the cultivation method. Alpha amylase
can be produced both by solid state and submerged fermentation technique (Prescott
and Dunn, 1987; Nielsen et al., 1995; Yovita et al., 2005). The liquid culture used in
submerged fermentation was usually preferable to solid state culture not only due to it
allowing better aeration and proper agitation but also the separation of enzyme from
the solid substrate is more difficult than submerged fermentation (Alazard and
Raimbault, 1981). Morphological variety is a typical feature of filamentous fungi. Its
3
morphology has distinct effects both on the enzyme production and rheological nature
of a fermentation broth. In submerged fermentation, two extreme types of morphology
are generally known, pellets and free filaments. Between these two extremes lies an
intermediate aggregated morphology called clumps (Wang et al., 2005). The
morphology of filamentous organism during enzyme production varies from round
pellets to free filaments depending upon the cultural conditions and strain genotype.
Traditional methods for strain improvement, such as ultra violet (UV)
radiation, use of alkylating agents like N-methyl N-nitro N-nitroso guanidine (NG),
ethyl methane sulphonate (EMS) and nitrous acid to obtain superior mutants have
been proved successful by subjecting the microorganisms to these mutagens, followed
by suitable selection and screening of the survivors (Szafraniec et al., 2003). However,
strain improvement is trial and error process involving laborious procedure. Rational
selection procedures are more efficient and usually have a biochemical basis (Elander
1982). In primary screening prior to laboratory fermentations, rational selection is
achieved by the use of techniques allowing visual identification of superior mutations.
The selection of alpha amylase producers using the size of the zone of hydrolysis of
starch is an example. However zonation can not in any way be correlated
quantitatively with the amount of alpha amylase produced because the hydrolytic
activity of other amylolytic enzymes such as glucoamylase. Therefore isolation of
improved producers of alpha amylase using starch plate can only be partially selective
(Kuek and Kidby, 1984). Mutant strains of Aspergillus oryzae were found to be best
for enzyme production compared to wild strain. It was studied a mutant strain of A.
oryzae showed more dextrinizing and saccharogenic activity than the parental strain.
4
In case of mutagenic application to the wild strain, better initial improvement can be
expected. A strain of A. oryzae treated with NG gave better enzyme production
compared to the parental strain. A best mutant for alpha amylase production can be
obtained by irradiating the fungal strain to the UV irradiation and then successive
treatment with mutagenic chemicals like NG, EMS etc. (Spohr et al., 1998; Qirang
and Zho, 1994; Azin and Noroozi, 2001)
Selection of suitable fermentation medium and initial pH is very important for
the enhanced alpha amylase production. All microorganisms require energy and
certain minerals for growth and metabolism. The energy for growth generally comes
from the oxidation of medium components. The presence of carbon, nitrogen sources
and mineral nutrients such as phosphorous, potassium, magnesium, and calcium are
essential for the growth of fungi as well as enzyme production (Hughes and Poole
1991).
The enzyme production has been greatly affected by the addition of different
carbon sources. The carbon sources affect not only the mode of amylase formation but
also the rate with which carbohydrates are metabolized (Dubey et al., 2000; Abdullah
et al., 2003). The influence of different carbon sources such as glucose, maltose,
fructose, galactose and sucrose on the alpha amylase production by A. oryzae was
studied and it was found that starch and maltose strongly increased enzyme
productivity by A. oryzae where as glucose led to very low productivity (Lachmund et
al., 1993; Carlsen and Nielsen., 2001). So it is important to select suitable carbon
source for the enhanced enzyme production. Fungal strains have been grown on
starch, maltodextrin, dextrin, maltose, amylopectin, glucose and dextran. All these
5
substrates exhibited good alpha amylase production. The optimum pH of fermentation
medium was found and fixed to 4.9 by using 100 mM citrate buffer for the enzyme
production. Various concentrations of soluble starch and soybean meal were used in
cultivating the organism. The highest enzyme activity was recorded with starch
(Omidiji et al., 1997; Moreira et al., 1999). A strain of Aspergillus sp. was able to
produce enzyme in mineral media supplemented with 1.0 % (w/v) starch or maltose as
carbon source. The alpha amylase production was found to be tolerant to a wide range
of initial pH values (4.0-10) and temperature (25-42°C). Aspergillus sp. isolated from
soil produced extracellular glucoamylase and alpha amylase using wheat starch as a
carbon source. The enzyme productivity was doubled by the addition of α-methyl-D-
glucoside to the medium (Junichi et al., 1988).
Different inorganic and organic nitrogen sources and their concentrations have
major influential impact on their ability to synthesize the enzyme as well as on the
growth of organism (Bailey and Ollis, 1977; Bajpai and Sharma, 1989; Hashim et al.,
1993). Both inorganic and organic nitrogen sources were tested for alpha amylase
production. Among the inorganic nitrogen sources, nitrate has been shown to be
inferior to ammonia. A mixture of ammonia and complex nitrogen sources such as
yeast extract or casein hydrolysate was found to be better than ammonia as nitrogen
source. Low concentration of casein hydrolysate resulted increase in alpha amylase
productivity (Pedersen and Nielsen, 2000). The organic nitrogen sources such as
peptone, yeast extract, tryptophan and corn steep liquor are widely used for enzyme
production. By the use of these nitrogen sources organism grew better and produced
higher levels of enzyme activity. However, urea and casein hydrolysate showed
6
marked effect on enzyme production by A. oryzae (Kammoun et al., 2008). Influence
of inoculum age and size on alpha amylase production should be optimized in depth
investigation before scaling up a high-yielding fermentation process (Bokosa et al.,
1992). The amount of inoculum introduced into the culture medium determines the
extent and quality of enzyme produced. So, there exists a correlation between amount
of inoculum and substrate concentration in context to alpha amylase production by A.
oryzae. Surfactants play an important role in increasing the enzyme production. Alpha
amylase activity was increased in the presence of surfactants because surfactants
increase the cell membrane permeability as a result enzyme secretion increased.
Different surfactants such as Tween 80, Triton X-100 and poly ethelyen glycols were
used to increase the permeability of cell membrane (Arnesen et al., 1998; Yoon et al.,
2005)
Fermenters of different working volumes may be used for the large scale alpha
amylase production as an industrially important enzyme under controlled conditions.
By optimizing the cultural conditions such as inoculum size, nutritional requirements,
temperature, pH, agitation, aeration, and dissolved oxygen etc. the enzyme production
can be enhanced by many fold (Gigras et al., 2002). Enzyme production commences
at a low rate during the logarithmic growth phase but reaches its maximum value
during the stationary phase towards onset of sporulation. Time course study and
agitation determines the efficacy of the batch process and subsequent product
formation. The pattern of accumulated reducing sugar after specific incubation time is
characteristic to the species (Matrai et al., 2000). Alpha amylase production at
different agitation rates (100-300) at 30°C were tested and maximum amount of
7
enzyme was obtained at 150-200 rpm after 72 h. According to Francis et al. (2002),
the maximum alpha amylase production was obtained after 120 h in a fermenter
operating at 300 rpm and airflow of 11/L/min in a limited dissolved oxygen
concentration. It was determined that the increase in agitation rate was not favorable
for enzyme production; despite of this an increase was verified in dissolved oxygen.
Enzyme production was superior with the A. oryzae NRRL 6270 at 30ºC after 96 h
when spore suspension used 1 x107 spores/ml.
Industrial enzymes produced in bulk generally require little downstream
processing and hence are relative crude preparations. The applications of enzyme in
pharmaceutical and clinical sectors etc. require high purity amylase. The enzyme in
purified form is also a prerequisite in studies of structure function relationships and
biochemical properties. The purification of enzyme is to remove as completely as
possible all the proteins except which possess the specific enzyme activity desired. A
frequently used method in enzyme purification is salt fractionation. Ammonium
sulfate is often used for this purpose because of its high solubility (700 g/l) which
permits the salting out of any protein. The properties of alpha amylase in culture broth
were examined by partially purified enzyme with 60 % ammonium sulfate. Alpha
amylase from Aspergillus sp. subjected to purification and characterization under
optimum conditions. The enzyme was purified by ammonium sulfate precipitation and
Sephadex G200 filtration. The purification of alpha amylase resulted 9.97 fold
purification. The optimum substrate (starch) concentration was 0.2 % (W/V) while the
optimum incubation temperature was 35°C. The purified enzyme had maximum
activity at pH 6.2, after 30 h of incubation (El-Safey and Ammar, 2002; Pimpa 2004).
8
Alpha amylase purified from the cultural broth of A. oryzae indicated 12.6 fold
purification and yield being 25.3 %. The molecular weight of alpha amylase from A.
oryzae was estimated to be 50 kDa. The purified enzyme was most active at pH 4.5
and temperature 55°C (Kariya et al., 2003).
The production and stability of the enzyme is very sensitive to pH and
temperature. Fungal alpha amylase was unstable above 45°C but at 25°C attack raw
starch granules more efficiently than enzyme from Bacillus amyloliquefaciens. The
optimum growth conditions for enzyme production by A. oryzae was pH 5.0 and 35oC
(Fairbairn et al., 1986; Jin et al., 1998). The enzyme retained 94 % activity in 1 h at
60°C. The alpha amylase is an unusual enzyme which converts starch to maltose in >
75 % yield. The purified enzyme, obtained in 11 % yield had optimal temperature and
pH 50-55°C and 5.0- 6.0, respectively. It may be of industrial value in the production
of low viscosity corn syrups (Hidaka et al., 1980).
Objectives
Specific objectives of present work are as follows
1-Isolation, identification and screening of A. oryzae strains.
2-Random mutagenesis by UV and chemicals to improve the fungal strain as well as
enzyme production.
3-Optimization of cultural conditions for the selected strain of A oryzae in shake flasks.
4-Scale up studies of enzyme production in a laboratory scale stirred fermenter.
5- Purification and characterization of alpha amylase.
9
REVIEW OF LITERATURE
Starch degrading amylolytic enzymes is of great importance in biotechnological
application ranging from food, fermentation, and textile to paper industries etc. Alpha
amylase is a key enzyme in metabolism of spacious diversity of living organisms which
utilize starch as carbon and energy sources. It can hydrolyze starch, glycogen and
related polysaccharides by randomly cleaving internal α-1,4-glucosidic linkages to
produce different sizes of oligosaccharides. Amylases are enzymes which hydrolyze the
starch molecules in to polymers consists of glucose units. Alpha amylase is ubiquitous
in distribution, with plants, bacteria and fungi being the major sources. Most of the
microbial alpha amylases belong to the family 13 glycosyl hydrolases, and they
contributed numerous common properties. But different reaction specificities have been
observed across the family members. Structurally alpha amylase possesses barrel
structures and is responsible for hydrolysis or formation of glycosidic bonds in the α-
conformation. Stability of alpha amylase has extensively been studied; pH and
temperature have very vital roles to play.
Alpha amylase acts on starch and breaking them up into sugars (hence the term
saccharification). Starch is a carbohydrate source consisting of two molecules amylose
and amylopectine. Amylose is formed from chains of glucose linked α1,4 and
amylopectine is formed from α1,4 linked chains of glucose with 1,6 linked branch
points. The amylases are enzymes that work by hydrolyzing the straight chain bonds
between the individual glucose molecules that make up the starch chain. A single
straight chain starch is called an amylose. A branched starch chain (which can be
10
considered as being built from amylose chains) is called an amylopectin. These
starches are polar molecules and have different ends.
Alpha amylase can be derived from several sources such as plants, animals and
microbes. The microbial enzyme meets the industrial demands a large number of them
are available commercially and have almost replaced chemical hydrolysis of starch
processing industry (Pandey et al., 2000). The major advantage of using
microorganisms for the amylase production is economical bulk production capacity
and microbes are also easy to manipulate to obtain enzymes of desired characteristics
(Lonsane and Ramesh, 1990). Alpha amylase has been derived from several fungi,
yeasts, bacteria and actinomycetes, however, enzymes from fungal and bacterial
sources have dominated applications in industrial sectors. Fungal sources are mostly
terrestrial isolates such as Aspergillus species. Mode of action, properties and product
of hydrolysis differ, some what and depend on the source of enzyme. Two types of
enzymes have been recognized called as liquefying and saccharifying. The main
difference between them is that the saccharifying enzyme produces a higher yield of
reducing sugar than liquefying enzyme. Many scientists carried out extensive work on
11
alpha amylase production. The enzyme production is dependent on the type of strain,
composition of media and methods of cultivation. Generally fungi secrete alpha
amylase (dextrinizing enzymes) although a few fungi have been known to secreted
alpha amylase and beta amylase (saccharifying enzymes). A. oryzae EI 212 secrete
alpha and beta amylase or both depending upon the composition of media and
fermentation conditions. The nature and amount of extracellular amylase produced by
Aspergillus species determine the efficiency of conversion of starch to
oligosaccharides.
Tokhadze et al. (1975) isolated 86 strains of the Aspergillus producing
maximum acid stable alpha-amylase. Repeated cultivation of the selected strains in
the Minoda agar medium along with sodium nitrate during submerged cultivation
showed a 3-fold increase in the alpha amylase production. Yabuki et al. (1977) studied
rapid induction in the alpha amylase production by A. oryzae using inducer such as
maltose. The mycelia were taken from 20 h old cultures and cultivated on the medium
containing peptone and glycerol. Afterwards these cultures were starved for 5 h; in
this case maltose was added as inducer. During first hour of induction, both extra and
intracellular alpha amylases were produced with the same rate (70-80/µg of cells/h).
After 1.5 h remarkable increase in alpha amylase production takes place and enzyme
production reached at optimum rate. No significant increase was occurred in the
weight of mycelia during 2 h of induction. When the purified samples of these intra
and extracellular enzymes were tested by using diethylaminoethylcellulose column
and techniques of gel filtration, both enzymes were showed similar properties in all
respects. Vallier et al. (1977) observed alpha amylase production after the lysis of
12
mycelia. For this purpose, mineral medium was used which consist of starch and
glucose. The lysis of mycelium seems to be due to the action of hydrolyzing enzyme
dextranase and levulanase on the cell wall. The pH of the media has great impact on
the lysis of cell wall and alpha amylase secretion. With increase in the pH of mineral
medium up to 8.8 the secretion of enzyme and lysis of mycelial wall were greatly
increased. This method makes it easy to get 3 times more enzyme production.
Sinha and Chakrabrty (1978) reported Aspergillus wentii hydrolysed the soluble
starch in to maltose. The optimum amylase production by using A. wentii was obtained
when fermentation medium consisted of Tryptophan as nitrogen source along with 1
% starch which was incubated for 72 h at 20°C and pH of medium was adjusted at 6.
The enzyme activity was greatly inhibited with the addition of 1mM sodium
iodoacetate. However, enzyme production was increased 3.51 to 6 mg/ml with the
addition of 10 mM sodium citrate. Varnavskaia et al. (1978) studied the impact of pH
on the protein conformation and alpha amylase activity produced by using Aspergillus
terricola. Dispersion of optical rotation technique showed that macromolecule of
alpha amylase consists of alpha helix and beta structures. The change in the values of
pH resulted in two conformational forms. When decrease in pH occurred from 4-2
alpha helix structure uncoiled and degradation of beta forms occurred with the
increase in the pH from 8-12.
Mahmoud et al. (1978) reported the use of different agricultural by-products and
wastes such as wheat bran, rice bran, cane molasses, corn bran, glucose syrup, corn
starch as a substitute of original carbon source in the fermentation medium for the
synthesis of alpha amylase by Aspergillus niger NRRL-337. The medium containing
13
rice bran showed maximum alpha amylase activity. The nitrogen source also
substituted by such type of material that makes the medium economic such as corn
steep liquor, corn steep precipitate, dried yeast and gluten-30 and 50. Corn steep
precipitates give highest alpha amylase production compared to other nitrogen
sources. From these results, it was concluded the medium containing rice bran 7.2 %,
corn steep precipitate 2.5 %, magnesium sulfate 0.1 %, potassium di hydrogen
phosphate 0.1 % and calcium carbonate 0.1 % showed maximum activity. The fungal
amylase was isolated and purified from this medium. The purified enzyme showed
optimal activity at 40°C and pH 4.3. Allen and Thoma (1978) studied alpha amylase
produced from A. oryzae acts on reducing ends, and maltotriose which was uniformly
labeled. The enzyme breaksdown the glycosidic bonds during enzyme substrate
formation.
Augustin et al. (1981) examined the activity and production of alpha amylase
and alpha glucosidase in the some members of ascomycetes, imperfect and mucoral
fungi. The factor of polysaccharide system which was responsible for the consumption
of alpha(1 to 4) glucans was described along with screening of the growth of
organism or fungi on soluble starch. Forty nine strains were tested for the production
of amylolytic activity and only twenty nine strains showed this activity. Kasim (1983)
investigated the biosynthesis of alpha-amylase and amyloglucosidase (EC.3.2.1.3) by
A. oryzae in submerged fermentation. For this purpose different sources of carbon and
nitrogen were tested. The medium which shows maximum production of alpha
amylase and glucoamylase was not very costly and consists of following components
in (%) corn steep liquor 3, magnesium sulfate 0.1, potassium dihydrogen phosphate
14
0.1, defatted rice bran 8 and calcium chloride 0.1. The pH of medium was adjusted at
5. The optimum conditions for enzymes production were incubation at 28°C for 96 h
and the inoculum consists of 0.5 % mycelial suspension. Erratt et al. (1984) reported
that starch was used as inducer for alpha amylase production from the A. oryzae.
When glucose was used as carbon source the production of both intra and extracellular
amylase was very low. While starch was used as carbon source increase in the activity
of alpha amylase was noticed. In glucose grown cultures intracellular activity of alpha
amylase increased 6.5 fold; however, 20 fold increase was observed in extracellular
activity. Regardless of type of carbon source used, the active protein react only those
antibodies which showed specificity only for alpha amylase and active protein have
molecular weight 52 500 +/- 1800.
Ustiuzhanina et al. (1985) studied the regularities in the biosynthesis of protease
and alpha amylase by using washed cells of selected strain of A. oryzae. The results
enabled us to compare the constitutive characters of protease and alpha amylase by
selected strain of A. oryzae. Carbon, nitrogen and sulfur play very important role in the
regulation of protease synthesis. However, in alpha amylase synthesis, merely carbon
source played an important role. Phosphorous was vital for the synthesis of both alpha
amylase and protease. Removal of phosphorous from the medium adversely affects the
production of both enzymes. The alpha amylase and protease production was
stimulated by the addition of celatin.
Hayashida and Teramoto (1986) reported that a protease negative mutant M33 of A.
ficum was obtained by treating A. ficum with MNNG. This strain showed highest alpha
amylase activity compared to parent strain in submerged fermentation at optimal
15
condition i.e. 30°C for 24 h. The molecular weight of purified enzyme was 54, 0000.
MacGregor (1988) studied two computerized methods which explain the sequence of
amino acid in the secondary structure of protein in alpha amylase which was produced
by A. oryzae. Alpha-amylase produced by A. oryzae, showed three dimensional
structures. The computerized methodology explained the position of amino acid and
gave the predictions about the structure of alpha amylase from different sources. It
was noticed all alpha amylase having known amino acid sequence possess same basic
structure, these alpha amylase possess barrel shape structure which was surrounded by
eight helices. The strong resemblance were found in those part of protein which take
part in binding the Ca+2 ions and active site of enzyme which play important role in
catalyzing the substrates hydrolysis. The active site was composed of amino acids
which were specifically found in the loop joining the adjacent helix. The changes in
the length and sequence of amino acid created the differences in binding the substrate
and produced modifications in the action pattern of alpha amylase from different
origins.
Ali and Abdel-Moneim (1989) reported that the best temperature for the
preservation of A. flavus var. columnaris alpha-amylase was -5°C followed by 5°C.
CaCl2 at 0.005 M had no effect on the activity in both temperatures. Repeated freezing
(-5°C) and thawing followed by freezing (-5° C) had no effect on stability of alpha-
amylase. On the other hand, 25°C was the lowest preservation temperature without
any effect on the stability of alpha-amylase. 0.005 M CaCl2 decreased the activity of
alpha amylase and reached a 100 % inhibition at 35th day. The fungal alpha amylase
had an optimum temperature of 55°C at pH 4.6, but had 60°C in buffer containing
16
0.005 M CaCl2 and 50°C in buffer containing 0.005 M Na2-EDTA. The addition of
0.01 M CaCl2 greatly increased the thermostability of alpha amylase at 40, 45, 50, 55
and 60°C for 30 min. Optimum pH for alpha amylase was 5, but in the presence of
0.01 M CaCl2 or Na2-EDTA 5.6. The enzyme was only stable for 4 h at 25°C.
Whereas, addition of 0.01 M CaCl2 showed a loss of 4 % compared to a 22 % loss in
the presence of 0.01 M Na2-EDTA after 4 h at 25°C and 65 % loss in the presence of
0.01 M CaCl2 together with 0.01 M Na2-EDTA in the beginning and a 100 % loss
after 4 h at 25°C. The optimum temperature for the activity of alpha-amylase at pH 5
was 50°C for the enzyme only but 55°C in the presence of 0.01 M CaCl2. However, at
pH 6 and 7 optimum temperature was 55°C for the activity of the enzyme only or with
0.01 M CaCl2. The presence of 0.01 M CaCl2 at pH 5, 6 and 7 resulted in increase of
enzyme activity at the temperatures above 50, 40 and 25°C, respectively. However,
0.01 M CaCl2 at pH 5 and 6 resulted in decreasing enzyme activity at temperatures
below 55 and 45°C, respectively.
Rousset and Schlich (1989) screened different species of A. niger for the
synthesis of amylolytic enzymes i.e., alpha amylase and glucoamylase by using the
submerged fermentation. Statistical analysis was used to explain the behaviour of
culture instead of explaining optimization of fermentation conditions. Principal
component analysis (PCA) was used to explain the affect of three agitation rates on
amylase production and the formation of many other factors which affect the growth
in indirect way. The result of Principal component analysis (PCA) describes the
transfer of oxygen at different agitation rate influences enzyme production and carbon
dioxide. The production of carbon dioxide was indirect growth measurement.
17
Maximum alpha amylase production was obtained at lower agitation speed while in
case of glucoamylase intermediate agitation speed gave maximum alpha amylase
production. Shah et al. (1991) optimized the conditions for the synthesis and recovery
of alpha amylase from A. oryzae. A. oryzae alpha amylase retained 100 %, 61 % and
58 % respectively when preserved for 12 months at 4°C, ambient temperature and
37°C. Harway (1991) has isolated thermophilic bacteria from the soil which was
preliminary enriched with 0.6 % starch broth at 55°C. These bacteria had ability to
hydrolyze the starch. Of the entire isolated cultures one was Bacillus coagulans, which
was best producer of alpha amylase. The maximum production was obtained in
optimal condition which consists of incubation temperature 55°C, 200 rpm agitation
speed, 48 h incubation period and broth extract starch agar medium.
Tsekova et al. (1993) studied the ability of Aspergillus genus for alpha amylase
production. When 3 % soluble starch was used in Czapek-Dox agar and in liquid
Czapek-Dox media maximum alpha amylase production was obtained. Sudo et al.
(1993) studied the fermentation medium containing all the components which were
necessary for the production of acid stable alpha amylase (asAA) by A. kawachii using
submerged fermentation. One hundred and thirty milligram of acid stable alpha
amylase per liter of medium was produced after 5 days of inoculation at 30°C in
submerged fermentation. Glycogen was present as stored polysaccharide. When the
amount of stored glycogen (CSG) decreased and inducer such as dextrin was present
synthesis of acid stable alpha amylase started. Maximum production of as AA was
obtained when amount of CGS reaches at zero. When the amount of CGS increased
production of acid stable alpha amylase tend to be decreased. The amount of glucose
18
in the medium and growth of mycelia was strongly influence by the concentration of
CGS. The quantity of acid stable alpha amylase was directly proportional to the
production of mycelia. Soccol et al. (1994) tested two species of Rhizopus for protein
enrichment of both cooked and raw cassava and also for the synthesis of
amyloglucosidase and alpha amylase in solid and submerged fermentation. The
protein enrichment and maximum enzyme synthesis were obtained in solid state
fermentation. Cooked cassava showed optimum production in solid state fermentation;
however, maximum synthesis of amyloglucosidase by R. oryzae was obtained when
raw cassava was used. Khoo et al. (1994) achieved fifty units per milliliter amylolytic
activity by using A. flavus in liquid medium containing topica starch. The culture
filtrate was subjected to electrophoretic analysis. This analysis showed filtrate contains
only one type of amylolytic enzyme named alpha amylase. The following factor
support the identification of alpha amylase (i) iodine stained starch quickly become
colourless (ii) starch digestion resulted in the formation of a mixture of glucose,
maltose, maltotriose and maltotetrose. Purification of enzyme was involve the use of
ammonium sulfate precipitation, ion exchange chromatography and gel
electrophoresis. The purified enzyme showed 52.5 ± 2.5 kDa molar mass with an
isoelectric point at pH 3.5. Characterization of enzyme showed the maximum activity
of purified enzyme was noticed at pH 6 and 55°C.
Omori et al. (1994) isolated acid labile alpha amylase (A-3) from A. kawachii in
barley koji. The enzyme was purified by using the different techniques such as ion
exchange chromatography and gel filtration. The changes in new alpha amylase
production was compared with two known alpha amylase represented as A-1 and A-2.
19
Sodium dodecyl sulfate poly acrylamide gel eletrophoresis showed A-3 has molecular
weight 56,000. The enzyme showed constant production at 40°C approximately for 54
h. However, in traditional method formation of A-3 was not detected after 36 h. In the
presence of 2 % citric acid in barley A3 was formed upto 36 h. The results indicated
production of A3 was influenced both by temperature and initial concentration of
citric acid. Chang et al. (1995) reported that alpha amylase produced by A. oryzae
was purified by passing through the different steps in a specific sequence such as
amylopectin affinity adsorption, DEAE-Sepharose ion exchange chromatography and
sephacryl S-200 HR gel filtration. After passing through these steps the enzyme
showed 16 fold increase in the purity and 45 % of enzyme was recovered. The
optimum conditions for purified enzyme was pH range 4-5, temperature 50°C and km
value for starch hydrolysis was 0.22 %. Incubation for 30 min at 50°C result 80 % lose
in enzyme activity. The heat denaturation constant and molecular weight by gel
filtration was 0.024/m and 52 kDa, respectively. The enzyme activity was inhibited by
using Mercuric ion (0.3m M), DNFB# (6mM), NBSI (6mM) and NAI (6mM). The
hydrolysis of maltoheptaose by the enzyme resulted in the formation of maltotriose
and maltotetraose.
Donmez and Melike (1996) isolated bacteria showing amylolytic activity from
different samples and grouped them on the basis of showing amylolytic activity in the
solid and liquid fermentation media. Of all the isolated strains Bacillus subtilis
produce 24 U/ml alpha amylase. Different carbon sources were added to the
fermentation media to check effect of these carbon sources on alpha amylase
production. The maximum activity of alpha amylase 360 U/ml was obtained in the
20
presence of dextrin and optimum temperature was 50°C for enzyme production.
However, if enzyme was incubated for 2 h at 100°C 23 % of this activity was lost.
Carlsen et al. (1996) tested the stability of alpha amylase produced by A. oryzae at
different pH. The enzyme showed highest stability at neutral pH (5-8); however,
beyond this pH range a great loss in the activity of enzyme was noticed. On line FIA
system was used and a rate constant was obtained by the empirical expression k = 1.19
× 107 [H+]1.99 (h−1) explained the inactivation of enzyme was greatly influenced by pH
values. The inactivated enzyme again obtained some of its activity at pH 6 and this
reactivation steps also obey the first order kinetics rules. The contamination of
protease in the protein sample was not result to the irreversible loss of activity.
Abou Zeid (1997) isolated filamentous fungi from cereals and screened to test
the alpha amylase producing potential. The strain which showed highest ability for
alpha amylase production was identified as A. flavus. The enzyme was purified by
using starch adsorption methodology. The polyacrylamide gel electrophoresis (PAGE)
indicated the molecular weight of A. flavus was 75, 000 ± 3,000. The optimum
temperature for purified enzyme was 7 and 30°C, respectively. The use of potassium
ions increased the activity of alpha amylase. However, magnesium ions did not
extremely influence the enzyme activity. The activity of alpha amylase was greatly
inhibited in the presence of manganese, zinc, copper and ferric ions. The hydrolysis of
native starch by A. flavus resulted in the formation of glucose and some other
oligosaccharides
Kajiwara et al. (1997) studied the production of acid stable alpha amylase from
A. kawachii during production of shochu-koji. From barley shochu-koji two types of
21
the acid stable alpha amylase (as AA) represented as as A-1 and as A-2 were puified.
The asA-1 and asA-2 showed different adsorption characteristics on raw starch. The
activity of as A-1 slowly increased during the process of shochu-koji production but
dropped after incubation of 24 h. Contrary to as A -1 the activity of as A-2 was
increased with increase in the incubation time. Temperature affect ratio of as A1 to
total as AA activity. When acid protease and as A-1 were incubated along with each
other and this sample was analyzed by SDS-PAGE. A known band was appeared in
the place of as A-1 band after 12 h of incubation. The unknown protein showed all the
characteristics which were present in as A-2. The result showed acid stable alpha
amylase was found in different form just like the glucoamylase produced by A.
awamori during the production of shochu-koji. Spohr et al. (1997) examined alpha
amylase producer strain of A. oryzae for the production of recombinant protein and
affect of growth on the production of protein. The comparison of these strains for
morphology and impact of morphology on the protein indicated the mutant strain
having denser mycelium, produce more alpha amylase compared to other strains.
Arnesen et al. (1998) cultivated thermophilic fungus in the presence of dextran
(having low molecular weight) along with Tween 80 or Triton X-100. The
fermentation was carried out in shake flasks for more than 120 h. The 2.7 fold increase
in the activity of alpha amylase was observed in medium containing Tween 80
compared to the medium with out Tween 80. The medium containing Tween 80
showed increase in the alpha amylase production after 48 h; while general protein
secretion was stimulated after 24 h of inoculation. The Tweeen 80 also influences the
production of biomass. The production of biomass increased gradually with the
22
increase in the concentration of Tween 80. Contrary to this Triton X-100 produce
reverse effect. It was noticed increase in the amount of Tween 80 resulted greater than
3 fold increase in the total extracelluar protein. The Tween 80 had no effect both on
the hyphal length and diameter. Glycosylation degree was also not effected by the
Tween 80. Anidyawati et al. (1998) purified three forms of alpha amylase to
homogeneous state by using the methodology of column chromatography. These
forms of alpha amylase were produced from A. awamori. These forms were
designated as Amyl1, Amyl 11, and Amyl 111. The SDS PAGE indicated these three
forms possess 49,000, 63,000 and 97,000 molecular weight, respectively. The
optimum pH for Amyl 11 and Amyl 111 was 5.5 while in the case of Amyl 1 the pH
was 4. Maltose and maltosetriose were formed by the hydrolyzing action of Amyl 1
on malto-tetraose-pentose,-hexaose,-heptose and β and γ-cyclodextrin. However Amyl
1 produces no hydrolyzing effects on raw corn starch, maltose, maltotriose,
isomaltotriose, isomaltosse, and α-cyclodextrin. Unlike Amyl 1 both Amyl 11, and
Amyl 111 have ability to hydrolyze maltotriose, raw corn starch and alpha, beta,
gamma cyclodextrin resulting in the formation of maltose along with minor products
of glucose and maltose. The range of soluble starch hydrolysis through Amyl 1, Amyl
II and Amyl III was 33, 35 and 38 %, respectively.
Jin et al. (1998) used A. oryzae for alpha amylase production and microbial
biomass protein (MBP) from starch processing waste water (SPW) in air lift
bioreactors. The production of MBP and fungal alpha amylase was carried out under
the optimized conditions i.e., pH 5 and 35°C. Bioproduct yield obtained from 12h
batch culture was 6.1 g/l. This yield consists of 55 EU/ml of alpha amylase and 38 %
23
protein. The enzyme showed stability at pH 5-9 and 25-35°C. Spohr et al. (1998)
tested three different strain of A. oryzae having the ability to form recombinant protein
with respect to growth and alpha amylase production. One was wild strain and the
second strain was a transfomant strain which consists of additional copies of alpha
amylase gene while third strain was morphological mutant. It was observed the
production and growth of organism were correlated. Comparison of production and
morphology of these strains indicated the variations in the morphology had direct
impact on enzyme production in submerged fermentation.
Moreira et al. (1999) isolated a fungal strain from the soil having the ability to
produce amylolytic enzymes. This strain was identified as A. tamari. A. tamari formed
both alpha amylase and glucoamylase in the mineral medium concomitant with carbon
source i.e., 1 % starch or maltose. The formation of alpha amylase and glucoamylase
indicated tolerance to wide range of initial fermentation medium pH (4-10) and
temperature (25 - 42°C). Ion exchange chromatography was used for the separation of
alpha amylase and glucoamylase. Partially purified alpha amylase and glucoamylase
showed maximum activities at pH 4.5 and 6 and stability at pH 4-7. The temperatures
at which enzymes showed highest activities was between 50 - 60°C.
Pedersen and Nielsen (2000) reported the effect of organic and inorganic
nitrogen sources on alpha amylase production by A. oryzae in continuous cultivations.
Both nitrogen sources were tested along with glucose. In case of inorganic nitrogen
source ammonia was better than nitrate. The comparison between organic and
inorganic nitrogen sources indicated organic nitrogen for example yeast extract or
casine hydrolysate was superior to ammonia. In the presence of 0.05 g/l casine
24
hydrolysate 35 % increase in alpha amylase production was found. The transcription
of the alpha amylase genes were not involved in the increase production of alpha
amylase the basic reason was the grater secretion of alpha amylase from the biomass.
Nguyen et al. (2000) optimized the composition of fermentation media for increasing
amylases production through Thermomyces lanuginosus by using the different ways.
The influence of different carbon and nitrogen sources was tested. The carbon and
nitrogen sources, which proved to be good substrates for the growth of T. lanuginosus
and exbhited maximal alpha amylase (92-125 U/ml) and glucoamylase (6-13 U/ml)
activites included starch, maltodextrin, dextrin, maltose, amylopectin, glucose dextran
and L-asparagine. L-asparagine at the level of 6.5 % was good for alpha amylase
production and 2 % L-asparagine was optimum for glucoamylase production. The pH
of medium was adjusted by using hundred millimolar citrate buffer for amylases
production. Response surface method (RSM) was used to find out the suitable
concentration of medium component for the synthesis of amylolytic enzymes. A
second order polynomial model was used at significance level 95 % (p<0.05) for alpha
amylase and glucoamylase. The selected composition of media was tested with respect
to synthesis of amylolytic enzymes.
Mariani et al. (2000) studied impact of Amaranth seed meal and the aeration on
the productiviy of alpha amylase by A. niger NRRL 3112. The assays for the selection
of fermentation media was carried out by using the rotary shaker at 250 rpm and 2.5
cm stroke. The selection of aeration conditions were carried out in New Brunswick
mechanically stirrer fermenter A fermentation medium containing 5.0g/l Amaranthus
cruentus seed meal produce 2750 U.Dun/ml alpha amylase with dry weight of 8.0 g/l
25
after 120 h, of inoculation. The optimum condition for alpha amylase production in
fermenter were fermentation period of 120 h, agitation rate 300 rpm and an air flow of
11/l/ min in limited concentration of dissolved oxygen. Although increase in agitation
speed increases the dissolve oxygen but it was not suitable for the formation of alpha
amylase. Morphology of A. niger such as long and branched hyphae was very
important to obtain the maximum alpha amylase production. Petrova et al. (2000)
reported the purification of wild and mutant strains of Thermomyces lanuginosus
ATCC 34626 a thermophilic fungus. The purification was carried out to homogeneity
by using the different techniques in a sequence such as precipitation with ice cold
propanol, anion exchange and molecular sieve chromatographic methods. The SDS-
PAGE results indicated purified alpha amylases (both with PI values of 3.0) have
molecular mass 58 kDa. The optimum pH for the activity of wild and mutant strains
was 5 and 4.5, respectively. 1 – Cyclohexyl - 3 - (2-morpholinyl – 4 - ethyl) -
carbodiimide (40 – 100 mM) and N- bromo succinimide (0.1 – 1mM) produce
inhibitory effect on the enzymes activity due to the presence of carboxylic groups and
tryptophan residues in the catalytic process.
Madihah et al. (2000) isolated and partially purified alpha amylase from the
fermentation of sago starch to solvent by C. acetobuylicum P262. The characterization
of partially purified enzyme showed the following optimal conditions. The highest
activity of alpha amylase was observed at pH 5.3 while enzyme showed stability from
pH 3-9. The highest activity of alpha amylase was found at 40°C; however, if alpha
amylase was placed at 60°C for 60 min merely 50 % of its original activity was
retained. The Km and Vmax values of alpha amylase for soluble starch were 0.31 g/l and
26
10.03 U/ml, respectively. Viswanathan and Surlikar (2001) designed the medium by
the use of fractional factorial method and Plackett-Burman design to study the
influence of component of Amaranthus paniculatas (Rajgeera) medium on alpha
amylase production by A. flavus. Fifteen components were used in developing the
medium. Out of these components only four i.e., CSL, NaCl, CaCl2 and NH4HPO4
were choosed on the basis of contrast coefficient values and selected as independent
variables for the Box-Behnken design. By using SPSS/PC +(version 7.5) statistical
analysis a polynomial multiple regression model was prepared. CSL, NaCl, CaCl2 and
NH4HPO4 increased the yield up to 81.3 % however, NaCl, CaCl2 influence the
product to the tune of 68.3 %. The comparison of control and optimized medium
exhibited 8 fold increase in production of enzyme in the optimized medium.
Carlsen and Nielsen (2001) tested the effect of different carbon sources such as
fructose, galactose, mannitol, glucose, glycerol, sucrose, and acetate on alpha amylase
production by A. oryzae in carbon limited chemostat cultures. A. oryzae was not able
to grow on such a medium which contain galactose as only carbon sources; however, a
combination of glucose and galactose allow the fungal strain to grow and produce
alpha amylase. Medium containing maltose and maltodextin indicated more alpha
amylase production during growth of A. oryzae compared to medium containing
glucose concentration less than10 mg/l. Sucrose, glycerol and mannitol showed low
alpha amylase production. Acetate alone did not show any production of enzyme but
acetate along with little quantity of glucose exhibited alpha amylase production. It was
observed alpha methyl-D-glucoside was acted as an inducer for alpha amylase
production but it was not as good as glucose.
27
Agger et al. (2001) evaluated the influential impact of formation of biomass on
the synthesis of alpha amylase by using the wild strain of A. oryzae and recombinant
strain of A. nidulans in submerged fermentation. It was noticed specific rate of alpha
amylase production was inversely proportional to the concentration of biomass
formation. When the concentration of biomass was increases 2-12 g dry weight/kg the
specific rate of enzyme production was decreased. However in case of recombinant
strain of A. nidulans in which gene creA was removed (which cause carbon catabolite
repression) no marked decrease in the specific rate of enzyme formation was observed.
The results indicated less alpha amylase production at high biomass formation was
due to slow mixing rate of vital components in viscous culture medium.
Ray (2001) isolated Penicillium sp possessing the ability to form alpha amylase
and xylanase in the presence of starch and xylan respectively, in fermentation. It was
noticed the optimum amylolytic activity and xylanolytic activity was obtained on 4th
and 6th day of fermentation respectively. The quality of alkalophilic strain of
Penicillium sp to hydrolyze starchy and hemicellulosic wastes made them a potent
strain for the large scale economic production of both enzymes using the cheap
substrates. Bogar et al. (2002) tested different strains of A. oryzae on spent brewing
grain (SBG) and corn fiber for alpha amylase production. A Plackett-Burman
experimental design was practiced to develop optimized media for alpha amylase
production using best producer strain. A. oryzae NRRL 1808 strain produced 4519 U
of alpha amylase/g of dry matter substrate in stationary 500 ml Erlenmeyer flask
culture after 72 h. The crude enzyme, in situ enzyme produced in solid substrate
fermentation material was economic biocatalytic product for animal feed and for the
28
production of bio alcohol from starchy substrate. Francis et al. (2002) investigated the
effect of spent brewing grains on alpha amylase production by A. oryzae NRRL 6270
when spent brewing grains utilized as sole carbon source. Maximum alpha amylase
production was obtained at 25°C. At 30°C almost similar results were obtained.
Optimum alpha amylase production [6870U/g dry substrate (gds)] was obtained in
solid state fermentation at 30°C after 96 h by the use of suspension containing 1×107
spores/ ml. Addition of any external carbon source in the spent brewing grain resulted
decreased in alpha amylase production.
Arnesen et al. (2002) used thermophilic fungus T. lanuginosus for alpha
amylase production in shake flasks. The fermentation medium contained carbon
source in the form of low molecular dextran. The fermentation was carried out up to
120 h. The results showed maximum alpha amylase activity after 96 h of inoculation
during stationary phase while the production of maximal biomass takes place after 48
h of fermentation. A same pattern was observed in the case of total extra cellular
protein. It was found many unidentified proteins and alpha amylase were de novo
synthesis by using pluse labeling techniques of proteins. The sequencing of alpha
amylase from T. lanuginosus using specific primmer and RT-PCR technique indicated
that transcription of alpha amylase was not start before the late growth phase and
reached at its highest value more than 24 h after maximum biomass was produced.
Gigras et al. (2002) used the central composite design along with 3 variables
i.e., starch, yeast extract, and di potassium hydrogen phosphate for alpha amylase
production by A. oryzae in shake flasks and bioreactor. The alpha amylase production
was 133U/ml in shake flasks while in case of bioreactor production was 161 U/ml.
29
However, there was great difference in the fermentation period of shake flasks and
bioreactor. The fermentation period for the maximum alpha amylase production by A.
oryzae in shake flasks was 120 h but in case of bioreactor this time period was reduced
to only 48 h. A high concentration of phosphate in the fermentation medium and use
of low inoculum size was essential to prevent the unnecessary foaming in bioreactor;
but managing the pO2 level and agitation rate was not compulsory for alpha amylase
production. The enzyme production increases with the increase in the pH of medium
and reached at its peak at pH above than 7.5. Thus in present study pH act as sign of
commencement or ending of the enzyme production.
Huang et al. (2003) developed a segregated model to explore the intrinsic
associations between growth, substrate consumption, cell differentiation and enzyme
formation by Bacillus subtilis in bioreactor. The segregated model represented three
different states of cell and the change from vegetative stage to sporangium and lastly
to mature spore. An age-based population balance model was used to explain the
maturity of sporangium in the direction of the formation of spores. Parameters in the
model were found out by placing the experimental data in the model. The model has
ability to describe the temporary behavior of B. subtilis in both batch and fed-batch
cultures.
Francis et al. (2003) optimized incubation temperature, initial moisture contents
and inoculum size by application of Box–Behnken design under the response surface
methodology for the highest production of alpha amylase by A. oryzae NRRL 6270.
The experimental data was added into a polynomial model to find out alpha amylase
production. A Plackett–Burman design was used to test the influence of nineteen
30
nutrient components on alpha amylase production by the A. oryzae. Soybean meal,
CaCl2 and Mg SO4 were chosen on the basis of their positive affect on alpha amylase
production. A Box–Behnken design was used to select the best condition for alpha
amylase production. Incubation temperature 30°C, initial moisture contents 70 % and
an inoculum size of 1×107 spores/g dry substrate were the optimum conditions for the
alpha amylase formation by A. oryzae NRRL 6270 on SBG. Under selected conditions
of solid state fermentation SSF, about 20 % enhancement in enzyme production was
found. Kariya et al. (2003) purified alpha amylase from culture broth of A. oryzae
MIB 316. The enzyme had ability to efficiently hydrolyzed amylopectin, amylose and
starch and break down maltopentose to produce a maltotriose and maltose. However,
maltose did not produce glucose. The N-terminal sequence of first 10 residues and
many other molecular characteristics were similar to Taka-amylase.
Kusuda et al., (2003) isolated alpha amylase from an immobile culture filtrate of
Tricholoma matsutak. The enzyme was purified to homogeneity by sequential steps of
Toyopearl-DEAE, gel filtration, and Mono Q column chromatography. The alpha
amylase showed 3580 fold purity and 10.5 % recovery. SDS-PAGE analysis resulted
in a single protein band. The characterization of purified alpha amylase showed that it
was most active at pH 5–6 and having stability between wide range pH i.e., 4–10. The
experimental results also indicated the alpha amylase was somewhat thermostable and
showed thermostability at 50°C while the optimal temperature was 60°C. The size-
exclusion chromatography and SDS-PAGE showed that purified alpha amylase had
molecular mass 34 kDa and 46 kDa, respectively. The mercuric ion did not inhibit the
activity of enzyme. Measurement of viscosity, TLC and HPLC analysis indicated
31
amylases from T. matsutake was of endo type. The specificity of alpha amylase was
tested by using amylose along with various polysaccharides. This alpha amylase
rapidly hydrolyzed the α-1,4 glucoside linkage in soluble starch and amylose A
(MW,2900), but was not able to hydrolyze the α-1,6 linkage and cyclic
polysaccharides e.g α- and β-cyclodextrin.
Kanwal et al. (2004) extracted alpha amylase from Malus pumila (apple) by
homogenizing the apple in buffer for alpha amylase. After extraction, the enzyme was
purified by passing sequential steps of purification. The crude extract exhibited 3.09
U/ml alpha amylase activity was subjected to ammonium sulfate precipitation. This
partially purified enzyme produces 4.76 U/ml and showed 5.01 U/mg specific activity.
The enzyme was further purified by gel filtration chromatography (Sephadex G-150).
After gel filtration chromatograph it produces of 5.025 U/ml and specific activity
38.95 U/ml along with 20-fold purification. SDS-PAGE of enzyme removed the
undesirable proteins and single band of enzyme was appeared. Molecular weight of
alpha amylase was 51,180 D which was finding out by Sephadex G-150 column.
Amylase exhibited optimal pH 6.8, incubation temperature 37°C, Km value 2.0x10-3
g/ml, λmax 540nm and incubation time for enzyme assay was ten min.
Apar and Ozbek (2004) studied the effects of temperature on the enzymatic
hydrolysis of starch from different sources such as corn, rice and wheat. Three
commercial alpha amylases produced from Bacillus sp. A oryzae and B. licheniformis
were employed for hydrolysis of starch. In every starch hydrolysis process, the
concentration of residual starch and the residual activity of alpha amylase in
percentage were determined at 50 and 60°C temperature based upon the processing
32
time in a stirred batch reactor. Mathematical models were developed for using
experimental data of residual starch concentration from each source. Some
inactivation models were used to understand the relation between temperature and
stability of enzyme during hydrolysis of starch from enzymes having different origins.
El-Safey and Ammar (2004) reported that the amylolytic family has great
importance due to its wide spectrum of application. Alpha amylase produced from
Aspegillus flavus var.columinaris was isolated and characterized. The enzyme was
purified by using ammonium sulfate precipitation and Sephadex G200 filtration
method. The purified enzyme showed 9.97 fold purification and 6471.6 (units/mg
port/ml) specific activity. The alpha amylase activity amplified with the enhancement
of enzyme concentration. The optimum condition for the production of alpha amylase
was 0.2% (w/v) starch, while the optimal temperature was 35°C. The purified alpha
amylase showed maximum activity at pH 6.2 after 30 h of incubation. Pimpa (2004)
reported that the highest alpha amylase production by Aspergillus sp. was obtained
after 24 h. Addition of suitable nitrogen sources and inorganic salts to the medium
appreciably increased the enzyme production. The maximum enzyme yield 36.5 U/ml
was obtained in the media containing wastewater, defatted soyabean 10 g/l, potassium
di hydrogen phosphate 10g/l, magnesium sulfate 5 g/l, zinc chloride 0.1 g/l. The alpha
amylase produced by Aspergillus sp. showed catabolic repression. The enzyme was
partially purified by subjecting into 60 % ammonium sulfate. The optimal pH and
temperature of partially purified enzyme was 5 and 50°C, respectively.
Chavez et al. (2004) screened different carbon sources namely sorghum, soluble
potato, corn and cassava starches as well as maltose for the concurrent cultivation and
33
production of both alpha amylase and glucoamylase by a novel Trichoderma sp. even
though maltose gave better results compared to other carbon sources with respect to
activity of alpha amylase (about 28,000 U/l) and alpha amylase production (about 390
U/l/h), cassava and corn starches showed maximum glucoamylase activities (17,000-
18,000 U/l) and production of enzyme was almost similar to those obtained with
maltose (about 100 U/l/h). Because of its capability to produce both alpha amylase and
glucoamylase, the Trichoderma sp used in this study proved to be beneficial in a direct
process of raw starch saccharification with no preliminary gelatinization.
Konsula and Kyriakides (2004) isolated a somewhat thermophilic Bacillus
subtilis strain, from fresh milk of sheep possess the ability to produce extracellular
thermostable alpha amylase. The medium containing low starch concentration showed
maximum alpha amylase production at 40°C. The enzyme exhibited highest activity at
135°C and pH 6.5. The thermostability of alpha amylase increased in the presence of
calcium or starch. This thermostable alpha amylase was employed for the hydrolysis
of different starches. Ammonium sulfate crude enzyme preparations as well as the
cell-free supernatant actively break down the starches. The use of the clear supernatant
as enzyme source was highly advantageous mainly because it decreases the cost of the
hydrolysis. When the reaction temperature increased up to 70°C, all of the substrate
showed higher rates of hydrolysis. Potato starch upon hydrolysis produced higher
concentration of reducing sugars compared to other starches at all tested temperatures.
Soluble and rice starch came at second and third position respectively, with respect to
reducing sugar liberating ability. However, in case of corn and oat starch alpha
amylase showed somewhat less affinity.
34
Ramachandran et al. (2004) investigated alpha amylase production by A. oryzae
in solid state fermentation (SSF).The substrate used was coconut oil cake (COC). Raw
COC was a good substrate and 1372 U/gds alpha amylase was produced in 24 h. As a
result of optimization of media component alpha amylase production was increased
(1827 U/gds) when solid state fermentation was carried out at 30°C for 72 h along
with media contained 68 % moisture contents. Addition of glucose and 0.5 % starch
further increased the alpha amylase production (1911 U/gds). However, maltose
repressed the alpha amylase production. Alpha amylase production increased upon
adding the organic and inorganic nitrogen sources. When peptone at the level of 1 %
was added in the fermentation media 1.7-fold increase in enzyme activity (3388
U/gds) was observed. The enzyme production and growth were correlated. The
activity became maximal when the fungal biomass was at its peak at 72 h.
Kunamneni et al. (2005) employed the response surface methodology to study
the collective impact of the nutritional parameters and to increase extracellular alpha-
amylase production in solid-state fermentation by T. lanuginosus. These nutritional
parameters consist of carbon source (soluble starch), nitrogen source (peptone) and a
concentrated mineral medium. A twenty three factorial central composite design using
response surface methodology was used to optimize the above three variables. The
best calculated values of these variables for optimal amylase production were soluble
starch 71.10 g/Kg, peptone 91.97 g/Kg and mineral salts solution 175.05 ml/Kg with
an estimated alpha amylase activity of 5.085 ´ 105 U/Kg of wheat bran. These
parameters were checked in the laboratory and ultimate alpha amylase activity
obtained, 4.946 ´ 105 U/Kg of wheat bran, was very near to the calculated value.
35
Kiran et al. (2005) isolated the thermophilic Bacillus sp. K-12 from soil samples
having the ability to produced amylolytic enzyme. Effects of different carbon sources
and chemicals on production of alpha amylase were checked in the laboratory.
Medium consist of 1 % starch showed maximum alpha amylase activity after 60 h of
fermentation. However manganese sulfate, zinc sulfate and EDTA showed inhibitory
effect on alpha amylase production by Bacillus sp. Haq et al., (2005a) reported the
choice of the appropriate surfactant for alpha amylase production by Bacillus subtilis
GCBM-25 during shake flasks. Various surfactants (laundry soap, detergent powder,
sulphonic acid, acyle benzene sulphonic acid, liquid soap, Tween 80, sodium silicate,
bath soap, sodium tripolyphosphate, sodium lauryl ether sulphate or sodium lauryl
sulphate) at rate 2.0 % (w/v) were screened for synthesis of enzyme. Among all the
surfactants, tested laundry soap proved to be superior with respect to alpha amylase
production (605 U/ml/min) after 44 h of fermentation using 4.0 % inoculum. The
enzyme production was enhanced (857 U/ml/min) with the addition of Millon soap at
rate 3.2 % (w/v) in the medium. However, addition of surfactants in the medium
reduced the thermostability from 70 to 50°C.
Haq et al. (2005) reported the use of agricultural starchy substrate for alpha
amylase production by Bacillus licheniformis. The use of agriculture by products
made the medium economic. Soluble starch, hordium, pearl millet, rice, corn, gram
and wheat starch were screened for the alpha amylase production by parental and its
mutant derivative. The mutant strain B. licheniformis GCUCM-30 exhibited 10 fold
more enzyme production compared to parental strain when1.5 % pear millet and 0.25
% of nutrient broth was added to fermentation medium.
36
Anto et al. (2006) reported the alpha amylase production by B. cereus MTCC
1305 in solid state fermentation using wheat bran and rice flake manufacturing waste
as substrates. Maximum alpha amylase activity (94±2) U/g was obtained when wheat
bran was used as a substrate. Optimal conditions for alpha amylase production were
inoculum size 10 % substrate moisture ratio 1:1 and glucose, (0.04 g/g). Addition of
different nitrogen sources (0.02 g/g) showed decrease in enzyme production. Optimal
alpha amylase activity was observed at 55°C and pH 5. Swain et al. (2006) reported
the alpha amylase production by B. subtilis isolated earlier from cow dung microflora.
The optimum temperature, pH and incubation period for amylase production were 50-
70°C, 5-9 and 36 h, respectively. Enzyme secretion was very similar in the presence of
any of the carbon sources tested (soluble starch, potato starch, cassava starch, wheat
flour, glucose, fructose, etc.). Yeast extract and ammonium acetate (1 %) as nitrogen
sources gave higher yield compared to other nitrogen sources (peptone, malt extract,
casein, asparagine, glycine, beef extract) whereas ammonium chloride, ammonium
sulfate and urea inhibited the enzyme activity. Addition of Ca+2 (10-40 mM) to the
culture medium did not result in further improvement of enzyme production, whereas
the addition of surfactants (Tween 20, Tween 40, Tween 80, and sodium lauryl
sulphate) at 0.02 % resulted in 2-15 % increase in enzyme production. There were no
significant variations in enzyme yield between shake flask and laboratory fermenter
cultures. The purified enzyme was in two forms with molecular mass of 18.0 ± 1 and
43.0 ± 1 kDa in native SDS-PAGE.
Kathireasan and Manivannan (2006) isolated Penicillium fellutanum from
coastal mangrove soil and screened out the sound effects of different variables such
37
as pH, temperature, incubation time, salinity, carbon and nitrogen sources in shake
flasks fermentation for alpha amylase production. The fermentation medium with no
addition of seawater and supplemented with maltose and peptone as carbon and
nitrogen source was incubated for 96 h, at pH 6.5 and temperature 30°C, gave
maximum alpha amylase production by P. fellutanum. Djekrif-Dakhmouche et al.
(2006) studied the alpha amylase production and optimization from A. niger ATCC
16404 .The statistical analysis has revealed that variation in agitation from 150 rpm -
200 rpm has no effect on the alpha amylase production but it increased biomass. As
far as variation in pH from 5 to 6 has positive effect on alpha amylase production
while its effect on the biomass was negative. The addition of starch at 10 g/l to the
fermentation medium (an inductive substrate and carbon source) stimulated the alpha
amylase production, while it has no effect on biomass production. Calcium chloride at
1 g/l (a structural and stabilizing element for the alpha amylase) solely affect the
enzyme production. The use of other salts (manganese, iron sulfates as well as
magnesium chloride) seemed to be increased alpha amylase production but did not
effect either the production of protein or biomass.
Prakasham et al. (2007) reported fractional factorial design of Taguchi
methodology for the optimization of medium along with eight variables soluble
starch, corn steep liquor, casein, potassium dihydrogen phosphate, magnesium sulfate,
initial pH, incubation temperature and inoculum size for the amylase production in
submerged fermentation by A. awamori. Considerable enhancement approximately
48% in acid amylase synthesis was observed. The optimized fermentation medium
included in (%) soluble starch 3, CSL 0.5, KH2PO4 0.125, casein 1.5 at pH 4 and
38
31°C. Shoji et al. (2007) reported a new submerged culture system of A. kawachii
NBRC4308 with the help of barley whose surface was wholly or partially covered
with husk. Both glucoamylase activity 150.8 U/ml and acid-stable alpha amylase
activity 7.7 U/ml were found in the supernatant in the presence of low concentration of
glucose.
Rao et al. (2007) investigated the formation of spores from B.
amyloliquefaciens B128 in shake flask cultivation. Fermentation media were
optimized by applying two steps approach. A rapid identification of an appropriate
carbon and nitrogen source was obtained by screening experimentation, and use of
response surface methodology (RSM). A five-level four-factor central composite
design was used to find out the highest spore yield at optimal level for lactose, tapioca,
ammonium sulfate and peptone. A noteworthy linear major effect was observed in the
case of topica and peptone, while lactose and ammonium sulfate produced no
important linear effect. Lactose-ammonium sulfate and lactose-peptone extensively
affected spore production. Optimum conditions for the alpha amylase production were
(g/l): lactose 12.7, tapioca 16.7, ammonium sulfate 1.8 and peptone 8. The predicted
spore production was 5.93 × 108 (no/ml). The real experimental results were in
concurrence with the prediction.
Suganuma et al. (2007) reported that highly humid climate of Japan facilitate the
growth of various molds. Among these molds A. oryzae was the most important and
popular in Japan, and has been used as yellow-koji in producing many traditional
fermented beverages and foods, such as Japanese sake, and soy sauce. The koji molds
black-koji and white-koji produce two types of alpha amylase, namely, acid-stable
39
(AA) and common neutral (NA).The latter enzyme was enzymatically genetically
similar to Taka amylase. In this review they investigated AA from three molds, A.
niger, A. kawachii and A. awamori, and the yeast Cryptococcus sp. regarding the
distinguishable properties between AA and NA. AA has many advantages in industrial
applications, such as its acid-stability, thermostability, and raw-starch digesting
properties. Bhanja et al. (2007) used Growtek bioreactor as modified solid state
fermenter to circumvent many of the problems associated with the conventional tray
reactors for solid state fermentation (SSF). A. oryzae IFO-30103 produced very high
levels of alpha amylase by modified solid state fermentation (mSSF) compared to SSF
carried out in enamel coated metallic trays utilizing wheat bran as substrate. High
alpha amylase yield of 15,833 U/ g dry solid in mSSF were obtained when the fungus
were cultivated at an initial pH of 6 at 32°C for 54 h whereas alpha amylase
production in SSF reached its maximal (12,899 U g–1 dry solid) at 30°C after 66 h of
incubation. With the supplementation of 1 % NaNO3, the maximum activity obtained
was 19,665 U g–1 dry solid (24% higher than control) in mSSF, whereas, in SSF
maximum activity was 15,480 U/ g dry solid in presence of 0.1 % Triton X-100 (20 %
higher than the control).
Tayeb et al. (2007) conducted the alpha amylase production using amplified
variants of B. subtilis (strain SCH) and of B. amyloliquefaciens (strain 267CH) in a
bioreactor with multiprotein-mineral media. The time course of fermentation in a
bioreactor revealed that the highest yield (about 8 x 104 U/ml within 6 h) by strain
SCH was obtained by applying: 3.5 % initial starch, 2 % additional starch after 19 h, 3
vvm aeration and 300 rpm agitation. The highest yield (about 19 x 104 U/ml within
40
100 h) by strain 267CH was obtained by applying: 2.5 % initial starch, 2 % additional
starch after 24 h, 3 vvm aeration, and 300 rpm agitation with the productivity after 60
h reaching only about 14 x 104 U/ml. Production occurred in both the logarithmic and
post logarithmic phases of growth. Maximum consumption of starch and protein
occurred during the first day of incubation. The optical density peak coincided with
enzyme production peak in case of strain SCH and preceded that of enzyme
production in case of strain 267CH. The alpha amylase produced by the two strains
was shown to be the liquefying and not both enzymes liquefied starch to a dextrose
equivalent of about 15-17 at 95°C hence they are classified among thermostable alpha
amylases. They exhibited broad pH and temperature activity profiles. The optimum
pH for activity was 4-7 for alpha amylase produced by strain SCH and 4-8 for alpha
amylase produced by strain 267CH while the optimum temperatures for their activities
were in the range of 37 -75°C at 0.5 % starch and in the range of 85 - 95°C at 35 %
starch.
Poornima et al. (2008) isolated different strains of actinomycetes and tested
these strain for their ability to synthesize the alpha amylase. Among all the strains, the
strain AE-19 showed best alpha amylase production. This strain was identified as
Streptomyces aureofasciculus and selected for subsequent studies. The highest alpha
amylase production was obtained in the presence of mannose and L-histidine as
carbon and nitrogen source, 0.05 % sodium chloride at temperature 45°C and pH 9.
These results indicated that strain can be successfully used for commercial alpha
amylase production after testing strain competence in large scale fermentations. Gupta
et al. (2008) studied the nutritional requirements of A. niger and the factors such as
41
incubation temperature, pH of medium, carbon and nitrogen sources, fermentaion
period, surfactants and concentration of metal ions. The experimental result showed
ideal carbon and nitrogen source for alpha amylase production was 0.5 % starch and
0.3 % peptone. The optimal pH, temperature and fermentation period were 5, 30°C
and 5th day, respectively. Different surfactant at varying level such as Tween-80,
Triton X-100 and Sodium dodecyl sulphate at 0.02, 0.002 and 0.0002 % concentration,
respectively exhibited enhanced alpha amylase productivity. The major purpose of the
present study was to employ an appropriate fungal strain for extracellular alpha
amylase production and find out the fermentation period for the synthesis of alpha
amylase and to determine the effects of external substances that might increased the
synthesis of alpha amylase.
Esfahanibolandbalaie et al. (2008) reported the effect of many chemical and
physical factors on alpha amylase production by A. oryzae in shake flasks
fermentation via an Adlof-Kuhner orbital shaker. The impact of varying pH of
medium ranging from 4-7 was studied. The maximum alpha amylase production was
obtained at pH 6.2. Carbon and nitrogen source has discernible effect on the enzyme
production. The corn starch at level of 15 g/l proved to be best carbon source for alpha
amylase synthesis while glucose represses the alpha amylase production. The medium
consist of corn starch, sodium nitrate as inorganic nitrogen resulted in significant
enzyme production. Among the organic nitrogen sources yeast extract at the level of
2.5g /l was excellent nitrogen source. The impact of different temperatures and
agitation speed from 20 to 40°C and 50 to 200 rpm, respectively was observed. The
maximum activity was obtained at 35°C and 180 rpm. Planchot and Colonna (2008)
42
purified A. fumigatus (Aspergillus sp. K-27) extracellular alpha amylase to
homogeneity by using anion-exchange DEAE-cellulose and affinity α-cyclodextrin-
Sepharose chromatography. The purified enzyme was glycoprotein in nature which
was found to contain 15 % carbohydrate. The purified alpha amylase exhibited an
isoelectric point of 3.7, and SDS PAGE estimated that purified enzyme possess a
molecular weight of 65,000. A large number of neutral hydrophobic residues were
present in an amino acid. The optimum enzyme activity was obtained at pH 5.5, and
the enzyme showed stability at 40°C. It hydrolyzed amylose and amylopectin, with
respective Km of 0.42 and 7.7 mg mL- 1 and kcat/K m of 3.4 and 2.5 mL mg -1 min-1.
The main end-products of maltohexaose, hydrolysis were glucose and maltose. While
intermediate products were maltotriose, maltotetraose, and maltopentaose having an α-
anomeric configuration. Although its capability to gradually degrade some α1-6
linkages, purified enzyme ought to be classified as an alpha-amylase.
Leman et al. (2009) reported alpha amylase from A. oryzae had only very little
effect on the side chain segments of the amylopectin molecules and the reason might
be enzyme hydrolysis the segments of internal chain. Singh et al., (2009) investigated
the effect of various agricultural by products as a substrate such as wheat bran, wheat
straw, rye, straw on the alpha amylase production by Humicola lanuginose in solid
state fermentation. Wheat bran proved to be good substrates for starch degrading
enzymes because highest alpha amylase production was observed when wheat bran
was used as a substrate. Various variables such as moisture content, incubation time
inoculum size and carbon source has marked effect on the enzyme production. It was
noted the optimum condition for the alpha amylase production by Humicola
43
lanuginose in SSF was incubation period 144 h, initial moisture content 90 %, initial
pH of medium 6, incubation temperature 50ºC , size of inoculum 20 % and soluble
starch as best carbon source.
Shafique et al. (2009) tested the five strains of fungi belonging to two
filamentous fungi A. niger and A. flavus for their ability to produce alpha-amylase.
The different chosen strains were cultivated on two different typed of media i.e.,
potato dextrose agar (PDA) and enzyme production medium (EPM), the pH of
medium was fixed at 3 level i.e., 4.5, 5.5 and 6.5. The efficiency or ability of strains
was estimated on the basis of the formation of hydrolysis zone. EPM medium at pH
4.5 was best for the highest activity of alpha amylase. Strain 74 and strain 198 of A.
niger and strain 209 and strain 231 of A. flavus gave best result on solid media; so
these strains were selected for the alpha amylase production in submerged
fermentation. All the selected strains showed highest activity of alpha amylase after 48
h in shake flasks.
44
Uses of alpha amylase
Starch is a major storage product of many economically important crops such as
wheat, rice, maize, tapioca, and potato. A large-scale starch processing industry has
emerged in the last century. In the past decades, we have seen a shift from the acid
hydrolysis of starch to the use of starch-converting enzymes in the production of
maltodextrin, modified starches, or glucose and fructose syrups. Currently, these
enzymes comprise about 30 % of the world's enzyme production. Besides the use in
starch hydrolysis, starch-converting enzymes are also used in a number of other
industrial applications, such as laundry and porcelain detergents or as anti-staling
agents in baking. A number of these starch-converting enzymes belong to a single
family: the alpha amylase family or family13 glycosyl hydrolases. This group of
enzymes share a number of common characteristics such as a (β/α)8 barrel structure,
the hydrolysis or formation of glycosidic bonds in the α conformation, and a number
of conserved amino acid residues in the active site. As many as 21 different reaction
and product specificities are found in this family.
Bread and chapatti industry
The quantities, taste, aroma and porosity of the bread are improved by using the enzyme
in the flour. More than 70 % bread in U.S.A, Russia and European countries contain
alpha amylase. Amylases play important role in bakery products (Goodwin and Mercer,
1972). For decades, enzymes such as malt and fungal alpha-amylases have been used in
bread-making. The significance of enzymes is likely to raise as consumers insist more
natural products free of chemical additives. For example, enzymes can be employed to
45
replace potassium bromate, a chemical additive that has been prohibited in a number of
countries. The dough for bread, rolls, buns and similar products consists of flour, water,
yeast, salt and possibly other ingredients such as sugar and fat. Flour consists of gluten,
starch, non-starch polysaccharides, lipids and trace amounts of minerals. As soon as the
dough is made, the yeast starts to work on the fermentable sugars, transforming them
into alcohol and carbon dioxide, which makes the dough rise. The major component of
wheat flour is starch. Amylases can degrade starch and produce small dextrins for the
yeast to act upon. The alpha-amylases degrade the damaged starch in wheat flour into
small dextrins, which allows yeast to work continuously during dough fermentation,
proofing and the early stage of baking. The result is improved bread volume and crumb
texture. In addition, the small oligosaccharides and sugars such as glucose and maltose
produced by these enzymes enhance the Maillard reactions responsible for the browning
of the crust and the development of an attractive baked flavour (Lundkvist et al., 2007).
Textile industry
Textile industries are extensively using alpha amylases to hydrolyze and solubilize the
starch, which then wash out of the cloth for increasing the stiffness of the finished
products. Fabrics are sized with starch. Alpha amylase is used as desizing agent for
removing starch from the grey cloth before its further processing in bleaching and
dyeing. Many garments especially the ubiquitions‛ Jean ’ are desized after mashing.
The desired fabrics are finally laundered and rinsed (Iqbal et al., 1997; Allan et al.,
1997).
46
Sugar and Glucose industries
Alpha amylase plays a very important role in the production of starch
conversion products of low fermentability. The presence of starch and other
polysaccharides in sugar cane creates problem throughout the sugar manufacturing
which is minimized or eliminated by the action of alpha amylase. The high quality
products depends upon the efficiency of the enzyme which lead to low production,
costs for the starch processor has increased (De-cordt et al., 1994; Ensari et al., 1996;
Hamilton et al., 1998). Many industries used alpha amylases for the production of
glucose. Enzyme hydrolyzed the starch and converted it into glucose. They hydrolyze
α-1,4 glucosidic linkage in the starch polymer in a random manner to yield glucose
and maltose (Akiba et al., 1998). Therefore, alpha amylase is extensively used in
many industries for the production of glucose (Shetty and Crab, 1990). This process
also gives the water-soluble dextrin.
Alcohol Industry
Alpha amylases convert starch in to fermentable sugars. Starches such as grain;
potatoes etc. are used as a raw material that helps to manufacture ethyl alcohol. In the
presence of amylases, the starch is first converted in to fermentable sugars. The use of
bacterial enzyme partly replaces malt in brewing industry, thus making the process
more economically significant. Alpha amylase can also carries out the reactions of
alcoholysis by using methanol as a substrate (Santamaria et al., 1999).
47
Paper industry
Starch paste when used as a mounting adhesive modified with additives such as
protein glue or alum, frequently, causes damage to paper as a result of its
embrittlement. Starch digesting enzymes, e.g. alpha amylase, in immersion or as a gel
poultice are applied to facilitate its removal. Alpha amylase hydrolyzed the raw starch
that is used for sizing and coating the paper instead of expensive chemically modified
starches. So, starch is extensively used for some paper size press publications (Okolo
et al., 1996).
Detergent, Building product and Feed industries
In detergent industries, the enzyme alpha amylase plays a vital role. It is widely used
for improvement of detergency of laundry bleach composition and bleaching with out
color darkening (Borchet et al., 1995; Atsushi and Eiichi, 1998). The addition of
enzyme stabilizes the bleach agent and preserves effectiveness of the bleach in laundry
detergent bar composition (Onzales, 1997; Mirasol et al., 1997) Modified starch is
used in the manufacture of gypsum board for dry wall construction. Enzyme modified
the starch for the industry use. Many starches or barely material are present in the
feed. So, the nutritional value of the feed can be improved by the addition of alpha
amylase.
Chocolate industry
Amylases are treated with cocoa slurries to produce chocolate syrup, in which
chocolate starch is dextrinizing and thus syrup does not become thick. Cocoa flavored
syrups having a high cocoa content and excellent stability and flow properties at room
48
temperature may be produced by using an amylolytic enzyme and a sufficient
proportion of Dutch process cocoa to provide a syrup pH of 5.5 to 7.5. The syrup is
made by alternate addition of cocoa and sweetener to sufficient water to achieve a
solids content of about 58 to 65 weight percent, adding an amylolytic enzyme, heating
to a temperature of about 175 -185°F for at least 10 to 15 min, raising the temperature
to about 200° F. and cooling. The stabilized cocoa flavored syrups may be added at
room temperature to conventional non-acid confection mixes for use in the production
of quiescently frozen chocolate flavored confections (Ismail et al., 1992)
49
MATERIALS AND METHODS
3.1: MATERIALS
The chemicals used in this study such as sodium potassium tartarate, 3,5-dinitro
salicylic acid, phenol, sodium metabisulphate, dihydrogen phosphate, manganese
sulphate, yeast extract, ferrous sulphate, magnesium chloride, diammonium sulphate,
starch, ferrous sulphate, acryleamide, bisacryleamide, trizmabase, Tris HCl, SDS,
glycine, bromophenol blue, β- mercaptoethanol, ammonium per sulphate, coomassie
brilliant blue R-250, TEMED, etc were of analytical grade and obtained from Sigma
(USA), BDH (UK), E-Merck (Germany), Acros (Belgium) and Fluka (Switzerland). All
other chemicals were of the highest possible purity.
3.2: METHODS
3.2.1: Isolation of organism:
The isolation of seventy eight Aspergillus oryzae cultures from soil samples collected
from different habitats such as textile wastes, garden compost etc was carried out by
serial dilution method (Clark et al., 1958). The soil samples were collected in sterile
polythene bags. One gram of the soil sample was dissolved in 100 ml of sterilized
distilled water. The soil suspension was then diluted up to 105-107 times.
Approximately 0.5 ml of this diluted suspension was transferred to the Petri plates
containing starch agar medium. The starch agar medium was prepared by dissolving
10 g of starch and 20 g of agar in 900 ml of distilled water and raising the volume up
to1000 ml. The pH of medium was adjusted to 4.8 by 0.1N HCl/NaOH. After raising
the volume to 1000 ml the medium was heated for 10 min to obtain a homogeneous
50
mixture. Approximately 10 ml of the medium was poured in separate test tubes. The
tubes were cotton plugged and sterilized in autoclave at 15 lbs/in2 pressure (121°C) for
15 min. After sterilization, the contents of each tube were transferred to the oven
sterilized (Model: UM-400 MEMMERT, Germany) Petri plates (at 180ºC for 2 h) and
allowed to solidify at room temperature.
The fungal cultures were further purified from bacterial contaminants by using
10 mg/l mixture of penicillin and streptomycin (1:1 ratio) in the plate medium. After
the addition of soil suspension, the Petri plates were rotated clockwise and counter
clockwise for uniform spreading of suspension on the medium. The plates were placed
in an incubator (Model: MIR-153, Sanyo Japan) at 30°C for 3-4 days for culture
development. The initial colonies forming clear zones of starch hydrolysis were
picked up and transferred to potato dextrose starch agar slants for culture maintenance.
The cultural and morphological characteristics of A. oryzae isolates were identified
according to Onion et al. (1986). The potato dextrose starch agar medium was
prepared by dissolving 39 g of PDA and 10 g of starch in 900 ml of distilled water and
raising the final volume up to 1000 ml. This was cooked for 10-15 min with constant
stirring until a clear solution formed. The pH of the medium was adjusted to 5.6 by
0.1N NaOH/HCl. Approximately 6-8 ml of the medium was poured in different test
tube. All the tubes were cotton plugged and sterilized in an autoclave at 15 lbs/in2
pressure (121°C) for 15 min. Afterwards, the tubes were kept in a slanting position (at
an angle of about 30°) to increase the surface area and allowed to solidify. The conidia
of isolated fungi were aseptically transferred to the slants containing potato dextrose
starch agar medium. The slants were incubated at 30°C in an incubator for 3-5 days
51
for maximum growth. The slants were stored in a refrigerator at 4°C for culture
maintenance.
3.3: Fermentation
3.3.1: Inoculum preparation
3.3.1.1: Conidial inoculum
Conidia from 3-4 day old slant cultures were used for inoculation. The conidial
suspension was prepared in sterilized 0.005 % dioctyl ester of sodium sulpho succinic
acid (Monoxal O.T). Ten milliliter of sterilized Monoxal O.T was transferred to each
slant having profuse conidial growth on its surface. An inoculating needle was used to
break the clumps of conidia. The test tube was shaken vigorously to make a
homogeneous suspension.
3.3.1.2: Conidial count
The numbers of conidia were counted with the help of a Haemacytometer. Each
milliliter of the suspension contained 2.6 ×10 6 CFU.
3.3.1.3: Vegetative inoculum
Fermentation medium of one hundred milliliter was transferred to a 1.0 L conical flask
followed by the addition of approximately 20-25 glass beads (2.0 mm dia.). The flask
was cotton plugged and sterilized. The four milliliter of the conidial suspension was
transferred aseptically to the flask, which was then incubated at 30°C on a orbital
shaking incubator (Model: 10X400.XX2.C, SANYO Gallenkamp, PLC, UK) at 200
rpm for 24 h.
52
3.3.2: Fermentation media
Different fermentation media (g/l) were evaluated for the alpha amylase production by
selected strain of Aspergillus oryzae at pH 6 (Hayashida et al., 1986; Spohr et al.,
1998; Nandakumar et al., 1999; Haq et al., 2002).
M1: Wheat bran 100, Zn SO4.7H2O 0.062, FeSO4 0.068, Cu SO4.7H2O 0.0008.
M2: Starch 10, yeast extract 3.0, MgSO4.7H2O, 0.005, CaCl2.2H2O 0.2, FeSO4
0. 1, Peptone 20, phosphate buffer 1000 ml.
M3: Starch 10, MgSO4.7H2O 0.005, CaCl2.2H2O 0.2, FeSO4 0.1, (NH4) 2SO4 2,
phosphate buffer 1000 ml.
M4: Starch 20, yeast extract 8.5, NH4Cl 1.3, MgSO4.7H2O, 0.12, CaCl2 0.06.
M5: Glucose monohydrate 4.84, (NH4)2SO4 4.84, KH2PO4 3.87, MgSO4.7H2O 3.75,
NaCl 1.80, CaCl2.2H2O 1.21, trace metal solution 0.12 ml.
M6: Glucose 50, NaNO3 3, KH2PO4 1.0, KCl 0.5, MgSO4.7H2O 0.2, FeSO4 0.01.
3.4: Shake flask studies
Twenty-five milliliter of fermentation media (M4 optimized) was transferred to
separate 250 ml cotton plugged conical flasks. The flasks were sterilized in an autoclave
for 15 min and cooled at room temperature. A one milliliter of inoculum was transferred
to each flask. The flasks were placed in the orbital shaking incubator for incubation at
30°C with shaking speed of 200 rpm. After 72 h of incubation, content of flasks were
filtered and filtrate was used for the estimation of enzyme while the residue was used
for the estimation of cell mass. All the experiments were run parallel in triplicates.
53
3.5: Fermenter studies
Scale up studies were carried out in a 7.5 L glass fermenter (Model: Bioflow-110
Fermenter/Bioreactor, USA) with a working volume of 5.0 L. The fermenter glass
vessel containing 4.7 L fermentation medium was sterilized in a stainless steel
autoclave (Model: KT-40 L, ALP, Japan) for 20 min at 15 lbs/in2 pressure (121ºC) and
cooled at room temperature. Vegetative inoculum was transferred to the vessel
through a hole at the top plate under aseptic conditions. The incubation temperature
was kept at 30°C, while the aeration and agitation rates were maintained at 1.0
L/L/min (vvm) and 200 rpm, respectively throughout the fermentation period. The air,
to be supplied was sterilized by passing through membrane filters (0.45 µm pore size).
Sterilized solution of 0.1 N HCl/ NaOH was used for pH adjustment. The sterilized
silicone oil 10 % (v/v) was used to control foam formed during the fermentation
process.
3.6: Nutritional and cultural requirements of Aspergillus oryzae
3.6.1: Fermentation media
Fermentation media play a very important role in the alpha amylase production as well
as for the growth of organism. Six different media were evaluated for the enzyme
production in shake flasks.
3.6.2: Incubation period
Incubation period has a vital role for the optimal alpha amylase production by
54
Aspergillus oryzae. The enzyme fermentation was carried out (8-96 h) at 30°C and
sample was collected every 8 h for the estimation of enzyme and dry cell mass in
present study.
3.6.3: Effect of initial pH
Maintenance of a favorable pH is one of the most important steps for successful
progression and termination of fermentation (Gigras et al., 2002). A range of different
pH (4-7) in shake flasks and (4-6.5) in fermenter was tested for alpha amylase
production.
3.6.4: Effect of temperature
The optimal incubation temperature is a function of the microbial strain and should be
determined for each set of conditions (Bhanja et al., 2007). The effect of different
temperature (25-50°C) on the biomass formation and production of enzyme was
investigated in present study.
3.6.5: Effect of volume
The effect of different volume of basal medium on the alpha amylase production by
Aspergillus oryzae was investigated. The amount of fermentation medium such as 5,
10, 15, 20, and 25 % (w/v) was evaluated in shake flask fermentation.
3.6.6: Effect of inoculum size
The size of inoculum is very important for alpha amylase production. Conidial
inoculum at varying concentration (2-12 % (v/v) during shake flasks and vegetative
inoculum (5.0-12.5 % v/v) during fermenter studies was investigated. The initial pH,
temperature, incubation time, agitation intensity, aeration were maintained constant.
55
3.6.7: Effect of agitation and aeration
Agitation and aeration are interrelated and had direct influence on the alpha amylase
production. Different agitation intensity (120-240 rpm) with air supply from 0.5-2.0
vvm was investigated for optimum alpha amylase production.
3.6.8: Evaluation of carbon sources
Carbon sources play a vital role for the growth as well as for the alpha amylase
production. Different additional carbon sources such as sucrose, glucose, lactose,
xylose, fructose, galactose, glycerol, mannitol and CMC were evaluated for the
production of alpha amylase by Aspergillus oryzae (Carlsen and Nielsen, 2001).
3.6.9: Evaluation of nitrogen sources
Different organic and inorganic nitrogen sources such as peptone, yeast extract, meat
extract, urea, casein, beef extract, corn steep liquor, ammonium nitrate, ammonium
sulfate, sodium nitrate, potassium nitrate etc, were evaluated for the enzyme
production as well as for the growth of organism.
3.7: Induction of mutation
3.7.1: Minimal inhibitory concentration of 2-deoxy-D-glucose
The parental strain was grown on starch agar medium along with 2-deoxy-D-glucose
(0.0-0.5 % w/v) at 30ºC in order to find out the minimal inhibitory concentration
(MIC), (Azin and Noroozi, 2001).
56
3.7.2: Ultraviolet (UV) irradiation
From the parental fungal isolate (5 day old culture), 1 ml of the conidial suspension
was transferred to a cotton wool plugged conical flask containing 25 ml of sterilized
M1 medium. The conidia were allowed to grow at 30°C on a shaking incubator with
200 rpm for about 6 h to get fresh growing fungal mycelia. Five milliliter of medium
containing mycelial suspension was transferred to a sterilized Petri plate and these
mycelia were exposed to ultraviolet (UV) irradiation for 15-75 min under the beam
(λ=253 nm and 220 V at 50 c/s) of UV lamp (Model: Mineral Light, UVS-12,
California, USA). The radiation dose given to the mycelial suspension was 1.2×102
J/m2/s. The distance between lamp and suspension was adjusted at 8 cm for each trial
to get more than 95 % death rate (Azin and Noroozi, 2001).
3.7.3: Nitroso guanidine treatment (NG)
NG was prepared in four different concentrations from 0.5-2.0 mg/ml. N-methyl-N-
nitro-N-nitroso guanidine (NG) was transferred to each sterilized centrifuged tube
containing 5 ml of conidial suspension and incubated at 30°C using a shaking water
bath (Model: WB-14, MEMMERT, Germany) for specific time interval to achieve a
death curve with sub-lethal level. After treatment with NG, 1 ml of cystein (1 %, w/v)
was added to terminate the reaction. The conidia were treated similarly except
replacing NG with sterile distilled water in control experiment. After fixed time
interval, the tubes were spun at 6000 g for 15 min. The supernatant was discarded to
remove NG from the fungal cells. Traces of NG were removed after three appropriate
washings with 0.1 M phosphate citrate buffer (pH 7.5). The treated conidia were
resuspended in the same buffer.
57
3.7.4: Nitrous acid treatment
A 0.07-0.3M solution of NaNO2 prepared in acetate buffer (0.2 M, pH 4.5) was added
to washed and spun conidia of A. oryzae (Carlton and Brown, 1981). The solution was
shaken thoroughly for specific time intervals. The one milliliter solution was
withdrawn and diluted 5 fold in phosphate buffer (0.2 M, pH 7.1) to stop the reaction.
A control was run similarly except replacing NaNO2 in acetate buffer with sterilized
saline water. After fixed time interval, the tubes were spun at 6000 g for 15 min. The
supernatant was discarded to remove nitrous acid from the fungal conidia and ten
milliliter of sterilized phosphate buffer was added to each tube. The tubes were re-
spun for the removal of traces of nitrous acid from conidia and repeated three times.
After washing the conidia were resuspended in same buffer.
3.7.5: EMS treatment
Different concentrations (25-150 µl) of ethyl methane sulphonate (EMS) were added
to individual centrifuge tubes containing 4 ml of conidial suspension and shaken to
form a homogeneous suspension. After specific time intervals the conidia were spun
and washed thrice in phosphate buffer. The EMS treated conidia was resuspended in
same buffer.
3.7.6: Selection of mutants
After treatment with mutagenic agents, about 100 µl of each suspension containing
treated conidia was aseptically transferred to the individual Petri plates containing
starch agar medium supplemented with (g/L); Triton X-100 (5.0), 2- deoxy-D-glucose
(above the MIC of parent strain). The plates were incubated at 30ºC and were
58
examined regularly after 3-4 day to study the growth pattern. The colonies were
selected qualitatively; showing the bigger zone of starch hydrolysis compared to
parental strain and was allowed to grow on PDA slants for culture maintenance. These
colonies were then tested quantitatively for enzyme production in shake flasks
fermentation.
3.8: Analytical techniques
After incubation, the fermented broth was filtered. The filtrate was used for the
estimation of total protein contents, and alpha amylase activity.
3.8.1: Estimation of alpha amylase
The estimation of alpha amylase was carried out according to the method of Rick and
Stegbauer (1974). “One unit of activity was that amount of enzyme, which in 10 min
liberates reducing group from 1 % Lintner‘s soluble starch corresponding to 1mg of
maltose hydrate.” The enzyme activity was determined by taking 1 ml of diluted
filtrate in a test tube. The one milliliter of starch solution (1 % w/v) was also added
into it. A blank was run parallel by replacing the filtrate with 1 ml of distilled water.
After incubation of 10 min at 40°C, the reducing sugar liberated was measured at 546
nm by the DNS method (Miller, 1959) using maltose as a standard.
3.8.2: Estimation of total protein contents
Total protein contents were determined by taking 0.1 ml of the filtrate with 5 ml of
Bradford reagent in a test tube and vortexes thoroughly. A blank containing 0.1 ml of
distilled water with 5 ml of the Bradford reagent was run parallel. The absorbance was
taken at 595 nm on a double beam UV/VIS scanning spectrophotometer (Model: CE-
59
7200, CECIL, England, UK) after 15 min of the reaction using bovine serum albumin
(BSA) as a standard. Protein contents were determined from the standard curve of
BSA (Bradford 1976).
3.8.3: Determination of mycelial morphology
Mycelial morphology was determined on an aliquot extended on the Petri plates
followed by pellet diameter (Moreira et al., 1996). For rounded pellets, if the diameter
was less than 0.5 mm, they were categorized as fine pellets, between 0.5-2 mm as
small pellets, between 2-3 as intermediate pellets while those above 3 mm were
referred to as large pellets.
3.8.4: Estimation of dry cell mass (DCM)
Dry cell mass was determine by filtering the culture broth through preweighed
Whatman filter paper No. 44. Mycelia were thoroughly washed with tap water and dry
in oven at 105°C for 2 h. The dry cell mass was weighed and calculated as g/l by
subtracting the initial weight from the final weight.
3.9: Statistical analysis
Treatment effects were compared by the method of Snedecor and Cochran (1980).
Post-Hoc Multiple comparison tests were applied under one-way ANOVA.
Significance has been presented in the form of probability (p<0.05) values.
3.10: Kinetic study
Kinetic parameters for batch fermentation were determined according to the method
describe by Pirt (1975) and Lawford and Rouseau (1993). The following parameters of
kinetics were studied:-
60
Specific growth rate
The value of specific growth rate i.e., µ (h-1) was calculated from plot of In (x) vs time
of fermentation.
Product yield co efficient
Product yield co efficient namely Yp/x was determined by the equation:
Yp/x=dP/dx
Volumetric rates
The volumetric rate of product formation Qp (U/l/h) was determined from the
maximum slope of enzyme produced vs time of fermentation. The volumetric rate for
biomass formation Qx (g cell mass /l/h) was determined from the maximum slope of
cell mass formation vs time of fermentation.
Specific rate constant
Specific rate constant for product formation was determined by the equation
qp =µ × Y p/x
3.11: Purification of alpha amylase
3.11.1: Separation of fungus from fermented broth
The fermented broth was spun at 9,000 g for 15 min at 4oC. The clear supernatant was
used for enzyme isolation and purification.
61
3.11.2: Ammonium sulfate precipitation
Solid ammonium sulfate was added to 1000 milliliter of crude cell free broth of alpha
amylase. The suspension was stirred for half an hour at 4ºC. After sufficient shaking,
the precipitates were collected by spining at 14,000 g for 20 min at 4ºC. To the
supernatant, added calculated amount of ammonium sulfate for 20-90 % (w/v)
saturation. Same procedure was applied to get the precipitates of all fractions. The
pelleted precipitate of each fraction was resuspended in 0.05 M Tris-HCl buffer, pH
7.5. The solution was dialyzed against the same buffer.
3.11.2.1 Anion- exchange chromatography
For the purpose of ion exchange chromatography, 0.4 g DEAE-Sephadex A-50
(Sigma, USA) was swollen in 100 milliliter of the 0.05 M Tris-HCl buffer, pH 7.5 in a
boiling water bath for 2 h. After cooling poured it into the column and made final bed
volume (1.5 × 10 cm). The dialyzed enzyme solution was applied to column that pre-
equilibrated with five column volumes of the 0.05 M Tris-HCl buffer, pH 7.5. A
stepwise NaCl gradient from 0 to 1 M in 150 ml of the same buffer was applied.
Fractions of 3 ml were collected at a flow rate 0.5 ml/min. The collected fractions
were assayed for protein at 280 nm and alpha amylase activity by performing enzyme
assay. The fractions containing enzyme activity were pooled, dialyzed and analyzed
on SDS-PAGE.
3.11.2.2: Gel filtration
Sephadex G-100 (Phamacia Fine Chemical), 2 g was swollen in 50 ml of 0.05 M Tris-
HCl buffer, pH 7.5 in a boiling water bath for 2 h. Poured the gel slurry along the side
62
of tilted column by taking care that no air bubble was entrapped. The column (1.5 ×
15.0 cm) was equilibrated with five column volumes of the 0.05 M Tris-HCl buffer,
pH 7.5 in order to stabilize the bed. The enzyme sample (5 ml) was eluted with the
same buffer; adjusting flow rate at 0.5 ml/min. The collected fractions were assayed
for protein and alpha amylase activity. The active enzyme fractions were pooled and
used for the determination of main characteristics of the enzyme.
3.11.3: Dialysis
The salts were removed by dialyzing precipitates and pooled samples by using 12,000
molecular weight cut off dialyzing bag, which was placed in one liter of the 0.05 M
Tris-HCl buffer (pH 7.5) for 3-4 h at 4ºC. The process was repeated 4-5 times until all
salts were removed from the enzyme solution.
3.11.4: Electrophoresis
The homogeneity of the purified enzyme was confirmed by sodium dodecyle sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) following the method of Hames
(1990).
3.11.5: Protein marker
The molecular weight of the alpha amylase was estimated by SDS- polyacrylamide gel
with protein marker (SMO 313).
3.12: Gel preparation
3.12.1: Separating gel:
The separating gel (12 % w/v) was prepared by adding 4 ml acrylamide (30 % w/v);
2.5 ml, 1.5 M Tris HCl (pH 8.8); 0.1 ml SDS (10 % w/v); 0.1 ml ammonium per
63
sulfate(10 % w/v); 6 µl TEMED ; 3.3 ml distilled water and poured in the gel
assembly leaving one inch vacant space at the top. Almost 100 µl of distilled water
was layered at the top of the gel to give a flat surface and to remove oxygen which
inhibited polymerization. The gel was allowed to polymerize for 30 min.
3.12.2 Stacking gel:
The stacking gel was prepared by adding 0.5 ml acrylamide (30 % w/v); 0.38 ml 1M
Tris HCl (pH 6.8); 0.03 ml, SDS 10 % (w/v); 0.004 ml APS (10 % w/v); 0.0003 ml
TEMED; 2.1 ml Distilled water. The water was removed from top of the separating
gel and stacking gel was poured in the gel assembly. Comb was inserted and gel was
allowed to polymerize at the room temperature for 10 min. When complete
polymerization took place, gel comb was taken out and valves were washed with tank
buffer four times by means of a syringe. After removing the bottom spacer the gel
assembly was settled in the gel chamber and made contact top and bottom with tank
buffer which was previously diluted in the ratio of 1:5 with distilled water.
The enzyme solution (6 µl) and loading buffer (4 µl) were denatured by heating
in boiling water bath for 3 min. The samples were loaded along the protein marker and
electrophoreses at a constant voltage of 150 v potential difference and 20 mA current
supplies for about 4 h.
3.13: Characterization of enzyme
Temperature and pH had great influence on the activity of alpha amylase. The enzyme
substrate complex was incubated at varying temperatures (25-70°C) and pH (3-7) and
the effect on the activity of purified enzyme was observed. Metal ions had marked
64
influence on the activity of enzyme so effect of different metal ions such as Mg SO4,
MnSO4, NaCl, NiCl2, Zn Cl2, CuCl2, COCl2, and FeSO4 on the activity of alpha
amylase was also studied.
3.14: Standard curves
3.14.1: Maltose
Anhydrous maltose (100 mg) was dissolved in a small quantity of distilled water and
volume was made up to 100 ml in a measuring flask. The stock solution (1 mg/ml)
was used to make 10 appropriate dilutions ranging from 0.1 to 1 mg/ml. The one
milliliter of each dilution was taken in separate test tubes followed by the addition of 2
ml of DNS reagent. A blank was run in parallel replacing the maltose dilution with 1
ml of distilled water. The tubes were incubated in a boiling water bath for 5 min prior
to cooling at room temperature. Absorbance was measured at 546 nm using a
spectrophotometer (Model: CECIL CE-7200 Aquarius, UK). A graph was plotted
taking the absorbance at the ordinate and sugar concentration at the abscissa (Fig 1).
3.14.2: Bovine serum albumin (BSA)
BSA (100 mg) was dissolved in approximately 90 ml of distilled water. The final
volume was raised up to 100 ml using a measuring flask. The stock solution (1 mg/ml)
was used to make 10 appropriate dilutions ranging from 100-1000 µl/ml. Each dilution
(0.1 µl) was taken in a separate test tube followed by the addition of 5 ml of BSA
reagent. A blank was also run in parallel by replacing BSA with distilled water. The
mixture was allowed to stand for 5-15 min for maximum coloration and optical
density was measured at 595 nm using a spectrophotometer (Model: CECIL CE-7200
65
Aquarius, UK). The curve was plotted by taking BSA concentration along x-axis and
optical density along y-axis (Fig 2).
3.15: Preparation of reagents/buffers
3.15.1: Dinitrosalicylic acid (DNS) reagent
Dinitrosalicylic acid (10.6 g) and sodium hydroxide (19.5 g) were dissolved in
approximately 600-800 ml of distilled water and gently heated in a water bath at 80°C
until a clear solution was obtained. Sodium potassium tartrate (306 g), phenol melted
at 60°C (7.5 ml) and sodium metabisulfate (8.3 g) were also added. After dissolving
the chemicals, final volume was raised up to 1416 ml with distilled water. The
solution was filtered through a large coarse sintered glass filter and stored at room
temperature in an amber colored bottle to avoid photo oxidation. It was stable for
about six months.
3.15.2: Bradford reagent
The coomassie brilliant blue hundred milligrams (G-250) was added in 50 ml of 95 %
(v/v) ethanol. This solution was poured into 100 ml of 85 % (w/v) phosphoric acid and
the final volume was raised up to 1.0 L with distilled water. After shaking filtered
through Whatman filter paper (No. 1) to obtain a clear solution. The reagent was
stored in an amber colored bottle to avoid photooxidation.
3.15.3: Starch Solution: The Lintner’s soluble starch 1g was dissolved in 100 ml of
acetate buffer (pH 5) and boiled until solution become transparent.
66
3.15.4: Acetate Buffer (pH 5.0)
Sol A: Acetic acid (12.06 ml) was added in approximately 900 ml distilled water and
final volume was raised up to 1 L
Sol B: Sodium acetate (27.22 g) was dissolved in approximately 900 ml distilled water
and final volume was raised up to 1 L.
Buffer solution (pH 5) was prepared by adding 148 ml of Sol A and 352 ml of Sol B
and raising the final volume up to 1 L.
3.15.5: Phosphate citrate buffer (pH 7.5)
The buffer was prepared by dissolving 922.5 g Na2HPO4 and 77.5 g citric acid (0.1 M)
in 700-800 ml distilled water and finally volume was raised to 1000 ml to get 0.1 M
phosphate citrate buffer having pH 7.5.
3.15.6: Preparation of 0.05 M Tris-HCl buffer (pH 7.5)
The Tris HCl buffer was prepared by dissolving 6.25 g of Tris in 700-800 ml of
distilled water and adjusted pH 7.5 with 5 N HCl with constant stirring. Finally
volume was raised to 1000 ml with distilled water.
3.15.7: Acrylamide bisacrylamide (30 % w/v)
The 30 % (w/v) acrylamide bisacrylamide was prepared by dissolving 29 g of
acrylamide and 10 g bisacrylamide in 1000 ml of distilled water. The solution was
filtered and stored at 4оC.
67
3.15.8: Separating buffer (1.5 M Tris HCl, pH 8.8)
The 1.5 M Tris HCl buffer was prepared by dissolving 181.5 g Trizma in 900 ml of
distilled water with constant stirring to adjust the pH 8.8 by adding concentrated HCl
(32 % v/v) drop wise. After pH adjustment raised the final volume upto 1000 ml with
distilled water.
3.15.9: Stacking buffer (1 M Tris HCl, pH 6.8)
The 1 M Tris HCl, buffer was prepared by dissolving 157.6 g Trizma base in 900 ml
of distilled water with constant stirring to adjust the pH at 6.8 by adding concentrated
HCl (32 % v/v) drop wise. After pH adjustment, the final volume was raised up to
1000 ml with distilled water.
3.15.10: Tank buffer (10 X, pH 8.3)
The Tank buffer was prepared by dissolving 3.94 g of Trizma base, 14.41 g of glycine
and 50 g of SDS in distilled water and raising the final volume up to 1000 ml. The
solution was stored at 4оC.
3.15.11: Gel loading buffer
The Gel loading buffer was prepared by mixing 1ml of Tris HCl (pH 6.8) 400g SDS,
200 ml glycerol, 2 g bromophenol blue dye, 1500 µl β-mercaptoethanol and raised the
volume up to 1000 ml and stored at 4оC.
3.15.12: SDS solution (10 % w/v)
The 10 % w/v SDS solution was prepared by dissolving 100 g of SDS in hot water.
The solution was stirred and final volume was made up to 1000 ml.
68
3.15.13: Ammonium per sulfate
The Ammonium per sulfate solution was prepared by dissolving 0.1 g of APS in
distilled water and raised the final volume up to 1 ml. Freshly prepared solution of
APS was used.
3.15.14: Staining and destaining solution
Staining solution was prepared by dissolving 2.5 g of Coomassie brilliant blue R-250
in 450 ml of methanol and then added 100 ml of acetic acid and raised the final
volume up to 1000 ml with distilled water. Destaining solution was prepared by
mixing 300 ml methanol, 100 ml acetic acid and volume was raised up to1000 ml with
distilled water.
69
Fig 3.1: Standard curve of maltose
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.20.0
0.2
0.4
0.6
0.8
1.0
1.2
Abs
orba
nce
Maltose conc. (mg/ml)
70
Fig 3.2: Standard curve of bovine serum albumin (BSA)
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.70.0
0.1
0.2
0.3
0.4
Abs
orba
nce
BSA (ug/ml)
71
RESULTS
4.1: IDENTIFICATION, ISOLATION AND SCREENING OF ORGANISM
Strains of Aspergillus oryzae were isolated for the of alpha amylase production from
different soil samples, by serial dilution method on the plates containing starch agar
medium. The strains were identified according to Onion et al. (1986). Colonies of A.
oryzae were greenish yellow, olive yellow or with different shades of green, typically
with dull brown shades with age. Colonies consisting of long conidiophores often
intermixed with aerial mycelium. Conidial heads radiate greenish yellow later
becoming light to dull brown. Conidiophores were hyaline, up to 4-5 mm in length,
mostly rough-walled. Vesicles were subglobose, 40-80 µm in diameter.
Conidiogenous cells uniseriate and biseriate. Phialides often directly borne on the
vesicle or on metulae, usually measuring 10-15 x 3-5 µm. Metulae 8-12 x 4-5 µm.
Metulae or phialides covering the entire surface or the upper three-fourths of the
vesicle. Conidia ellipsoidal when young, globose to subglobose when mature, 4.5-8.0
µm in diameter, green, smooth to finely rough walled.
Screening of A. oryzae isolates for alpha amylase production was carried out in
shake flasks. The seventy eight isolates were screened out for their enzyme
synthesizing ability (Table 4.1). The range of enzyme activity of the wild isolates is
given in Table 4.1.1. Of all the isolates examined, the isolate No. 30 gave maximum
enzyme production and assigned the code IIB-30. This strain was selected for
subsequent studies.
72
Table 4.1: Isolation and screening of Aspergillus oryzae for the alpha amylase production
Isolate No. Enzyme activity(U/ml)
Dry cell mass (g/l) Mycelial morphology
1 103±0.3 9.5±0.5 Large pellets 2 60±0.1 6.3±0.3l Small pellets 3 20±0.2 3.5±0.2 Small pellets 4 41±0.5 4.5±0.1 Large pellets 5 90±0.1 8.5±0.1 Small pellets 6 29±0.2 3.9±0.1 Large pellets 7 118±0.1 10.0±0.2 Small pellets 8 13±0.3 2.9±0.3 Small pellets 9 3.0±0.1 2.0±0.1 Small pellets
10 73±0.1 9.0±0.1 Small pellets 11 100±0.2 9.5±0.2 Small pellets 12 83±0.4 7.3±0.3 Small pellets 13 111±0.1 2.7±0.1 Large pellets 14 40±0.75 3.9±0.2 Small pellets 15 81±0.15 7.5±0.2 Small pellets 16 11±0.1 2.9±0.7 Small pellets 17 35±0.1 4.0±0.1 Small pellets 18 50±0.2 5.7±0.2 Small pellets 19 96±0.1 7.8±0.1 Small pellets 20 7.0±0.1 2.0±0.1 Small pellets 21 .1.0±0.1 1.5±0.1 Small pellets 22 57±0.2 6.0±0.1 Small pellets 23 33±0.2 3.0±0.1 Small pellets 24 17±0.3 2.7±0.3 Small pellets 25 109±0.1 7.0±0.2 Large pellets 26 69±0.3 6.3±0.3l Small pellets 27 87±0.1 4.9±0.1 Small pellets 28 26±0.1 1.6±0.1 Small pellets 29 45±0.1 5.1±0.1 Small pellets 30 130±0.1 13±0.1 Large pellets 31 53±0.2 3.1±0.2 Small pellets 32 12±0.2 1.9±0.1 Small pellets 33 60±0.4 4.2±0.1 Small pellets 34 70±0.1 7.3±0.2 Small pellets 35 120±0.2 10.3±0.1 Large pellets 36 91±0.1 5.1±0.3 Large pellets 37 2.0±0.2 1.3±0.1 Small pellets 38 111±0.3 5.5±0.1 Large pellets 39 14±0.1 2.1±0.2 Small pellets
73
40 97±0.5 9.2±0.1 Small pellets 41 39±0.1 5.0±0.2 Large pellets 42 9.0±0.1 2.0±0.1 Small pellets 43 79±0.2 7.0±0.1 Large pellets 44 99±0.2 7.3±0.2 Large pellets 45 20±0.2 2.7±0.3 Small pellets 46 47±0.1 6.0±0.2 Small pellets 47 6.0±0.1 2.0±0.1 Small pellets 48 63±0.2 11.9±0.1 Large pellets 49 78±0.4 5.2±0. Large pellets 50 13±0.1 3.6±0.2 Small pellets 51 83±0.3 6.5±0.1 Large pellets 52 114±0.1 5.2±0.1 Large pellets 53 59±0.1 5.9±0.1 Small pellets 54 39±0.5 4.9±0.2 Small pellets 55 112±0.1 5.9±0.2 Large pellets 56 93±0.2 6.7±0.1 Large pellets 57 15±0.05 2.11±0.05 Small pellets 58 61±0.4 5.0±0.4 Large pellets 59 10±0.1 3.0±0.1 Small pellets 60 102±0.1 6.0±0.1 Large pellets 61 2.0±0.5 1.3±0.5 Small pellets 62 58±0.2 4.3±0.2 Small pellets 63 27±0.5 1.9±0.5 Small pellets 64 77±0.2 6.5±0.2 Large pellets 65 110±0.5 7.3±0.5 Large pellets 66 108±0.1 8.5±0.1 Large pellets 67 11±0.15 1.9±0.1 Small pellets 68 30±0.5 2.8±0.5 Small pellets 69 64±0.1 3.9±0.1 Small pellets 70 87±0.2 5.7±0.2 Small pellets 71 98±0.1 7.9±0.1 Large pellets 72 21±0.4 2.1±0.4 Small pellets 73 6.0±0.1 1.3±0.1 Small pellets 74 51±0.1 4.3±0.1 Small pellets 75 115±0.5 9.5±0.5 Large pellets 76 16±0.2 2.1±0.2 Small pellets 77 44±0.2 3.8±0.2 Small pellets 78 63±0.1 5.7±0.1 Small pellets
The mean difference is significant at the level of 0.05, ± indicates the standard deviation (SD) among the three parallel replicates in each column. Incubation time 72 h, pH 6.0, incubation temperature 30°C, agitation rate 160 rpm
74
Table 4.1.1: Sub grouping of alpha amylase producing isolates of A. oryzae
Number of isolates Range of enzyme activity (U/ml)
35 1-50 30 51-100 13 101-150
The one culture gave a maximum of 130U/ml alpha amylase production and it was coded as A. oryzae IIB-30 this culture was selected for mutation through UV radiations.
75
4.2: STRAIN IMPROVEMENT
4.2.1: PHYSICAL MUTAGENESIS
4.2.1.1: SCREENING OF UV TREATED ISOLATES
The different isolates were obtained by irradiating the conidia of A. oryzae IIB-30 with
different doses of UV light (15-75 min) in order to increase the alpha amylase
production. The data of Table (4.2) shows the screening of UV treated isolates for the
enzyme production. A total of 32 strains were isolated by observing bigger zones of
starch hydrolysis in Petri plate compared to parental strain. Of all the isolates tested,
the strain isolated after 45 min of UV irradiation with a zone diameter of 1.5 cm gave
maximum enzyme production (160±2 U/ml). The mutant strain gave maximum
production was assigned the code UV-23 and selected for further studies. The number
of survivors and range of enzyme production of the UV treated isolates is given in
Table 4.2.1 and 4.2.2, respectively.
76
Table 4.2: Screening of UV isolates of A. oryzae IIB-30 for alpha amylase production* UV irradiated
isolates Exposure time(min)
Enzyme activity (U/ml)
Dry cell mass (g/l)
Mycelial morphology
UV-1 15 120±1.0 12.0±0.1 Large pellets UV-2 90±0.3 10.5±0.1 Large pellets UV-3 70±0.2 9.60±0.1 Small pellets UV-4 101±0.5 11.7±0.1 Large pellets UV-5 93±0.4 10.7±0.1 Small pellets UV-6 113±0.7 11.8±0.1 Large pellets UV-7 126±1.5 12.9±0.2 Large pellets UV-8 140±1.2 13.2±0.3 Large pellets UV-9 99±1.0 11.3±0.2 Small pellets
UV-10 106±0.9 11.8±0.2 Large pellets UV-11 87±0.4 10.2±0.1 Small pellets UV-12 30 100±1.0 11.0±0.2 Large pellets UV-13 93±0.8 10.3±0.1 Small pellets UV-14 117±1.1 11.3±0.1 Large pellets UV-15 73±0.9 9.60±0.1 Small pellets UV-16 129±1.4 13.1±0.2 Large pellets UV-17 82±0.6 10.0±0.1 Small pellets UV-18 63±0.1 8.50±0.1 Small pellets UV-19 109±0.8 12.1±0.2 Large pellets UV-20 45 76±0.7 9.0±0.1 Small pellets UV-21 136±1.3 13.2±0.2 Large pellets UV-22 123±1.2 12.7±0.3 Large pellets UV-23 160±2.0 14.8±0.4 Large pellets UV-24 28±0.1 5.20±0.1 Small pellets UV-25 39±0.1 6.60±0.1 Small pellets UV-26 60 90±0.3 10.8±0.2 Small pellets UV-27 78±0.5 13.1±0.2 Small pellets UV-28 80±0.6 10.7±0.1 Small pellets UV-29 115±1.0 12.0±0.2 Large pellets UV-30 75 132±1.2 13.1±0.3 Large pellets UV-31 53±0.2 7.5±0.1 Small pellets UV -32 43±0.1 7.2±0.1 Small pellets
The mean difference is significant at the level of 0.05, ± indicates the standard deviation (SD) among the three parallel replicates in each column. Incubation time 72 h, pH 6.0, incubation temperature 30°C, agitation rate 160 rpm
77
Table 4.2.1: UV treated survivors at different exposure time
Exposure time Total number of survivors 15 38 30 25 45 17 60 13 75 2.0
Table 4.2.2: Range of alpha amylase activity of UV isolates
Number of strains Range of enzyme activity (U/ml)
3 1-50 15 51-100 14 101-150
The one isolate gave maximum production of alpha amylase 160 U/ml and it was coded as UV-23. This strain was selected for mutation through NG.
78
4.3: CHEMICAL MUTAGENESIS
4.3.1: SCREENING OF NG TREATED ISOLATES
The UV treated mutant was further subjected to chemical treatment in order to
increase the alpha amylase production. The A. oryzae UV-23 was exposed to different
concentrations of NG (0.5-2.0 mg/ml). The data of Table (4.3) shows the screening of
NG treated isolates for the enzyme production. A total of 18 strains were isolated by
observing bigger zones of starch hydrolysis in Petri plate compared to parental strain.
Of all the isolates tested, the strains isolated after treatment with 1.5 mg/ml NG with a
zone diameter of 1.9 cm gave maximum (270±0.1 U/ml) enzyme production which is
two fold than UV-23. The mutant strain exhibiting highest enzyme production assigns
the code NG-15 and selected for further studies. When the concentration of NG
increased, the number of survivors decreased as shown in the Table 4.3.1. At 2.0
mg/ml concentration of NG 90 % death rate was observed further increase in
concentration cause complete death of organism. The number of survivors and range
of enzyme production by NG treated isolates is given in table 4.3.1 and 4.3.2,
respectively.
4.3.2: SCREENING OF NITROUS ACID TREATED ISOLATES
The NG mutant strain A. oryzae NG-15 were further treated with different
concentrations of nitrous acid (0.1-0.4 M). Selective NA treated strains were isolated
on the basis of qualitative screening showing bigger zones of starch hydrolysis than
the parental strain. The NA treated strains were screened for enzyme production
79
(Table 4.4). Out of which mutant strain NA-17 gave maximum alpha amylase
production (285±0.1 U/ml). When the concentration of nitrous acid was increased, the
number of the survivors was decreased as shown in the Table 4.4.1. Range of enzyme
production of the nitrous acid treated isolates is given in Table 4.4.2.
4.3.3: SCREENING OF EMS TREATD ISOLATES
The nitrous acid treated mutant NA-17 was subjected to the varying concentration (25-
150 µl/ml) of ethyl methane sulphonate (EMS).A total of twenty-six EMS treated A.
oryzae strains were obtained at 90 % death rate, which were screened for alpha
amylase production (Table 4.5). The mutant EMS-18 showed 1.5-fold higher enzyme
yield (347±1.2U/ml) compared to NA-17. As the concentration of EMS was increased,
the number of survivors was decreased (Table 4.5.1.) The range of enzyme production
of EMS treated isolates is given in Table 4.5.2
80
Table 4.3: Screening of NG treated A. oryzae UV-23 isolates for alpha amylase production *
NG treated isolates
NG concentration (mg/ml)
Enzyme activity(U/ml)
Dry cell mass(g/l)
Mycelial morphology
NG-1 0.5 160±0.2 14.9±0.1 Large pellets NG-2 109±0.8 11.5±0.3 Small pellets NG-3 136±0.3 12.8±0.2 Large pellets NG-4 150±0.1 13.9±0.1 Large pellets NG-5 96±0.2 12.7±0.2 Small pellets NG-6 111±0.1 11.9±0.1 Small pellets NG-7 87±0.6 10.4±0.2 Small pellets NG-8 123±0.2 12.2±0.2 Large pellets NG-9 1.0 201±1.0 15.0±1 Large pellets
NG1-0 145±0.4 11.6±0.2 Large pellets NG-11 34±0.1 3.60±0.1 Small pellets NG-12 220±0.3 14.3±0.3 Large pellets NG-13 83±0.5 8.30±0.3 Small pellets NG-14 1.5 132±0.5 11.6±0.1 Small pellets NG-15 270±0.1 15.6±0.4 Large pellets NG-16 63±0.4 5.60±0.1 Small pellets NG-17 2.0 103±0.3 10.5±0.1 Small pellets NG-18 49±0.1 6.20±0.4 Small pellets
The mean difference is significant at the level of 0.05, ± indicates the standard deviation (SD) among the three parallel replicates in each column.
*Incubation time 72 h, temperature 30°C, pH 6.0, agitation rate 160 rpm
81
Table 4.3.1: NG treated survivors of A. oryzae
NG concentration(mg/ml) Total number of survivors 0.5 31 1.0 20 1.5 11 2.0 2.0
Table 4.3.2: Range of alpha amylase activity of NG isolates
Number of isolates Range of enzyme activity(U/ml) 2 1-50 4 51-100 8 101-150 1 151-200 2 201-250 1 251-300
The one culture gave maximum production of alpha amylase 270 U/ml and it was coded as NG 15. This culture was selected for mutation with nitrous acid.
82
Table 4.4: Screening of nitrous acid treated strains of A. oryzae NG-15 for the alpha amylase production*
Nitrous acid
treated isolates conc. of nitrous
acid(M) Enzyme
activity(U/ml) Dry cell mass(g/l)
Mycelial morphology
NA-1 0.1 261±0.2 15.0±0.1 Large pellets NA-2 256±0.7 15.2±0.2 Large pellets NA-3 259±0.1 14.0±0.1 Large pellets NA-4 218±0.6 14.3±0.3 Large pellets NA-5 247±0.1 14.9±0.1 Large pellets NA-6 258±0.5 13.8±0.8 Large pellets NA-7 250±0.7 13.7±0.9 Large pellets NA-8 262±0.3 13.9±0.3 Large pellets NA-9 266±0.3 12.7±0.1 Large pellets
NA-10 0.2 259±0.0.2 11.9±0.3 Small pellets NA-11 248±0.6 13.0±0.3 Large pellets NA-12 230±0.6 13.8±0.1 Large pellets NA-13 256±0.1 14.9±0.1 Large pellets NA-14 168±0.2 9.90±0.4 Small pellets NA-15 0.3 209±0.5 11.6±0.05 Large pellets NA-16 196±0.4 11.3±0.1 Small pellets NA-17 285±0.1 16.1±0.1 Large pellets NA-18 39±0.4 2.60±0.2 Small pellets NA-19 0.4 58±0.7 3.80±0.2 Small pellets NA-20 109±0.5 6.80±0.2 Small pellets NA-21 153±0.1 10.6±0.3 Small pellets
The mean difference is significant at the level of 0.05, ± indicates the standard deviation (SD) among the three parallel replicates in each column.
*Incubation time 72 h, temperature 30°C, pH 6.0, agitation rate 160 rpm
83
Table 4.4.1: Nitrous acid treated survivors of A. oryzae
Nitrous acid concentrations (M) Total number of survivors 0.1 39 0.2 32 0.3 27 0.4 24
Table 4.4.2: Range of alpha amylase activity of nitrous acid treated isolates
Number of isolates Range of enzyme activity (U/ml) 1 1-50 1 51-100 1 101-150 3 151-200 6 201-250 9 251-300
The one isolate gave a maximum production of alpha amylase 285 U/ml and it was coded as NA17.This strain was further treated with EMS.
84
Table 4.5: Screening of EMS treated A. oryzae NA17 for the alpha amylase production *
EMS treated
isolates EMS
conc.(µl/ml) Enzyme
activity (U/ml) Dry cell mass(g/l)
Mycelial morphology
EMS_1 25 255±0.1 14.7±0.1 Large pellets EMS-2 198±0.2 13.9±0.1 Large pellets EMS-3 250±0.3 14.2±0.2 Large pellets EMS-4 230±0.2 14.1±0.1 Large pellets EMS-5 275±0.2 15.6±0.1 Large pellets EMS-6 200±1.0 12.3±1 Small pellets EMS-7 273±0.1 15.2±0.1 Large pellets EMS-8 50 240±0.5 13.2±0.2 Large pellets EMS-9 168±0.05 14.1±0.1 Small pellets
EMS-10 290±0.3 12.3±0.3 Large pellets EMS-11 197±0.2 15.3±0.1 Small pellets EMS-12 100±0.7 12.4±0.4 Small pellets EMS-13 75 187±0.4 11.7±0.2 Small pellets EMS-14 300±0.8 12.1±0.1 Large pellets EMS-15 87±1 15.9±1 Small pellets EMS-16 118±0.1 7.20±0.2 Small pellets EMS-17 147±0.5 10.6±0.5 Small pellets EMS-18 100 347±0.1 16.9±0.1 Large pellets EMS-19 106±0.1 11.6±0.2 Small pellets EMS-20 47.0±0.2 5.8±0.2 Small pellets EMS-21 93±0.4 2.6±0.1 Small pellets EMS-22 125 101±1 5.8±1 Small pellets EMS-23 208±0.1 13.3±0.4 Small pellets EMS-24 287±0.1 14.1±0.1 Large pellets EMS-25 150 201±0.3 12.6±0.3 Large pellets EMS-26 126±0.2 10.2±0.2 Small pellets
The mean difference is significant at the level of 0.05, ± indicates the standard deviation (SD) among the three parallel replicates.
*Incubation time 72h, temperature 30°C, pH 6.0, agitation rate 160rpm
85
Table 4.5.1: EMS treated survivors of A. oryzae
EMS concentrations (µl) Total number of survivors 25 45 50 32 75 21
100 5 125 3 150 2
Table 4.5.2: Range of α-amylase activity of EMS treated isolates
Number of isolates Range of enzyme activity (U/ml) 1 1-50 2 51-100 5 101-200 4 151-200 6 201-250 6 251-300 1 301-350
The one isolate gave a maximum production of alpha amylase 347 U/ml and it was coded as EMS-18. This strain was selected for optimization
86
4.4: OPTIMIZATION OF CULTURAL CONDITIONS IN SHAKE
FLASKS
4.4.1: SCREENING OF CULTURE MEDIA
The six different fermentation media (cited in literature for the production of alpha
amylase by different strains) were evaluated for the alpha amylase production by wild
and mutant strains of A. oryzae (Fig 4.1). Of all the media tested, M 4 medium gave
highest units of alpha amylase 168±2 (wild) and 385±2 (mutant). The dry cell mass
was 14.9±2 and 16.8±2.4 g/l, respectively. The rest of the fermentation media gave not
significant enzyme production for both strains as compared to M4 medium. The
enzyme production was found to be highly significant (p≤0.05) in M4 medium so, it
was selected in subsequent studies.
4.4.2: RATE OF ALPHA AMYLASE PRODUCTION
The effect of incubation period on the alpha amylase production both by wild and
mutant strains of A. oryzae was optimized (Fig 4.2). The fermentation was carried out
for 96 h and enzyme production was calculated after every 8 h. The production of
enzyme was increased with the increase in the incubation period and reached
maximum at 72 h after inoculation by both the wild (168±2 U/ml) and mutant (386±2
U/ml) strains. The dry cell mass was 14.9±2 and 16.8±2.4 g/l, respectively. Further
increase in incubation period did not show any increase in the formation of enzyme
rather it was decreased. The enzyme production was highly significant (p≤0.05) after
72 h. So, it was selected in subsequent studies.
87
4.4.3: EFFECT OF INCUBATION TEMPERATURE
Figure 4.3 shows the effect of varying incubation temperature (25-50°C) on the alpha
amylase production by both wild and mutant strains of A. oryzae. Both the wild
(166±2 U/ml) and mutant (386±3 U/ml) strains gave maximum enzyme production at
30°C. The dry cell mass was 14.2±1.0 and17.8±2.40 g/l, respectively. When
temperature of medium was increased from 30°C, the enzyme production was
reduced. At 50°C, the enzyme production became not significant (p≤0.05) as
compared to other temperatures.
4.4.4: EFFECT OF DIFFERENT INITIAL pH
The effect of different initial pH of the fermentation medium on the alpha amylase
production was investigated (Fig 4.4). The initial pH of the fermentation medium was
adjusted at 4-7 in shake flasks. The enzyme production following growth of organism
by both wild and mutant strains was found to be significant (p≤0.05) at pH 5. As the
pH of the medium was increased from 5, there was gradual reduction in the enzyme
formation by both wild and mutant strains of A. oryzae. At alkaline pH, the enzyme
production was extremely low. Thus, pH 5 was selected for of alpha amylase
production.
4.4.5: EFECT OF DIFFERENT VOLUMES OF MEDIUM
Figure 4.5 depicted the effect of different volumes of the basal medium such as 5, 10,
15, 20 and 25 % (v/v) in 250 ml Erlenmeyer flask on alpha amylase production by
both the wild and mutant strains of A. oryzae. The maximum enzyme production by
88
wild (189±1) and mutant (416±3) strains were observed when 10 % volume (25
ml/250 ml flask) was used. As the volume of fermentation medium was increased, the
enzyme production was decreased gradually. The enzyme production was significant
at 10 % volume of fermentation medium so it was selected for further studies.
4.4.6: EFFECT OF DIFFERENT INOCULUM SIZES
Effect of size of inoculum (2-12 % v/v) on the alpha amylase production by wild and
mutant strains of A. oryzae was evaluated (Fig 4.6). The inoculum size of 4 % v/v
(1ml=2.6Χ106CFU) yielded maximum enzyme production both by wild (191±1.04)
and mutant (418±3) strains. The dry cell mass was 14.7±1.0 and 17.9±2.0 g/l,
respectively. Beyond this level the enzyme production was decreased gradually. The
enzyme production was found to be significant (p≤0.05) at 4 % level hence; it was
optimized for maximum enzyme production.
89
Fig 4.1: Screening of fermentation media for the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18*
0
200
400
600
800
1000
M1 M2 M3 M4 M5 M6
Fermentation media
Enzy
me
activ
ity (U
/ml)
0
5
10
15
20
25
30
DC
M (g
/l)
Enzyme activity of wild strain IIB-30 (U/ml) Enzyme activity of mutant strain EMS-18 (U/ml)
DCM of wild strain IIB-30(g/l) DCM of mutant strainEMS-18 (g/l)
Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p≤ 0.05.
*Incubation time 72 h, temperature 30°C, pH 6.0, agitation rate 160 rpm
90
Fig 4.2: Rate of fermentation for the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 *
0
200
400
600
800
1000
0 8 16 24 32 40 48 56 64 72 80 88 96
Time (h)
Enzy
me
activ
ity (U
/ml)
0
5
10
15
20
25
30
DC
M (g
/l)
Enzyme activity of wild strain IIB-30 (U/ml) Enzyme activity of mutant strain EMS-18 (U/ml)
DCM of wild strain IIB-30 (g/l) DCM of mutant strain EMS-18 (g/l)
Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p≤ 0.05.
*Incubation temperature 30°C, pH 6.0, agitation rate 160 rpm
91
Fig 4.3: Effect of incubation temperature on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 *
0
200
400
600
800
1000
25 30 35 40 45 50
Temperature°C
Enzy
me
activ
ity (U
/ml)
0
5
10
15
20
25
30
DC
M (g
/l)Enzyme activity of w ild strain IIB-30 (U/ml) Enzyme activity of mutant strain EMS-18 (U/ml)
DCM of w ild strain IIB-30 (g/l) DCM of mutant strain EMS-18 (g/l)
Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p≤ 0.05.
*Incubation time 72 h, incubation temperature 30°C, pH 6.0, agitation rate 160 rpm
92
Fig 4.4: Effect of different initial pH of fermentation medium on the of alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 *
0
200
400
600
800
1000
4 4.5 5 5.5 6 6.5 7
pH
Enzy
me
activ
ity (U
/ml)
0
5
10
15
20
25
30
DC
M (g
/l)Enzyme activity of wild strain IIB-30 (U/ml) Enzyme activity of mutant strain EMS-18 (U/ml)
DCM of wild strain IIB-30 (g/l) DCM of mutant strain EMS-18 (g/l)
Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p≤0.05.
*Incubation time 72 h, incubation temperature 30°C, agitation rate 160 rpm.
93
Fig 4.5: Effect of different volumes of media on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 *
0
200
400
600
800
1000
5 10 15 20 25
Volume of media (%)
Enzy
me
activ
ity (U
/ml)
0
5
10
15
20
25
30
DC
M (g
/l)Enzyme activity of wild strain IIB-30 (U/ml) Enzyme activity of mutant strain EMS-18
DCM of wild strain IIB-30 (g/l)) DCM of mutant strain EMS-18 (g/l)
Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p≤ 0.05.
*Incubation time 72 h, incubation temperature 30°C, agitation rate 160 rpm
94
Fig 4.6: Effect of different inoculum sizes on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 *
0
200
400
600
800
1000
2 4 6 8 10 12
Inoculum (%)
Enzy
me
activ
ity (U
/ml)
0
5
10
15
20
25
30
DC
M (g
/l)
Enzyme activity of wild strain IIB-30 (U/ml) Enzyme activity of mutant strain EMS-18 (U/ml)
DCM of wild strain IIB-30 (g/l) DCM of mutant strain EMS-18 (g/l)
Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p≤ 0.05.
*Incubation time 72 h, pH 5.0, incubation temperature 30°C, agitation rate160 rpm
95
4.5: OPTIMIZATION OF NUTRITIONAL REQUIREMENTS OF A.
ORYZAE IN SHAKE FLASKS
4.5.1: EFFECT OF STARCH FROM DIFFERENT SOURCES
Figure 4.7 depicted the effect of starch from different sources such as wheat, corn, rice
and sweet potato at the concentration of 1 % on the alpha amylase production by both
wild and mutant strains of A. oryzae. Of all the starches tested, corn starch gave
maximum enzyme production by both the wild (190±2.0) and mutant (419±3.0)
strains. The dry cell mass was 14.6±2.0 and 16.8±2.0 g/l, respectively. All other
starches gave less significant results (p≤0.05 as compared to corn starch. Therefore
corn starch was selected for subsequent studies.
4.5.2: EFFECT OF DIFFERENT CONCENTRATIONS OF CORN STRACH
The effect of different concentration of starch (1.0-5.0 % w/v) was investigated for the
alpha amylase production by both the wild and mutant strain of A. oryzae (Fig 4.8).
The maximum enzyme production by both strains wild (201±1.04) and mutant
(436±2.0) was obtained at the level of 2 %. (w/v) The dry cell mass was 15.3±1.0 and
18.1±1.9 g/l, respectively. Beyond this concentration the enzyme production was
decreased. The enzyme production was significant (p≤0.05) at 2 % (w/v) so; it was
selected for further studies.
4.5.3: EVALUATION OF ADDITIONAL CARBON SOURCES
The effect of different carbon sources such as glucose, sucrose, xylose, lactose,
fructose, galatose, caboxy methyl cellulose, glycerol and mannitol was evaluated for
the alpha amylase production by wild and mutant strains of A. oryzae in present course
96
of study. Of all the carbon sources tested lactose showed considerable increase in the
enzyme production by both the wild (235±1.04) and mutant strains (460±2.0)
compared to others (Fig 4.9). The dry cell mass was 15.9±1.0 and 18.8±2.1 g/l,
respectively. These carbon sources were added to the fermentation media at the
concentration of 0.5 %.( w/v) Therefore, lactose as additional carbon source was
selected and its various concentrations were also tested for the enzyme production (Fig
4.10). The concentration of the lactose was kept from 0.5-2.5 % (w/v). The lactose at
the concentration of 1.5 %( w/v) in case of wild (260±2.0 U/ml) and 1.0 % (w/v) in
case of mutant (490±2.3 U/ml) was found to be the best for the enzyme production.
Further increase in the concentration of lactose resulted decrease in the enzyme
production. At 2.5 % (w/v) concentration of lactose the enzyme production was not
significant (p≤0.05) as compared to other concentrations.
4.5.4: EVALUATION OF INORGANIC NITROGEN SOURCES
Various inorganic nitrogen sources such as (NH4)2SO4, NH4NO3, NaNO3 and
KNO3 was evaluated for the alpha amylase production by the wild and mutant strains
of A. oryzae (Fig 4.11). The nitrogen sources were added to the fermentation media at
the level of 0.1 % (w/v). Of all the nitrogen sources examined (NH4)2SO4 gave
maximum enzyme production by both the wild (274±2 U/ml/) and mutant (503±3
U/ml) strains. Therefore, various concentrations of (NH4)2SO4 were also evaluated for
the enzyme production (Fig 4.12). The concentration of the (NH4)2SO4 was kept 0.1-
0.5 %.(w/v). The (NH4)2SO4 at the concentration of 0.3 % (w/v) was found to be
significant (p≤0.05) for the enzyme production both by the wild (284±1.04) and
mutant (525±3.0) strains. The dry cell mass was 16.9±1 and 19.6± 2 g/l, respectively.
97
Further increase in the concentration of (NH4)2SO4 was resulted decrease in the
enzyme production.
4.5.5: EVALUATION OF ORGANIC NITROGEN SOURCES
Different organic nitrogen sources such as peptone, meat extract, Corn steep liquor
(CSL), urea, casein and beef extract were evaluated for the alpha amylase production
(Fig 4.13). The nitrogen sources were added to the fermentation media at the
concentration of 0.1 % (w/v). Of all the nitrogen sources tested, peptone gave
maximum enzyme production by both the wild (298±1.U/ml) and mutant (542±2.9
U/ml) strains. The dry cell mass was 17.5±1.0 and19.5±2.0 g/l, respectively. The CSL
was proved as the second best nitrogen source for enzyme production by both strains.
The least alpha amylase production was observed when casein was used as an organic
nitrogen source. Variations in concentrations of peptone were also effective for alpha
amylase production. Therefore, various concentrations of peptone (0.1-0.5 % w/v)
were also evaluated for the enzyme production by both strains (Fig 4.14). Peptone at
the concentration of 0.2 % w/v found to be significant (p≤0.05) for the enzyme
production. Further increase in the concentration of peptone was resulted decrease in
the enzyme production.
4.5.6: EFFECT OF SURFACTANTS
The effect of various surfactants such as Tween 80, Triton X-100, Sodium dodecyl
sulphate (SDS), Di-octyl ester of sodium sulpho succinic acid (Monoxal O.T), Poly
ethylene glycol (PEG) and sodium lauryl sulphate was investigated for the alpha
amylase production by the A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-
18 (Fig 4.15). The surfactants were added to the fermentation media at the
98
concentration of 0.05 % (v/v). Of all the surfactants examined, Tween 80 gave
maximum enzyme production by both the wild (312±2.0 U/ml) and mutant (573±2.0
U/ml) strains. Therefore, various concentrations of Tween 80 were also evaluated for
the enzyme production (Fig 4.16). The concentrations of the Tween 80 was kept as
0.05 - 0.25 % (v/v). The Tween 80 at the concentration of 0.1 % (v/v) was found to be
significant (p≤0.05) for the enzyme production both by the wild (320±2.0 U/ml) and
mutant (589±3.0 U/ml) strains. The dry cell mass was 16.9 and 19.6 g/l, respectively.
Further increase in the amount of Tween 80 was resulted decrease in the enzyme
production. Hence, Tween 80 at the concentration of 0.1 % (v/v) was selected for
further studies.
99
Fig 4.7: Effect of raw starch from different sources on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18.*
0
200
400
600
800
1000
Wheat starch Corn starch Rice starch Sweet potatostarch
Starch (1%)
Enzy
me
activ
ity (U
/ml)
0
5
10
15
20
25
30
DC
M (g
/l)
Enzyme activity of wild strain IIB-30(U/ml) Enzyme activity of mutant strainEMS-18 (U/ml)
DCM of wild strain IIB-30 (g/l) DCM of mutant strain EMS-18 (g/l)
Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p≤ 0.05.
* Incubation time 72 h, incubation temperature 30°C, pH 5.0, agitation rate 160 rpm
100
Fig 4.8: Effect of different concentrations of starch on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18.*
0
200
400
600
800
1000
1 2 3 4 5
Concentration of starch (%)
Enzy
me
activ
ity (U
/ml)
0
5
10
15
20
25
30
DC
M (g
/l)Enzyme activity of w ild strain IIB-30 (U/ml) Enzyme activity of mutant strain EMS-18 (U/ml)
DCM of w ild strain IIB-30 (g/l) DCM of mutant strain EMS-18 (g/l)
Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p≤ 0.05.
* Incubation time 72 h, incubation temperature 30°C, pH 5.0, agitation rate 160 rpm
101
Fig 4.9: Evaluation of additional carbon sources on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18.*
0
200
400
600
800
1000
Glucos
e
Sucros
e
Xylose
Lacto
se
Fructose
Galatos
eCMC
Glycero
l
Mannito
l
Control
Carbon sources (0.5%)
Enzy
me
activ
ity (U
/ml)
0
5
10
15
20
25
30
DC
M (g
/l)Enzyme activity of wild strain IIB-30 (U/ml) Enzyme activity of mutant strain EMS-18 (U/ml)
DCM of wild strain IIB-30 (g/l) DCM of mutant strainEMS-18 (g/l)
Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p≤ 0.05.
* Incubation time 72 h, incubation temperature 30°C, pH 5.0, agitation rate 160 rpm
102
Fig 4.10: Effect of different concentrations of lactose on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18.*
0
200
400
600
800
1000
0.5 1 1.5 2 2.5
Lactose concentrations (%)
Enzy
me
activ
ity (U
/ml)
0
5
10
15
20
25
30
DC
M (g
/l)Enzyme activity of w ild strain IIB-30 (U/ml) Enzyme activity of mutant strainEMS-18 (U/ml)
DCM of w ild strain IIB-30(g/l) DCM of mutant strain EMS-18 (g/l)
Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p≤ 0.05.
* Incubation time 72 h, incubation temperature 30°C, pH 5.0, agitation rate 160 rpm
103
Fig 4.11: Evaluation of inorganic nitrogen sources on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18.*
0
200
400
600
800
1000
Ammoniumsulfate
Ammoniumnitrate
Sodium nitrate Potassiumnitrate
Control
Inorganic nitrogen sources (0.1%)
Enzy
me
activ
ity (U
/ml)
0
5
10
15
20
25
30
DC
M (g
/l)
Enzyme activity of wild strain IIB-30 (U/ml) Enzyme activity of mutant strain EMS-18 (U/ml)
DCM of wild strain IIB-30 (g/l) DCM of mutant strain EMS-18 (g/l)
Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p≤ 0.05.
* Incubation time 72 h, incubation temperature 30°C, pH 5.0, agitation rate 160 rpm
104
Fig 4.12: Effect of different concentrations of ammonium sulfate on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18.*
0
200
400
600
800
1000
0.1 0.2 0.3 0.4 0.5
Ammonium sulfate concentrations (%)
Enzy
me
activ
ity (U
/ml)
0
5
10
15
20
25
30
DC
M (g
/l)
Enzyme activity of wild strain IIB-30 (U/ml) Enzyme activity of mutant strain EMS-18 (U/ml)
DCM of wild strain IIB-30 (g/l) DCM of mutant strainEMS-18(g/l)
Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p≤ 0.05.
* Incubation time 72 h, incubation temperature 30°C, pH 5.0, agitation rate160 rpm
105
Fig: 4.13: Evaluation of organic nitrogen sources on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 *
0
200
400
600
800
1000
Meatextract
CSL Urea Casein Beefextract
Peptone Control
Organic nitrogen sources (0.1%)
Enzy
me
activ
ity (U
/ml)
0
5
10
15
20
25
30
DC
M (g
/l)
Enzyme activity of wild strain IIB-30 (U/ml) Enzyme activity of mutant strain EMS-18 (U/ml)
DCM of wild strain IIB-30 (g/l) DCM of mutant strain EMS-18 (g/l)
Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p≤0.05.
* Incubation time 72 h, incubation temperature 30°C, pH 5.0, agitation rate160 rpm
106
Fig 4.14: Effect of different concentrations of peptone on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 *
0
200
400
600
800
1000
0.1 0.2 0.3 0.4 0.5
Concentrations of peptone
Enzy
me
activ
ity (U
/ml)
0
5
10
15
20
25
30
DC
M (g
/l)Enzyme activity of wild strain IIB-30 (U/ml) Enzyme activity of mutant strain EMS-18 (U/ml)
DCM of wild strain IIB-30 (g/l) DCM of mutant strain EMS-18 (g/l)
Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p≤0.05.
* Incubation time 72 h, incubation temperature 30°C, pH 5.0, agitation rate160 rpm
107
Fig 4.15: Effect of different surfactants on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18.*
0
200
400
600
800
1000
Tween80
TritonX
-100
Sodium
dodyc
yl su
lphate
Monoxa
l O.T
Polyethy
lene gl
ycol
Sodium
laury
sulph
ate
Control
Surfactants (0.05%)
Enzy
me
activ
ity (U
/ml)
0
5
10
15
20
25
30
DC
M (g
/l)
Enzyme activity of w ild strain IIB-30 (U/ml) Enzyme activity of mutant strain EMS-18 (U/ml)
DCM of w ild strain IIB-30 (g/l) DCM of mutant strain EMS-18 (U/ml)
Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p≤ 0.05.
* Incubation time 72 h, incubation temperature 30°C, pH 5.0, agitation rate 160 rpm
108
Fig 4.16: Effect of different concentrations of Tween 80 on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18.*
0
200
400
600
800
1000
0.05 0.1 0.15 0.2 0.25
Concentrations of Tween 80 (%)
Enzy
me
activ
ity (U
/ml)
0
5
10
15
20
25
30
DC
M (g
/l)Enzyme activity of wild strain IIB-30 (U/ml) Enzyme activity of mutant strain EMS-18 (U/ml)
DCM of wild strain IIB-30 (g/l) DCM of mutant strain EMS-18 (g/l)
Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p≤ 0.05.
* Incubation time 72 h, incubation temperature 30°C, pH 5.0, agitation rate 160 rpm
109
4.6: OPTIMIZATION OF CULTURAL CONDITIONS IN STIRRED
FERMENTER
4.6.1: RATE OF ALPHA AMYLASE PRODUCTION
The rate of fermentation of both the wild (IIB-30) and mutant (EMS-18) strains of A.
oryzae for the alpha amylase production was investigated in stirred fermenter (Fig
4.17). The time course aliquots were withdrawn after every 8 h aseptically and
subjected to enzyme estimation (U/ml) and dry cell mass determination (g/l) up to 96
h of fermentation period. It was found that the enzyme production was increased
gradually and reached its maximum (335 U/ml) and (608 U/ml) after 64 h for wild and
48 h of fermentation for mutant respectively. The dry cell mass was (18.2) and (19.8),
g/l respectively. A significant finding of present experiment was that fermentation
period was reduced to 16 h in case of wild and 24 h in case of mutant from 72 h in
shake flask studies. Rapid decline in enzyme production was seen in case of wild and
mutant strain when incubation period was increased from optimum time period.
Data obtained from above experiment was subjected to kinetic analysis for
calculations of µ (h-1)max (specific growth rate), qp (unit product produced/g cell/h), Qp
(enzyme produced/l/h), Qx (g cell mass formation/l/h), Yp/x(enzyme produced/g cell
mass formation). The kinetic evaluation of results also revealed that optimum
fermentation period for enzyme production was 64 h in case of wild and 48 h in case
of mutant strains of A. oryzae (Table 4.6).
110
4.6.2: EFFECT OF pH
Effect of different initial pH (4-6.5) of fermentation medium by both wild and mutant
strains of A. oryzae was investigated in stirred fermenter (Fig 4.18). At pH 5, the
maximum enzyme production by both wild (342U/ml) and mutant (626U/ml) strains
were observed. The dry cell mass was 18.7 and 22.5 g/l, respectively. With the
increase of pH, a decrease in enzyme production was observed. At alkaline pH the
production was extremely low.
The kinetic parametric study indicated that the yield of the enzyme by biomass
formation as well as the rates of enzyme formation was significant at pH 5 (Table 4.7).
Thus, pH 5 was selected for the production of enzyme by both wild and mutant strains
of A. oryzae.
4.6.3: EFFECT OF AERATION LEVELS
Figure 4.19 showed the effect of rate of aeration on the alpha amylase production by
wild and mutant strain of A. oryzae. The rate of aeration varied from 0.5-2 vvm in
stirred fermenter. Enzyme production by wild strain was found maximum i.e. 350
U/ml when aeration rate was set at 1.0 vvm while mutant strain gave maximum
enzyme activity (660 U/ml) at 1.5 vvm. The dry cell mass was 18.9 and 23.1 g/l,
respectively. Any variation beyond this optimum level, gave less enzyme production.
The kinetic analysis of above parameters revealed that the values of Yp/x, Qp,
Qx were significant at an air supply of 1.0 vvm (wild) & 1.5 vvm (mutant). Therefore,
1.5 vvm was optimized for further studies for enhanced enzyme production.
111
4.6.4: EFFECT OF DISSOLVED OXYGEN
Figure 4.20 showed the effect of different levels (5-20 % v/v) of dissolved oxygen on
alpha amylase production by wild and mutant strains of A. oryzae. Dissolved oxygen
at the level of 15 % (v/v) gave the maximum enzyme production by wild (362U/ml)
and mutant (687U/ml) strains. The dry cell mass was 19 and 23.6 g/l, respectively.
Beyond this level, a decrease in enzyme production was recorded.
Data obtained from above experiment was subjected to kinetic analysis for
calculations of Qp (enzyme produced/l/h), Qx (g cell mass formation/l/h),
Yp/x(enzyme produced/g cell mass formation). The kinetic evaluation of results also
revealed that optimum level of dissolved oxygen in fermenter was 15 % (v/v) for
enzyme production by A. oryzae IIB-30 and its mutant derivatives A. oryzae EMS-18
(Table 4.9).
4.6.5: EFFECT OF INOCULUM SIZE
Effect of different sizes of inoculum was investigated for alpha amylase production by
both the wild (IIB-30) and mutant (EMS-18) strains of A. oryzae in stirred fermenter
(Fig 4.21). The size of vegetative inoculum was varied from 5-12.5 %, (v/v) and
fermentation was carried out. The maximum alpha amylase production by both wild
(372 U/ml) and mutant (718 U/ml) strains was observed at the inoculum size of 10 %
(v/v). The dry cell mass was 19.2 and 24.1 g/l, respectively. Beyond this concentration
the enzyme production decreased gradually.
112
All kinetic parameters showed 10 % (v/v) vegetative inoculum to be optimum
for the enzyme production. Thus the inoculum size of 10 % (v/v) was used in further
studies for the enzyme production in stirred fermenter.
4.6.6: EFFECT OF AGITATION INTENSITY
The effect of rate of agitation on the alpha amylase production by wild and mutant
strains of A. oryzae was investigated in stirred fermenter. The rate of agitation was
varied from 120-240 rpm (Fig 4.22). Maximum enzyme production was obtained
when agitation was maintained at 200 rpm. Further increase or decrease in agitation
speed resulted in decrease enzyme production by both the strains.
Evaluation of kinetic parameters Yp/x, Qp, Qx indicated that production yield by
wild and mutant strains was found optimum when agitation speed of impeller was set
at 200 rpm (Table 4.10). Thus agitation speed of 200 rpm was selected for further
studies.
113
Fig 4.17: Comparison of rate of alpha amylase production by wild (IIB-30) and mutant strain of A. oryzae (EMS-18) in stirred fermenter*
0
200
400
600
800
1000
0 8 16 24 32 40 48 56 64 72 80 88 96
Time (h)
Enzy
me
activ
ity (U
/ml)
0
5
10
15
20
25
30
DC
M (g
/l)
Enzyme activity of wild strain IIB-30 (U/ml) Enzyme activity of mutant strainEMS-18 (U/ml)
DCM of wild strain IIB-30 (g/l) DCM of mutant strainEMS-18(g/l)
* Incubation temperature 30°C, pH 5.0, agitation rate 160 rpm, aeration 1vvm
114
Table 4.6: Kinetic evaluation of rate of fermentation for the alpha amylase production by A. oryzae IIB-30 and its mutant derivatives in stirred fermenter
Kinetic parameters wild Mutant µ
Yp/x Qp Qx qp
0.2 55000 5583 0.30
11000
0.22 185714 10133 0.33
40857
Kinetic parameters: Yp/x= enzyme produced/g cell mass formation.
Qp=enzyme produced/l/h. Qx= g cell mass formation/l/h.
µ(h-1)max= specific growth rate.
115
Fig 4.18: Effect of initial pH of media on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 *
0
200
400
600
800
1000
4 4.5 5 5.5 6 6.5
Initial pH
Enzy
me
activ
ity (U
/ml)
0
5
10
15
20
25
30
DC
M (g
/l)
Enzyme activity of wild strain IIB-30 (U/ml) Enzyme activity of mutant strain EMS-18 (U/ml)
DCM of wild strain IIB-30 (g/l) DCM of mutant strain EMS-18
*Incubation time 48 h, temperature 30°C, agitation intensity 160 rpm, aeration 1vvm
116
Table 4.7: Kinetic evaluation of different pH values of media for the alpha amylase production by A. oryzae IIB-30 and its mutant derivative in stirred fermenter
Kinetic parameters: Yp/x= enzyme produced/g cell mass formation.
Qp=enzyme produced/l/h. Qx= g cell mass formation/l/h.
pH 4 4.5 5.0 5.5 6.0 6.5 Kinetic
parametes wild Mutant Wild Mutant Wild Mutant Wild Mutant Wild Mutant Wild Mutant
Yp/x Qp Qx
20915 4333 0.20
28032 9016 0.25
21487 5166 0.23
30353 9633 0.28
216785700 0.31
36066 10433 0.37
21551533330.25
33604 10016 0.33
19607 4166 0.19
27822 8550 0.30
182883333 0.17
24642 6900 0.28
117
Fig 4.19: Effect of different aeration levels on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 *
0
200
400
600
800
1000
0.5 1 1.5 2
Aeration levels (vvm)
Enzy
me
activ
ity (U
/ml)
0
5
10
15
20
25
30
DC
M (g
/l)Enzyme activity of wild strain IIB-30 (U/ml) Enzyme activity of mutant strain EMS-18 (U/ml)
DCM of wild strain IIB-30 (g/l) DCM of mutant strain EMS-18(g/l)
*Incubation time 48 h, incubation temperature 30°C, agitation intensity 160 rpm
118
Table 4.8: Kinetic evaluation of different aeration for the alpha amylase production by A. oryzae IIB-30 and its mutant derivative in stirred fermenter
Aeration (vvm)
0.5 1.0 1.5 2.0
Kinetic parameters
Wild Mutant Wild Mutant Wild Mutant Wild Mutant
Yp/x
Qp
Qx
18518
4500
0.23
27899
9483
0.31
19050
5833
0.31
28571
10466
0.36
18881
5016
0.26
310928
11000
0.38
18181
4000
0.22
28416
10183
0.35
Kinetic parameters: Yp/x= enzyme produced/g cell mass formation.
Qp=enzyme produced/l/h. Qx= g cell mass formation/l/h.
119
Fig 4.20: Effect of different level of dissolved oxygen on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 *
0
200
400
600
800
1000
5 10 15 20
Dissolve oxygen level (%)
Enzy
me
activ
ity (U
/ml)
0
5
10
15
20
25
30
Dry
cel
l mas
s (g/
l)Enzyme activity of wild strain IIB-30 (U/ml) Enzyme activity of mutant strain EMS-18 (U/ml)
DCM of wild strain IIB-30 (g/l) DCM of mutant strain EMS-18 (g/l)
*Incubation time 48 h, initial pH 5.0, incubation temperature 30°C, agitation intensity 160 rpm, aeration 2.0 vvm.
120
Table 4.9: Kinetic evaluation of different levels of dissolved oxygen for the alpha amylase production by A. oryzae IIB-30 and its mutant derivative in stirred fermenter
Dissolve oxygen
(%)
5.0 10 15 20
Kinetic parameters
Wild Mutant Wild Mutant Wild Mutant Wild Mutant
Yp/x
Qp
Qx
19052
4750
0.24
28903
10000
0.31
19937
5033
0.25
31840
10983
0.38
20133
6033
0.31
32258
11450
0.39
19655
5350
0.26
29110
10666
0.33
Kinetic parameters: Yp/x= enzyme produced/g cell mass formation.
Qp=enzyme produced/l/h. Qx= g cell mass formation/l/h.
121
Fig 4.21: Effect of different inoculum size on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 *
0
200
400
600
800
1000
5 7.5 10 12.5
Inoculum (%)
Enzy
me
activ
ity (U
/ml)
0
5
10
15
20
25
30
DC
M (g
/l)Enzyme activity of wild strain IIB-30 (U/ml) Enzyme activity of mutant strain EMS-18 (U/ml)
DCM of wild strain IIB-30 (g/l) DCM of mutant strain EMS-18 (g/l)
*Incubation time 48 h, incubation temperature 30°C, agitation intensity 160 rpm, initial pH 5.0.
122
Table 4.10: Kinetic evaluation of different inoculum sizes for the alpha amylase production by A. oryzae IIB-30 and its mutant derivative in stirred fermenter
Inoculum (%)
5.0 7.5 10 12.5
Kinetic parameters
Wild Mutant Wild Mutant Wild Mutant Wild Mutant
Yp/x
Qp
Qx
19375
4800
0.18
29570
10500
0.36
25396
5333
0.21
29792
11483
0.38
25486
6200
0.32
31093
11966
0.40
21549
5100
0.23
28506
9950
0.32
Kinetic parameters: Yp/x= enzyme produced/g cell mass formation.
Qp=enzyme produced/l/h. Qx= g cell mass formation/l/h.
Fig 4.22: Effect of different agitation intensity on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 *
123
0
200
400
600
800
1000
120 160 200 240
Agitation intensities (rpm)
Enzy
me
activ
ity (U
/ml)
0
5
10
15
20
25
30
DC
M (g
/l)
Enzyme activity of wild strain IIB-30 (U/ml) Enzyme activity of mutant strain EMS-18 (U/ml)
DCM of wild strain IIB-30 (g/l) DCM of mutant strain EMS-18 (g/l)
*Incubation time 48 h, incubation temperature 30°C, pH 5.0.
124
Table 4.11: Kinetic evaluation of different agitation speeds for the alpha amylase production by A. oryzae IIB-30 and its mutant derivative in stirred fermenter
Agitation intensity
120 rpm 160rpm 200 rpm 240 rpm
Kinetic parameters
Wild Mutant Wild Mutant Wild Mutant Wild Mutant
Yp/x
Qp
Qx
19254
4966
0.17
30120
10500
0.26
19473
6166
0.31
31818
11933
0.39
29215
6416
0.33
39622
12500
0.41
19444
5166
0.26
30338
11666
0.36
Kinetic parameters: Yp/x= enzyme produced/g cell mass formation.
Qp=enzyme produced/l/h. Qx= g cell mass formation/l/h.
4.7: Purification of alpha amylase
125
4.7.1: Ammonium sulfate precipitation
Table 4.12 shows the data of ammonium sulfate precipitation at different saturation
concentrations (20-90 % w/v) for the alpha amylase recovery from cell free broth. The
specific activity of broth was 208.3 U/mg of protein before treating with ammonium
sulfate. At 20-40 % (w/v) saturation, no activity was found. At 70 % (w/v) saturation
concentration the maximum specific activity (280.9 U/mg of protein) with 1.3 fold
purification was obtained. Above 70 % (w/v) both of these values were again
decreased and at 90 % (w/v) no pellet was obtained
4.7.2: Stepwise purification:
Table 4.13 shows the successive steps of purification of alpha amylase from mutant
strain of A. oryzae EMS-18 to homogeneity by ammonium sulfate precipitation
followed by using Sephadex DEAE and Sephadex G-100 columns.
4.7.2.1: Ammonium sulfate precipitation
The initial specific activity of cell free crude broth was 208.3 U/mg of protein then it
increased (280.9 U/mg of protein) with first purification step of ammonium sulfate (70
% w/v).
4.7.2.2: Anion- exchange chromatography
The dialyzed enzyme solution was loaded on prepared Sephadex DEAE column. Fig
4.23 shows the stepwise gradient elution pattern of alpha amylase when elution buffer
of Tris HCl (0.05 M, pH 7.5) containing NaCl (0-1.0 M) was used. The pooled distinct
peak was obtained showing enzyme activity (10113.1U) at the 0.30 M concentration
of NaCl. The specific activity (561.8U/mg of protein) was observed as shown in Table
126
4.13. The molecular weight was found to be as 48 kDa by applying the few fractions
on SDS-PAGE. Fig 4.25
4.7.2.3: Gel filtration
Sephadex G-100 was finally used as finishing step of purification. Upon loading
dialyzed sample, twenty five fractions were eluted with Tris HCl (0.05 M, pH 7.5)
buffer. Fig 4.24 also shows the elution pattern in the form of distinct peaks. The
active fractions containing 5963.1U enzyme activity were pooled up, dialyzed.
However, the specific activity (1987.7 U/mg of protein) and fold purification (9.5)
were recorded as shown in Table 4.13.
Table 4.12: Purification summary of alpha amylase produced by mutant strain of A. oryzae EMS-18 by using ammonium sulfate
127
Ammonium sulfate
fractionation (%)
Total units (U)
Total protein (mg)
Specific activity (U/mg)
Recovery or % yield
Fold purification
Crude broth 25000 120 208.3 100 1.0
0-20 - - - - -
0-40 - 110 - - -
0-50 18500 100 185 74 0.8
0-60 17300 90 192.2 69.2 0.92
0-70 22479 80 280.9 89.9 1.3
0-80 16200 78 207.6 64.8 0.99
0-90 - - - - -
Table 4.13: Step wise purification profile of alpha amylase produced by mutant strain of A. oryzae EMS-18.
128
Purification steps
Enzyme activity
(U)
Total protein (mg)
Specific activity (U/mg)
Recovery or % yield
Fold purification
Crude broth 25000 120 208.3 100 1
Ammonium sulphate
fractionation (70%)
22479 80 280.9 89.9 1.3
DEAE Sephadex
chromatography
10113.1 18 561.8 40.4 2.6
Sephadex G-100
5963.1 3 1987.7 23.8 9.5
129
Fig 4.23: Elution pattern on Sephadex – DEAE
Fig 4.24: The elution profile on Sephadex G-100
130
0
0.5
1
1.5
2
2.5
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Fractions
Abs
orba
nce
( 280
nm
)
0
1000
2000
3000
4000
5000
6000
7000
Enzy
me
activ
ity (U
)
Absorbance Enzyme activiity (U)
Molecular weight
131
Electrophoretic mobility of purified alpha-amylase with reference to mobilities of
protein marker (SMO 313) Fractions was analyzed on SDS-polyacrylamide gel. The
mobility of the purified alpha-amylase corresponded to a molecular weight of 48kDa
(Fig 31).
1 2 3
Fig4.26. SDS-PAGE analysis of pooled fractions of ion exchange
chromatography and ammonium sulfate fractionation
Lane 1; Marker, lane 2; Ammonium sulfate fractionation, lane 3; pooled fractions
of ion exchange chromatography.
4.8: CHARACTERIZATION
4.8.1: TEMPERATURE OPTIMA OF PURIFIED ALPHA AMYLASE
116kDa66.2
45.0 35.0
25.0
18.4 14.4
48kDa
132
Figure 4.26 shows the effect of temperature on the activity of purified alpha amylase
by mutant strain of A. oryzae. The enzyme substrate complex was incubated at
different incubation temperature such as 25, 30, 35, 40, 45, 50, 55, 60, 65, 70°C. The
activity of enzyme was increased with increase in temperature and found optimum at
40°C (1560±3.2 U/ml). Further increase in the incubation temperature resulted
decrease in the activity of enzyme. At 70°C the activity of purified enzyme is not
significant (p ≤0.05). Thus, the temperature 40°C was selected for optimum activity of
alpha amylase.
4.8.2: EFFECT OF TIME OF INCUBATION ON THE ACTIVITY OF
PURIFIED ALPHA AMYLASE
Figure 4.27 shows the effect of incubation time of enzyme substrate complex on the
activity of purified alpha amylase. The enzyme substrate complex was incubated for
varying time intervals (10-70 min). The enzyme activity was found to be optimum
(1568±2.5) after 30min of incubation. Further increase in the time of incubation
resulted decrease in the activity of enzyme. The 30 min incubation time of reaction
mixture was highly significant (p≤0.05) as compared to other temperatures which
were tested so; it was selected for further studies.
4.8.3: EFFECT OF DISTILLED WATER AND BUFFER ON THE ACTIVITY
OF PURIFIED ALPHA AMYLASE
Figure 4.28 shows the effect of distilled water and different buffers on the activity of
alpha amylase. Different buffers and distilled water such as citrate, acetate and
phosphate were used in reaction mixture. The maximum enzyme activity (1592±3.2)
133
was obtained in acetate buffer. The other buffers and distilled water show non
significant results. Thus, acetate buffer was selected for further studies.
4.8.4: EFFECT OF pH ON THE ACTIVITY OF PURIFIED ALPHA AMYLASE
Figure 4.29 shows the effect of acetate buffer pH of reaction mixture (enzyme
substrate complex) for the activity of purified alpha amylase. The enzyme substrate
complex was incubated at pH 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5 and 7. The enzyme activity
was found to be optimum at pH 5. Further increase in the pH resulted decrease in the
activity of enzyme. At neutral pH, the activity of enzyme was extremely low. The
acidic pH 5 of reaction mixture was significant (p≤0.05) as compared to other pH and
selected for subsequent studies.
4.8.4: EFFECT OF METAL IONS ON THE ACTIVITY OF PURIFIED ALPHA
AMYLASE
The residual activities were determined after incubation of purified enzyme with 5
mM metal ions (Fig 4.30). The result showed CuCl2, Zn Cl2, BaCl2, FeSO4, Mg SO4,
NiCl2 and NaCl has inhibitory effect on the activity of enzyme. While CaCl2, COCl2,
MnSO4 have stimulatory effect on the activity of enzyme.
Fig 4.26: Effect of temperature on the activity of purified alpha amylase by mutant strain of A. oryzae EMS-18
134
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
10 20 30 40 50 60 70 80
Temperature °C
Enzy
me
activ
ity (U
/ml)
Enzyme activity (U/ml)
Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p≤ 0.05.
Fig 4.27: Effect of time of incubation on the activity of purified alpha amylase by mutant strain of A. oryzae EMS-18
135
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
0 10 20 30 40 50 60 70
Time (min)
Enzy
me
activ
ity (U
/ml)
Enzyme activity (U/ml)
Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p≤ 0.05.
Fig 4.28: Effect of different buffers and distilled water on the activity of purified alpha amylase by mutant strain of A. oryzae EMS-18
136
0
400
800
1200
1600
2000
2400
Distilled water Citrate buffer Acetate buffer Phosphate Buffer
Distilled water & buffers
Enzy
me
activ
ity (U
/ml)
Enzyme activity (U/ml)
Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values in vary significantly at p≤ 0.05.
Fig 4.29: Effect of different pH on the activity of purified alpha amylase by mutant strain of A. oryzae EMS-18
137
0
400
800
1200
1600
2000
2400
0 1 2 3 4 5 6 7 8
pH of acetate buffer
Enzy
me
activ
ity (U
/ml)
Enzyme activity (U/ml)
Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p≤ 0.05.
Fig 4.30: Effect of metal ions on the activity of purified alpha amylase by mutant strain of A. oryzae EMS-18
138
0
400
800
1200
1600
2000
2400
Magnes
ium su
lphate
Magnes
e sulph
ate
Sodium
chlorid
e
Nickle
chlor
ide
Zinc ch
loride
cupro
us ch
loride
Calcium ch
loride
Cobalto
us ch
loride
Ferrou
s chlo
ride
Barium
chlorid
e
Control
Metal ions (5mM)
Enzy
me
activ
ity (U
/ml)
Enzyme activity (U/ml)
Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p≤ 0.05.
Discussion
139
Isolation, identification and screening of a potent strain are the vital steps of alpha
amylase production. In this connection, seventy eight strains of A. oryzae were
isolated from soils of different habitats by serial dilution method (Clark et al., 1958);
identified according to Onion et al. (1986) and tested for enzyme production in
submerged fermentation. Of all the isolates tested, strain no. 30 gave maximum alpha
amylase production. The strain no.30 was selected for further studies and assigned the
code IIB-30. The IIB-30 was subjected to physical (UV radiation) and chemical
mutagenesis (NG, HNO2, EMS) to enhanced the enzyme productivity. The isolates
obtained after UV irradiation were thirty two in number and screened for enzyme
production out of which isolate no.23 showed better enzyme productivity compared
to parental strain and was assigned the code UV-23. Perhephs it was due to the fact
that UV irradiation possibly changed structure of DNA by photolysis i.e, formation of
pyrimidine dimers. The structural alteration in DNA was associated with the activity
of the enzyme. Thymidine-thymidine dimer probably promoted mycelial growth and
subsequently enzyme activity, which resulted in greater secretion of enzyme from the
mycelial cells (Ali et al., 2002). UV mutant of fungi showed more enzyme production
compared to parental strain as reported by Azin and Noroozi, (2001); Rubinder et al.
(2002); Ellaiah et al. (2002) and Karanam and Medicherla, (2008).
The UV-23 mutant was subjected to N-methyl N-nitro N-nitroso guanidine (NG)
to induce mutagenesis at various concentrations. The eighteen NG treated colonies
were picked up on the basis of starch hydrolysis zones diameter larger than the UV-23
and further screened in shake flasks for enzyme production. Out of which, one mutant
NG15 gave 2 fold increase in alpha amylase production. Probably it was due to the
140
fact that treatment with NG resulted alkylation of guanine residues which formed
permanent lesions within the structure of DNA and causes mutations (Drazic and
Delac, 1970). The NG-15 was further subjected to alternate treatment with nitrous acid
and EMS for further improvement in the enzyme production. EMS-18 gave 2.6 fold
alpha amylase production than the parental culture. UV, NG and nitrous acid were
commonly used for strain improvement as reported by Azin and Noroozi (2001) and
Rubinder et al. (2002).
The six different media were evaluated for alpha amylase production by both
wild and mutant strain of A. oryzae out of which M4 medium (g/l); starch 20, yeast
extract, 8.5, NH4Cl 1.3, MgSO4.7H2O 0.12, CaCl2 0.06 was found best for maximum
enzyme production. Yeast extract and ammonium chloride serve as inorganic and
organic nitrogen source respectively, in M4 medium. Yeast extract is a complex
nitrogen source containing free amino acids and peptides and therefore, was
considered an ideal source for enzyme production. The production and stability of
enzyme are significantly affected by the supplementation of metal ions in the
fermentation medium because the metal ions act as activators for enzyme activity (Lin
et al., 1997; Noorwez and Satyanarayana, 2000). M4 medium contained the ions such
as Ca+2 Cl-, Mg+2 and SO4-which were vital for the growth of fungus and enzyme
production. Calcium and chloride ions act as stabilizer, binder, activator and
stimulator (Donell et al., 1975; Chambert et al. 1999). All the other media gave less
significant results as compared to M4 medium due to the deficiency of any constituent
in those media necessary for growth as well as for the enzyme production or it was
because of repressor effect of any component of the media on the growth of organism.
141
The alpha amylase production was increased with the increase in the
incubation period and found to be maximal after 72 h of inoculation by both strains.
The results indicated that enzyme was secreted early in active growth phase and
reached maximum towards the end of exponential growth phase. The enzyme activity
appreciably decreased after 72 h. However, in fermenter the enzyme production found
to be optimum after 64 h (wild) and 48 h (mutant). Probably it was due to denaturation
of enzyme because of interaction with other compounds in the fermentation medium
and also due to the depletion of the nutrients and formation of other by products such
as proteases in the fermentation medium (Ramesh and Lonsane, 1990; Kirshna and
Chandrasekaran, 1996). However, in case of fermenter, the reduction in the
fermentation period compared to shake flasks perhaps due to the fact that growth
factors like pH, temperature, agitation were more accurately controlled which made
the environment favourable for growth of organism and enzyme production (Gigras et
al., 2002).
The kinetic parametric results depicted that the volumetric rate of product and
cell mass formation was highly significant after 64 h of inoculation (wild) and 48 h
(mutant). The value of Yp/x was highly significant by both wild and mutant strains in
fermenter. The maximum enzyme production was obtained after 96 h of inoculation
(Kasim 1983; Nguyen et al., 2000; Francis et al., 2002). So, present finding was a
significant improvement on that reported by these scientists as there was a reduction in
fermentation time that would lead to lower energy requirements for the process and
thus make enzyme production more economical.
142
The effect of varying the incubation temperature on the enzyme production was
investigated. The enzyme production was found to be optimal at 30ºC. Higher
temperatures resulted decreased enzyme production as reported by Dakhmouche et al.
(2006); Bhanja et al. (2007) and Shafique et al. (2009). From shake flask and
fermenter experiments investigating the effect of pH, enzyme production was found to
be optimum at pH 5. Further increase in pH had an adverse effect on enzyme
production which is not un expected as it is known that enzymes are usually very
sensitive to small changes in pH; H+ion concentration also has a significant effect on
the growth of mycelium and hence enzyme production (Kasim 1983; Stamford et al.,
2001 and Gupta et al., 2008).
Optimization of the volume of fermentation medium is also necessary for air
supply nutrient supply, growth of microorganism and enzyme production. The
different volumes of the fermentation medium were evaluated in 250 ml Erlenmeyer
flasks by both wild and mutant strains of A. oryzae in present study. The maximum
enzyme production was obtained in 10 % of the fermentation medium. As the volume
of the medium was increased, the enzyme production was decreased. Probably it was
due to the fact that decrease in the agitation speed of medium, reduced air supply and
consequently enzyme production. At low volume of fermentation medium, the enzyme
production was also decreased. It might be due to nutrient present in the fermentation
medium were inadequate for the growth of strains of A. oryzae and hence, for enzyme
production (Mimura and Shinichi, 1999; Ivanova et al., 2001).
The size of inoculum has direct effect on the growth of organism and enzyme
production as reported by Allan et al. (1996) and Shafique et al. (2009). Different
143
inoculum sizes were tested for enzyme production in shake flasks and fermenter. Of
all the inoculum size tested, 4 % and 10 % of inoculum containing 2.6×106 CFU/ml
was found to be optimum for the best enzyme production in shake flasks and
fermenter. As the inoculum size was further increased, the enzyme production was
decreased. Possibly it was due to the fact that over growth of A. oryzae produced
anaerobic conditions during the fermentation and it consumed majority of substrate for
growth and metabolic processes, hence enzyme production was reduced. As the
inoculum size was decreased, the enzyme production was also decreased. The reason
might be inadequate amount of mycelia produced at low amount of conidia which in
due course decreased enzyme production. The kinetic parametric study indicated that
the yield of the enzyme by biomass formation as well as the rates of enzyme formation
was significant at 10 % inoculum size in fermenter.
Starches from different sources such as corn, rice, wheat and sweet potato were used
in the present study. The corn starch gave maximum enzyme production. The effect
of different concentrations of corn starch was evaluated. Of all the concentrations
tested starch at the concentration of 2 % gave maximum enzyme production. Beyond
this concentration decrease in the enzyme production was take place.Perhephs it was
because of that a high starch concentration, when attacked by alpha amylase during
fermentation might have undergone degradation resulting into the accumulation of
reducing sugars. It might lead to the enhancement in sugar concentration of the
substrate and catabolic repression of enzyme synthesis (Dvadtsatova et al., 1976;
Gigras et al., 2002; Ajer Dharani, 2004; Krishna and Chandrasekaran, 1996).
144
The effect of addition of different carbon sources such as glucose, sucrose, xylose,
lactose, fructose, galactose, caboxy methyl cellulose, glycerol and mannitol were
evaluated for enzyme production. Of all the carbon sources tested lactose gave
maximum enzyme production. Lactose along with starch was proved to be good
carbon source in the present study. Perhaps starch and lactose act as complex
carbohydrate sources and were gradually metabolized by a microorganism which
enhanced the accumulation of inducible alpha amylase in fermentation media (Nguyen
et al., 2000; Calik and Ozdamar, 2001). Thus lactose was selected as additional
carbon source for the enzyme production and its various concentrations were tested.
Lactose at the concentration of 1 % was found to be best for the enzyme production.
Further increase or decrease in the concentration of lactose was resulted decrease in
enzyme production. Possibly it was due to the fact that lower level of carbon was
inadequate for the growth as well as enzyme production and excess carbon was
equally detrimental and cause catabolic repression (Carlsen and Nielsen, 2001; Gupta
et al., 2008).
Different inorganic nitrogen sources such as ammonium sulfate, ammonium
nitrate, sodium nitrate and potassium nitrate were evaluated for the enzyme
production. Of all the inorganic nitrogen sources tested ammonium sulfate at 0.3 %
gave maximum enzyme production. The different additional organic nitrogen sources
such as meat extract, corn steep liquor, urea, casein, beef extract and peptone were
also evaluated for the enzyme production. Peptone with inorganic nitrogen sources
gave the maximum enzyme production as reported by Pedersen and Nielsen (2000)
and Gupta et al. (2008). The effect of different surfactants such as Tween 80, Triton
145
X-100, sodium dodecyl sulfate, Monoxal, O.T and poly ethylene glycols were tested
for the enzyme production. Of all the surfactants tested Tween 80 gave the maximum
enzyme production. There are chances that Tween 80 not only increased the
permeability of cell but also have stimulatory effect on the enzyme production
compared to other surfactants as reported by Arnesen et al. (1998).
A general requirement for a bioreactor is the provision of aeration system that
can maintain a high dissolve oxygen level. Optimum supply of oxygen is very
essential for aerobic fermentation; in this connection rate of agitation and different
volume of air supply was studied for the enzyme production in stirred fermenter. The
enzyme production was increased as the agitation intensity was increased and found to
be maximal at 200 rpm. Change in the rate of agitation resulted reduction in enzyme
production. Probably higher stirring speed above than 200 rpm resulted in mechanical
and oxidative stress, excessive foaming, disruption and physiological disturbance of
cells, while lower stirring speed seemed to limit oxygen levels along with the lacking
of homogeneous suspension of the fermentation medium and breaking of the clumps
of cells. The enzyme production increased with the increase of aeration and reached
maximum at 1.0 vvm (wild) & 1.5 vvm (mutant). The anaerobic condition available to
microorganism greatly disturbed the physiology and metabolism of organism because
of this at low level of air supply the productivity of enzyme was greatly inhibited. In
addition another toxic by product were produced in the fermentation medium with
little titer of enzyme activity, while higher concentration rates have some detrimental
effects on the growth of microorganism and subsequently enzyme production during
bioprocess time (Ionita et al., 2001).
146
Alpha amylase was purified by ammonium sulfate, and successive chromatography
techniques including anion exchange and gel filtration in the present study. Different
saturation concentrations of ammonium sulfate were used. The fold purification was
1.3 at 70 % saturation concentration. At this concentration most of the protein having
maximum enzyme activity. Possibly it was due to the fact that hydrophobic groups
predominate in the interior of protein but some were on the surface. As the
concentration of salt increased water was removed and the protein thus exposing the
hydrophobic patches on one protein molecule can interact with those on another
resulting in the aggregation of desire protein (enzyme). The enzyme solution
(dialyzed) was further purified using Sephadex- DEAE column. The positively
charged proteins were removed as contaminants. A linear gradient elution pattern
indicated that maximum peak was achieved at the 0.30 M concentration of NaCl
elution buffer as reported by Kusuda et al. (2003). After anion exchange, the dialyzed
active fraction was loaded on Sephadex G-100. The pattern of elution was used to
determine the molecular weight of alpha amylase as 48 kDa on SDS-PAGE
(Anidyawati et al., 1998; Chang et al., 1995). The comparison of successive
purification steps starting from specific activity of crude broth (208.3 U/mg) to final
finishing technique of gel filtration (1987.7U/mg) indicated the 9.5-fold increase in
specific activity.
For characterization of purified alpha amylase, the optimization of temperature,
incubation time, different buffers, pH and metal ions were studied. Among different
temperature the maximum activity was observed at 40°C. Probably it was due to the
fact that reaction rate initially increased as the temperature rised, due to increased
147
kinetic energy of reacting molecules. However, as the temperature was increased the
kinetic energy of enzyme exceeds the energy barrier. It resulted in the breaking of
weak hydrogen bonding and hydrophobic bonds that maintain the structure of enzyme.
The inactivation of enzyme at low temperature and thermal denaturation at high
temperature might cause decrease in activity. The effect of different buffer and pH
were analyzed by carrying out the enzyme assay along with different buffers and pH
Acetate buffer at pH 5 gave the maximum enzyme activity. At pH below and above
optimal level, a decline in activity was possibly due to the structural unstability of
protein (Kusuda et al. 2003). Most of alpha amylase is known to be metalloenzymes;
supplementation of metal ion improved the activity of enzyme. Effect of metal ion on
the activity of enzyme was observed; in the presence of CaCl2 maximum activity of
enzyme was obtained. Perhaphs it may be possible the affinity of Ca+2 to alpha
amylase was much stronger than any other ions and Ca+2 stabilize the enzyme activity
while the other metals such as CuCl2, ZnCl2, BaCl2, FeSO4, Mg SO4, NiCl2 and NaCl
has inhibitory effect on the enzyme activity probably these metal block binding sites
of enzyme or enzyme contain number of metals and displacement of these ions by
another metal ions, either with some change or similar size result in inhibition of
enzyme activity (Abou Zeid 1997).
CONCLUSION
148
In the present study, seventy eight strains of Aspergillus oryzae were isolated for the
enzyme production. The improvement in enzyme production was achieved by
subjecting parental strain to successive physical (UV) and chemical (NG, NA, EMS)
mutagens. The mutant gave 2.6 fold more production compared to parental strain in
term of enzyme production. Many factors need to be considered by alpha amylase
production to obtain economically most favourable process. The most important
among them were physical factors and culture medium. The optimization of process
parameters were under taken in shake flasks and fermenter. All fermentation were
carried out following growth of organism at 200 rpm (30°C) for 72 h in shake flasks,
64 h (wild) and 48 h (mutant) in fermenter.The time required for maximal enzyme
production was reduced in fermenter as compared to shake flasks by both wild and
mutant strain. Fermentation medium containing (g/l); corn starch 20, lactose 10,
ammonium sulfate 3, peptone 2, yeast extract 8, ammonium chloride 1.3, calcium
chloride 0.06,magnesium sulphate 0.12 and Tween-80 1.0 at pH 5 was selected. In
case of fermenter inoculum size (10 %), aeration (1.5vvm) dissolved oxygen level (15
%) were found optimum for maximum enzyme production. A total of 9.5 fold
purification and 23.8 % recovery were obtained. Gel electrophoresis indicated
molecular weight of A. oryzae alpha amylase to be 48 kDa.
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