EXPRESSION AND BIOCHEMICAL CHARACTERIZATION OF … · Amplifikasi sebanyak 1741 pasang bes fragmen...
Transcript of EXPRESSION AND BIOCHEMICAL CHARACTERIZATION OF … · Amplifikasi sebanyak 1741 pasang bes fragmen...
EXPRESSION AND BIOCHEMICAL CHARACTERIZATION OF
MALTOGENIC AMYLASE FROM Bacillus lehensis G1
SAMSON PACHELLES
A thesis submitted in fulfilment of the
requirements for the award of the degree of
Master of Engineering (Bioprocess)
Faculty of Chemical Engineering
Universiti Teknologi Malaysia
MAY 2013
iv
ACKNOWLEDGEMENT
First and foremost, I would like to extend my sincere appreciation to my
supervisor, Prof. Dr. Rosli Md. Illias for his stern yet valuable advicse as well as his
guidance throughout the duration of this research. I would also like to thank and
acknowledge the assistance of the laboratory staff of the Department of Bioprocess
Engineering, UTM and my laboratory colleagues.
I would like to express my gratitude to Malaysia Genome Institute (MGI) for
the financial aid since the research was part of a project under Genomics and
Molecular Biology Initiatives Programmes (07-05-16-MGI-GMB12).
Last but not least, I would like to convey my absolute appreciations to my
parents and families for their continual support, love and patience.
v
ABSTRACT
This study was carried out for the expression and characterization of
maltogenic amylase (MAG1) from Bacillus lehensis G1. Amplification of 1741 base
pair gene fragments encoding MAG1 and expression in Escherichia coli have been
successful. The expression of MAG1 was optimized at 30°C and 0.5 mM isopropyl
β-D-1-thiogalactopyranoside (IPTG) for a period of 12 hour post-induction time.
Purification of the crude enzyme was done using the ACTAprime System which uses
the concept of affinity chromatography. The optimum temperature and pH of the
purified MAG1 were 40 °C and pH 7.0 respectively. The enzyme did not show
potent thermostability and was stable at pH ranging from 7.0 to 9.0. The purified
MAG1 has a preference towards cyclodextrin (CD) specifically β-CD. Starches were
least favored by MAG1 followed by pullulan. Only the metal ion Mn2+
increased the
activity of MAG1 while K2+
, Li2+
and Mg2+
slightly affect its activity. The metal ion
Ca2+
significantly reduced MAG1 activity while Fe2+
, Co2+
, Zn2+
, Cu2+
, Pb2+
and Ni2+
drastically reduced the activity of MAG1. In terms of additives, only 2-
mercaptoethanol (2-ME) managed to enhance the activity of MAG1 while
ethylenediaminetetraacetic acid (EDTA) and Tween20 did not affect its activity.
Drastic reduction of MAG1 activity was caused by phenylmethylsulfonyl fluoride
(PMSF), sodium dodecyl sulfate (SDS), methanol and ethanol. The hydrolysis
pattern of MAG1 was studied using CDs and maltooligosaccharides. The hydrolysis
of CDs resulted in the formation of maltose but no glucose was detected. MAG1 was
also able to linearize the CDs. MAG1 did not react with glucose, maltose and
maltotriose but did react with maltotetraose, maltopentaose, maltohexaose and
maltoheptaose to produce mainly maltose and maltotriose. The Km and Vmax of
MAG1 towards β-CD were 6.358 mg/mL and 91.63 µmol/min respectively.
vi
ABSTRAK
Penyelidikan ini telah dijalankan untuk ekspresi dan mencirikan enzim
maltogenic amylase (MAG1) yang diperolehi daripada Bacillus lehensis G1.
Amplifikasi sebanyak 1741 pasang bes fragmen yang mengkodkan MAG1 dan
ekspresi dalam Escherichia coli telah berjaya. Ekspresi MAG1 adalah optimum pada
suhu 30°C dengan induksi 0.5 mM isopropil β-D-1-tiogalaktopiranosid (IPTG)
selama 12 jam. Penulenan enzim telah dijalankan menggunakan sistem ACTAprime
yang menggunakan konsep kromatografi afiniti. Suhu dan pH optimum bagi enzim
yang telah ditulenkan adalah pada 40 °C dan pH 7.0. Enzim ini tidak menunjukkan
kestabilan yang positif pada suhu tinggi dan stabil pada pH dari 7.0 ke 9.0. MAG1
yang telah ditulenkan lebih cenderung kepada siklodekstrin (CD) terutamanya β-CD.
Kanji kurang digemari oleh MAG1 diikuti oleh pullulan. Aktiviti MAG1 telah
dipertingkatkan oleh Mn2+
manakala K2+
, Li2+
dan Mg2+
tidak terlalu memberi kesan
kepada aktiviti MAG1. Aktiviti MAG1 telah direncat oleh Ca2+
dan direncat dengan
mendadak oleh Fe2+
, Co2+
, Zn2+
, Cu2+
, Pb2+
and Ni2+
. Selain daripada itu, 2-
merkaptoetanol (2-ME) juga meningkatkan aktiviti MAG1 tetapi aktivitinya tidak
dipengaruhi oleh asid atelindiamintetraasetik (EDTA) dan Tween20. Perencatan
yang mendadak pada aktiviti MAG1 adalah disebabkan oleh fenilmetilsulfonil
florida (PMSF), natrium dodecil sulfat (SDS), metanol dan etanol. Hasil hidrolisis
oleh MAG1 dikaji dengan menggunakan CD dan maltooligosakarida. Hidrolisis CD
menghasilkan maltosa sebagai produk utamanya tetapi tiada penghasilan glukosa
dapat dikesan. MAG1 juga mampu menukar CD kepada bentuk linearnya. MAG1
tidak bertindak balas dengan glukosa, maltosa dan maltotriosa tetapi bertindak balas
dengan maltotetraosa, maltopentosa, maltoheksosa dan maltoheptosa untuk
menghasilkan maltosa dan maltotriosa. Nilai Km dan Vmax bagi MAG1 terhadap β-
CD adalah 6.358 mg/mL dan 91.63 µmol/min.
vii
TABLE OF CONTENTS
CHAPTER TITLE PAGE
TITLE
DECLARATION ii
DEDICATION iii
ACKNOWLEDGMENT iv
ABSTRACT v
ABSTRAK vi
TABLE OF CONTENTS vii
LIST OF TABLES xi
LIST OF FIGURES xiii
LIST OF SYMBOLS/ABBREVIATIONS xvii
LIST OF APPENDICES xx
1 INTRODUCTION 1
1.1 Introduction 1
1.2 Objectives of Research 3
1.3 Scopes of Research 3
2 LITERATURE REVIEW 4
2.1 Starch 4
2.2 Starch Degrading Enzyme 7
2.3 Amylases 8
viii
2.4 Alpha-amylase 9
2.5 Maltogenic Amylase 13
2.6 Biochemical Characteristics of
Maltogenic Amylase 17
2.7 Reaction Patterns of Maltogenic Amylase 19
2.7.1 Hydrolysis Pattern of Maltogenic
Amylase 19
2.7.2 Transglycosylation Pattern of Maltogenic
Amylase 20
2.7.3 Coupled Reactions of Maltogenic Amylase 22
2.8 Maltogenic Amylase as an Anti-Staling Agent 24
3 METHODOLOGY 26
3.1 Bacterial Strains 27
3.2 Chemicals 27
3.3 Preparation of Bacterial Glycerol Stock 27
3.4 Medium and Agar Preparation 28
3.4.1 Horikoshi Broth and Agar 28
3.4.2 Luria-Bertani Broth and Agar 28
3.5 General DNA Technique 29
3.5.1 Agarose Gel Preparation 29
3.5.2 Genomic DNA Extraction 29
3.5.3 Quantification of DNA using
Spectrophotometer 30
3.6 Polymerase Chain Reaction (PCR) for the
Gene of Maltogenic Amylase 31
3.6.1 Primer Design 31
3.6.2 PCR Mixture and Protocol 31
3.6.3 Purification of PCR Product 32
3.7 Plasmid Extraction 33
3.8 Digestion of DNA and Plasmid 34
3.9 Ligation of DNA to Plasmid 34
3.10 Preparation of Competent Cell using TSS Method 34
ix
3.11 Transformation into E. coli Hosts 35
3.12 Screening for Positive Transformant 36
3.13 Expression of Recombinat Maltogenic Amylase 36
3.14 Cell Lysis using Sonicator 36
3.15 Purification of Recombinant Maltogenic Amylase 37
3.15.1 System Wash 37
3.15.2 Connecting Column 38
3.15.3 Selecting the Program for Purification 38
3.15.4 Collecting Samples 38
3.16 Analysis of the Purification Result using
SDS-PAGE 39
3.17 Western Blot 39
3.18 Protein Determination 40
3.19 Assay for Maltogenic Amylase 41
3.20 Biochemical Characterization of Recombinant
Maltogenic Amylase 41
3.20.1 Optimum Temperature and pH 41
3.20.2 Thermal and pH Stability 41
3.20.3 Susbstrate Specificity 42
3.20.4 Effect of Metal Ions and Additives 42
3.20.5 Hydrolysis Pattern of the Recombinant
Maltogenic Amylase 43
3.20.6 Analysis of Hydrolysis Pattern using TLC 43
3.20.7 Determination of Kinetic Parameters 43
4 RESULTS AND DISCUSSION 45
4.1 Cloning of Recombinant Maltogenic Amylase 45
4.1.1 Amplification of Maltogenic
Amylase Gene 45
4.1.2 Cloning of Maltogenic Amylase Gene 46
4.2 Bioinformatic Analysis of Maltogenic
Amylase Gene 47
4.2.1 Full Sequence Analysis of Maltogenci
x
Amylase Gene 47
4.2.2 Hydrophobicity of the Recombinant
Maltogenic Amylase 57
4.3 Expression of Maltogenic Amylase 59
4.3.1 Expression at Different Temperature and
Different Concentration of IPTG 60
4.3.2 Expression in Different Expression Hosts 64
4.4 Western Blot 66
4.5 Purification of the Crude Recombinant
Maltogenic Amylase 67
4.6 Biochemical Characterization 70
4.6.1 Effect of Temperature and Temperature
Stability 70
4.6.2 Effect of pH and pH Stability 72
4.6.3 Substrate Specificity 74
4.6.4 Effect of Metal Ion 76
4.6.5 Effect of Additives 77
4.6.6 Hydrolysis Pattern 78
4.6.6.1 Hydrolysis of Cyclodextrin (CD) 78
4.6.6.2 Hydrolysis of
Maltooligosaccharides 80
4.7 Kinetic Parameter of Recombinant
Maltogenic Amylase 82
5 CONCLUSION 83
5.1 Conclusions 83
5.2 Recommendations 84
REFERENCES 85
Appendices A - E 93 - 105
xi
LIST OF TABLES
TABLE NO. TITLE PAGE
2.1 Differences of amylose and amylopectin 6
2.2 The members in the α-amylase family 10
2.3 Recently discovered maltogenic amylase from different
sources with its basic biochemical characteristic 18
2.4 Example of donor, acceptor and products that have been
studied for the transglycosylation reaction of
maltogenic amylase 21
3.1 PCR primers for the amplification of maltogenic amylase
from B. lehensis G1 31
3.2 PRC components used to obtain the gene of
maltogenic amylase 32
4.1 Nucleotides distribution table of MAG1 50
4.2 Amino acids distribution table of MAG1 51
4.3 Identity comparison of MAG1 with other maltogenic
amylase from different sources 55
xii
4.4 Hydrophobicity scores at the conserved regions
of MAG1 58
4.5 Activity of MAG1 expressed in E. coli BL21 at
different temperature and IPTG concentration 62
4.6 Activity of MAG1 expressed in different host 65
4.7 Purification table of recombinant maltogenic
amylase expressed at the optimized conditions 69
xiii
LIST OF FIGURES
FIGURE NO. TITLE PAGE
2.1 General structure of starch 5
2.2 Schematic representation of the action pattern of
amylolytic enzymes on starch (amylopectin) polymers 11
2.3 Conserved sequence regions in the α-amylase family 12
2.4 The first three dimensional structure of the α-amylase 13
2.5 Structure of cyclodextrin 14
2.6 Schematic diagram on substrate preference of dimeric
maltogenic amylase 15
2.7 Ribbon diagram for a monomer of maltogenic amylase
from Thermus sp. 16
2.8 The ribbon diagram representation of dimeric maltogenic
amylase from Thermus sp. 16
2.9 Differences of amino acids in conserve regions for
TpMA and ThMA 20
xiv
2.10 Proposed models of coupled hydrolysis and
transglycosylation by maltogenic amylase from
Bacillus stearothermophilus ET1 22
2.11 Schematic drawings of products from reaction of acarbose
with maltogenic amylase from Thermus sp. 23
3.1 Overview of methodology 26
4.1 Gel electrophoresis of genomic DNA of
B. lehensis G1 extraction 46
4.2 Gel electrophoresis of amplified PCR product of the
maltogenic amylase gene 46
4.3 Gel electrophoresis of double-digestion
(Bam HI and Xho I) of the cloning vector pET21a 47
4.4 Nucleotide and deduced amino acid sequence of MAG1 50
4.5 Multiple alignment MAG1 with other amylolytic enzymes 54
4.6 Phylogenetic analysis of MAG1 based on amino
acid sequences 56
4.7 Hydrophobicity analysis of MAG1 57
4.8 SDS-PAGE electrophoresis of MAG1 expressed in
E. coli BL21 at 37 °C with 1 mM IPTG 60
4.9 SDS-PAGE of MAG1 expressed at 30°C with
different concentration of IPTG 62
4.10 SDS-PAGE of MAG1 expressed at 25°C with different
xv
concentration of IPTG 63
4.11 SDS-PAGE of MAG1 expressed at 20°C with
different concentration of IPTG 63
4.12 SDS-PAGE of MAG1 expressed in Origami 2 at 30°C
with different concentration of IPTG 65
4.13 SDS-PAGE of MAG1 expressed in Origami B at 30°C
with different concentration of IPTG 66
4.14 Western blot of MAG1 expressed at the 30°C and
0.5 mM IPTG 67
4.15 SDS-PAGE of the purified MAG1 68
4.16 Effect of temperature on the activity of MAG1 70
4.17 Temperature stability of MAG1 72
4.18 Effect of pH on the activity of MAG1 73
4.19 pH stability of MAG1 74
4.20 Relative activity of MAG1 on different substrate 76
4.21 Effect of metal ions on the activity of MAG1 77
4.22 Effect of additives on the activity of MAG1 78
4.23 TLC analysis of MAG1 hydrolytic activity on CD 79
4.24 TLC analysis of MAG1 hydrolytic activity on
xvi
Maltooligosaccharides 81
4.25 Partial multiple alignment of MAG1 with other
amylolytic enzymes showing the conserve region IV 81
4.26 Lineweaver-Burk plot for MAG1 using β-CD as
a substrate 82
xvii
LIST OF SYMBOLS/ABBREVIATION
Asn - Asparagine
Asp - Aspartic acid
BCIP - 5-Bromo-4-Chloro-3-Indolyl Phosphate
Blast - Basic Local Alignment Search Tool
Bp - base pair
DNA - Deoxyribonucleic acid
EC - Enzyme Commision
EDTA - ethylene diamine tetraacetic acid
eg. - example
et al. - and frinds
EtOH - Ethanol
g/L - gram per litter
Glu - Glutamate
His - Histidine
HPAEC - High Performance Anion Exchange
Chromatography
HPLC - High Performance Liquid
Chromatography
IPTG - IsoPropyl β-D-1-ThioGalactopyranoside
kb - kilo base
KCl - potassium chloride
KI - potassium iodide
LMW - Low Molecular Weight
MeOH - Methanol
mg/mL - milligram per milliliter
xviii
mM - millimolar
NaCl - sodium chloride
NaOH - sodium hydroxide
NBT - Nitro Blue Tetrazolium chloride
nm - nano meter
PAGE - polyacarylamide gel electrophoresis
PMSF - phenylmethanesulfonyl fluoride
rpm - revolutions per minute
SDS - sodium dedocyl sulphate
Ser - serine
TBE - Tris/Borate/EDTA
TBS - Tris Buffered Saline
TBSTT - Tris Buffered Saline Tween-20 Triton
TE - Tris/EDTA
TLC - Thin Layer Chromatography
TSS - Transformation and Storage Solution
U/mL - unit per milliliter
UV - ultraviolet
UV-Vis - ultraviolet visible
v/v - volume per volume
w/v - weight per volume
α - alpha
β - beta
γ - gamma
µL - micro litter
% - percentage
°C - degree Celsius
Ca2+
- calcium ion
Co2+
- cobalt ion
Cu2+
- copper ion
Fe2+
- iron ion
K+
- potassium ion
Li2+
- lithium ion
Mg2+
- magnesium ion
xix
Mn2+
- manganese ion
Ni2+
- nickel ion
Pb2+
- lead ion
Zn2+
- zinc ion
H2SO4 - sulfuric acid
KH2PO4 - monopotassium phosphate
MgSO4 - magnesium sulfate
Tm - melting temperature
xx
LIST OF APPENDICES
APPENDIX TITLE PAGE
A Medium and Buffer 92
B Standard Curve for Maltogenic Amylase Assay 102
C Standard Curve for Bradford Assay 103
D pET-21a(+) VECTOR (PROMEGA) 104
E Publication 105
CHAPTER 1
INTRODUCTION
1.1 Introduction
Alpha-amylase (EC 3.2.1.1.) is one of the enzymes that have been
investigated most thoroughly and used most widely in the starch hydrolytic industry.
These amylase are endo-acting amylases which means the enzyme hydrolyze α-1,4-
glycosidic linkage of internal starch polymer randomly. This in turn resulted in
oligosaccharides with varying lengths and α-dextrins that retains its α-1,6-glycosidic
linkages. There are various amylolytic enzymes such as pullulanase (EC 3.2.1.41;
Kuriki et al., 1988), cyclodextrin glucanotransferase (EC 2.4.1.19; Sakai et al,.
1987), cyclodextrinase (EC 3.2.1.54; Kitahata et al, 1983), and maltogenic amylase
(EC 3.2.1.133; MAase; Kim et al,. 1992) in which these enzymes exhibits novel
enzymatic properties that clearly differentiate it from those of α-amylase, being
capable of hydrolyzing poly- and/or maltooligosaccarides such as pullulan and
cyclodextrins (CDs) as well as its extensive transglycosylation activity.
Maltogenic amylases which belong to the glycoside hydrolase family 13
(GH13) are very unique where these enzymes have multi-substrate specificity
towards starch, pullulan, and CDs, which is modulated by dimerization of the
2
enzyme. Other than its typical-α-amylase hydrolytic activity, maltogenic amylase is
also able to perform a transglycosylation activity via formation of various glycosidic
linkages such as α-1,3- and α-1,6-linkages to produce branched oligosaccharides
from substrate such as liquefied starch (Park et al., 2000). This enzyme is named
maltogenic amylase because in general it will hydrolyze CD into mainly maltose and
glucose.
The enzyme activity was found to be cell-bounded which means that the
enzyme is intracellular (Kolcuoğlu et al., 2010). It was proven by the predicted
amino acid sequence with no sign of signal sequence necessary for secretion of the
enzyme outside the cell. In order to exploit its benefits, maltogenic amylase has long
been discovered and isolated such as from B. licheniformis (Kim et al., 1992), B.
stearothermophilus (Cha et al., 1998), and a Thermus species (Kim et al., 1999).
From previous researches on maltogenic amylase, it has been proven that
maltogenic amylase is able to produce branched side chains when reacted with
amylopectin cluster (Kim et al., 2006). Moreover, this enzyme hydrolyzes acarbose,
a potent α-amylase inhibitor (Park et al., 1998). The combined activities of
maltogenic amylase which are hydrolysis and transglycosylation have been utilized
in the production of branched oligosaccharides and various modified sugars (Kim et
al., 1994; Lee et al., 1995). Maltogenic amylase is also a highly effective anti-staling
agent used for bread making in industrial bakeries.
Different source of enzymes exhibits their own unique characteristic even
though they have some similarities in terms of function, substrate they act upon or
their structure. Finding the enzymes of highly useful characteristic is very important
as it can contribute in variety of industries. This is where maltogenic amylase from
Bacillus lehensis G1 becomes an interest. Maltogenic amylase has very useful
industrial applications and exploring another source of this enzyme can give us more
option to consider.
3
Amount of this enzyme that can be obtained from the wild microorganism is
normally limited and their characteristics are unknown. Therefore, their applicability
to meet with the conditions of a reaction criteria such as high temperature is still
unclear. We intend to clone and over-expressed the maltogenic amylase in
expression system such as Escherichia coli (E. coli) and characterize the
recombinant enzyme. This will contribute in research to improve the characteristic of
the enzyme such as thermostability by protein engineering. It is an important
characteristic for industrial application where a thermostable enzyme is preferred.
Other than that, an efficient way to produce this enzyme is crucial in order to obtain a
low cost production.
1.2 Objectives of Research
The objectives of this research are to clone and express maltogenic amylase
from Bacillus lehensis G1 (MAG1) in E. coli and to characterize the purified
recombinant maltogenic amylase.
1.3 Scopes of Research
1.3.1 Amplification of maltogenic amylase gene using polymerase chain
reaction (PCR) method
1.3.2 Cloning and expression of maltogenic amylase gene in E. coli
expression system
1.3.3 Purification and characterization of recombinant maltogenic
amylase
REFERENCES
Aiyer, P. V. (2005). Amylases and their applications. African Journal of
Biotechnology. 4(13): 1525-1529.
Ao, Z., Simsek, S., Zhang, G., Venkatachalam, M., Reuhs, B. L. and Hamaker, B. R.
(2007). Starch with a slow digestion property produced by altering its chain
length, branch density, and crystalline structure. Journal of Agricultural and
Food Chemistry. 55(11): 4540-4547.
Banks, W. and Greenwood. (1975). Starch and its components. Edinberg
University Press.
Baneyx, F. (1999). Recombinant protein expression in Escherichia coli. Current
Opinion in Biotechnology. 10: 411-421.
Barfoed, H. C. (1976). Enzymes in starch processing. Cereal Food World. 21: 588-
604.
Bessette, P. H., Åslund, F., Beckwith, J. and Georgiou, G. (1999) Efficient folding of
proteins with multiple disulfide bonds in the Escherichia coli cytoplasm.
Proceedings of the National Academy of Sciences. 96(24): 13703-13708.
Buedenbender, S. and Schulz, G. E. (2009). Structural base for enzymatic
cyclodextrin hydrolysis. Journal of Molecular Biology. 385: 606–617
Cha, H. J., Yoon, H. G., Kim, Y. W., Lee, H. S., Kim, J. W., Kweon, K. S., Oh, B. H.
and Park, K. H. (1998). Molecular and enzymatic characterization of a
maltogenic amylase that hydrolyzes and transglycosylates acarbose. European
Journal of Biochemistry. 253: 251–262.
Cho, H. Y., Kim, Y. W., Kim, T. J., Lee, H. S., Kim, D. Y., Kim, J. W., Lee, Y. W.,
Lee, S. B. and Park, K. H. (2000). Molecular characterizationof a dimeric
intracellular maltogenic amylase of Bacillus subtilis SUH4-2. Biochimica et
Biophysica Acta. 1478: 333–340.
86
Coutinho, P. M. and Henrissat, B. (1999). Recent advances in charbohydrate
engineering. Cambridge: Royal Society of Chemistry. 3-12.
Donovan, R. S., Robinson, C. W. and Glick, B. R. (1996). Review: Optimizing
inducer and culture conditions for expression of foreign proteins under the
control of the lac promoter. Journal of Industrial Microbiology. 16: 145-154.
Fogarty, W. M. (1983). Microbial amylases. In Fogarty, W.M. Microbial Enzymes
and Biotechnology. (Ed.) (pp. 1-92). London: Applied Science Publishers.
Fogarty, W. M. and Kelly, C. T. (1980). Amylase, amyloglucosidase and related
glucanases. Microbial Enzymes and Bioconversions. Rose, A.H(Ed). (pp. 115-
170). London: Academic Press.
Fogarty, W. M. and Kelly, C. T. (1990). Recent advances in microbial amylases. In
Fogarty, W. M. and Kelley, C. T. Microbial Enzymes and Biotechnology. (pp.
71-132). London: Elsevier Applied Science Publishers.
Gasser, B., Maurer, M., Rautio, J., Sauer, M., Bhattacharyya, A., Saloheimo, M.,
Penttilä, M. and Mattanovich, D. (2007). Monitoring of transcriptional
regulation in Pichia pastoris under protein production conditions. BMC
Genomics. 8: 179.
Gerrard, J. A., Every, D., Sutton, K. H. and Gilpin, M. J. (1997). The role of
maltodextrins in the staling of bread. Journal of Cereal Science. 26: 201.
Goesaert, H., Slade, L., Levine, H. and Delcour, J. A. (2009). Amylase and bread
firming – an integrated view. Journal of Cereal Science. 1-8.
Gomes-Ruffi, C. R., da Cunha, R. H., Almeida, E. L., Chang, Y. K. and Steel C. J.
(2012). Effect of the emulsifier sodium stearoyl lactylate and of the enzyme
maltogenic amylase on the quality of pan bread during storage. LWT – Food
Science and Technology. 49(1): 96-101.
Gray, J. A., and BeMiller, J. N. (2003). Bread staling: molecular basis and control.
Food Science and Food Safety. 2: 1–21.
Hong, F., Meinander, N. Q. and Jönsson, L. J. (2002). Fermentation strategies for
improved heterologous expression of laccase in Pichia pastoris. Biotechnology
and Bioengineering. 79(4): 438-449.
Horikoshi, K. (2006). Starch-degrading Enzymes. In Horikoshi, K. (Ed).
Alkaliphiles. (pp. 143-174). Japan: Springer Berlin Heidelberg.
87
Jahic, M., Wallberg, F., Bollok, M., Garcia, P. and Enfors, S. O. (2003). Temperature
limited fed-batch technique for control of proteolysis in Pichia pastoris
bioreactor cultures. Microbial Cell Factories. 2: 6.
Jakubovics, N. S., Smith, A. W. and Jenkinson, H. F. (2002). Oxidative stress
tolerance is manganese (Mn2+
) regulated in Streptococcus gordonii.
Microbiology. 148: 3255–3263.
Janeček, S. (1997). α-Amylase family: molecular biology and evolution. Progress in
Biophysics and Molecular Biology. 67: 67-97.
Jespersen, H.M., MacGregor, E.A., Henrissat, B., Sierks, M.R. and Svensson, B.
(1993). Starch- and glycogen-debranching and branching enzymes: Prediction
of structural features of the catalytic (β/α)8-barrel domain and evolutionary
relationship to other amylolytic enzymes. Journal of Protein Chemistry. 12(6):
791-805.
Kim, I.C., Cha, J.H., Kim, J.R., Jang, S.Y., Seo, B.C., Cheong, T.K., Lee, D.S., Choi,
Y.D. and Park, K.H. (1992). Catalytic properties of the cloned amylase from
Bacillus licheniformis. The Journal of Biological Chemistry. 31: 22108–22114.
Kim, I. C., Yoo, S. H., Lee, S. J., Oh, B. H., Kim, J. W. and Park, K. H. (1994).
Synthesis of branched oligosaccharides from starch by two amylases cloned
from Bacillus licheniformis. Bioscience, Biotechnology and Biochemistry. 58:
416–418.
Kim, J. W., Kim, Y. H., Lee, H. S., Yang, S. J., Kim, Y. W., Lee, M. H., Kim, J. W.,
Seo, N. S., Park, C. S. and Park K. H. (2007). Molecular cloning and
biochemical characterization of the first archaeal maltogenic amylase from the
hyperthermophilic archaeon Thermoplasma volcanium GSS1. Biochimica et
Biophysica Acta. 1774: 661–669.
Kim, J. S., Cha, S. S., Kim, H. J., Kim, T. J., Ha, N. H., Oh, S. T.,Cho, H. S., Cho,
M. J., Kim, M. J., Lee, H. S., Kim, J. W., Choi, K. Y., Park, K. H. and Oh, B.
H. (1999). Crystal structure of a maltogenic amylase provides insights into a
catalytic versatility. The Journal of Biological Chemistry. 274: 26279–26286.
Kim, T. J., Kim, M. J., Kim, B. C., Kim, J. C., Cheong, T. K. and Kim, J. W. (1999).
Modes of action of acarbose hydrolysis and transglycosylation catalyzed by a
thermostable maltogenic amylase, the gene for which was cloned from a
Thermus strain. Applied and Environmental Microbiology. 65:1644–51.
88
Kim, T. J., Nguyen, V. D., Lee, H. S., Kim, M. J., Cho, H. Y., Kim, Y.W., Moon, T.
W., Park, C. S., Kim, J. W., Oh, B. H., Lee, S. B., Svensson, B. and Park, K. H.
(2001). Modulation of the multisubstrate specificity of Thermus maltogenic
amylase by truncation of the N-terminal domain and by a salt-induced shift of
the monomer/dimer equilibrium. Biochemistry. 40: 14182–14190.
Kim, T. J., Park, C. S., Cho, H. Y., Cha, S. S., Kim, J. S., Lee, S. B., Moon, T. W.,
Kim, J. W., Oh, B. H. and Park, K. H. (2000). Role of the glutamate 332
residue in the transglycosylation activity of Thermus maltogenic amylase.
Biochemistry. 39: 6773–6780.
Kim, Y. H. (2006). Cloning, characterization and application of Thermoplasma
volcanium GSS1 maltogenic amylase. Masters thesis. Seoul National
University, Seoul, Korea.
Kitahata, S. (1988). Handbook of amylases and related enzymes. Pergamon, Tokyo.
154-163.
Kolcuoğu, Y., Colak, A., Faiz, O. and Belduz, A. O. (2010). Cloning, expression and
characterization of high thermo- and pH-stable maltogenic amylase from a
thermophilic bacterium Geobacillus caldoxylosilyticus TK4. Process
Biochemistry.
Kuriki, T., Park, J. H., Okada, S. and Imanaka, T. (1988). Purification and
characterization of thermostable pullulanase from Bacillus stearothermophilus
and molecular cloning and expression of the gene in Bacillus subtilis. Applied
and Environmental Microbiology. 54 (11): 2881-2883.
Kyte, J. and Doolittle, R. (1982). A simple method for displaying the hydrophatic
character of a protein. Journal of Molecular Biology.157: 105-132.
Lee, H. S., Auh, J. H., Yoon, H. G., Kim, M. J., Park, J. H., Hong, S. S., Kang, M.
H., Kim, T. J., Moon, T. W., Kim, J. W., and Park, K. H. (2002). Cooperative
action of α-glucanotransferase and maltogenic amylase for an improved
process of isomaltooligosaccharide (IMO) production. Journal of Agricultural
and Food Chemistry. 50: 2812–2817.
Lee, H. S., Kim, M. S., Cho, H. S., Kim, J. I., Kim, T. J., Choi, J. H., Park, C., Lee,
H. S., Oh, B. H. and Park, K. H. (2002). Cyclomaltodextrinase, neopullulanase,
and maltogenic amylase are nearly indistinguishable from each other. Journal
of Biological Chemistry. 277: 21891–21897.
89
Lee, S. J., Yoo, S. H., Kim, M. J., Kim, J. W., Seok, H. M. and Park, K. H. (1995).
Production and characterization of branched oligosaccharides from liquefied
starch by the action of Bacillus licheniformis amylase. Starch. 47: 127–134.
Lee, Y. H., Kim, M. J., Baek, J. S., Lee, H. S., Cha, H. J., and Lee, S. B. (2003).
Preparation and characterization of maltosyl-sucrose isomers produced by
transglycosylation of maltogenic amylase from Bacillus stearothermophilus. .
Journal of Molecular Catalysis B: Enzymatic. 26: 293–305.
Li, D., Park, J. T., Li, X., Kim, S., Lee, S., Shim J. H., Park, S. H., Cha, J., Lee, B.
H., Kim, J. W. and Park, K. H. (2010). Overexpression and characterization of
an extremely thermostable maltogenic amylase, with an optimal temperature of
100 °C, from the hyperthermophilic archaeon Staphylothermus marinus. New
Biotechnology. 27(4): 300-307.
Li, D., Park, S. H., Shim, J. H., Lee, H. S., Tang, S. Y., Park, C. S. and Park, K. H.
(2004). In vitro enzymatic modification of puerarin to puerarin glycosides by
maltogenic amylase. Carbohydrate Research. 339: 2789–2797.
Li, X., Li, D., Yin, Y. and Park, K. H. (2009). Characterization of a recombinant
amylolytic enzyme of hyperthermophilic archaeon Thermofilum pendens with
extremely thermostable maltogenic amylase activity. Applied Microbiology
and Biotechnology. 85: 1821-1830.
Lin, H., Kim, T., Xiong, F. and Yang, X. (2007). Enhancing the production of Fc
fusion protein in fed-batch fermentation of Pichia pastoris by design of
experiments. Biotechnology Progress. 23: 621–625.
Liu, B., Wang,Y. and Zhang, X. (2006). Characterization of a recombinant
maltogenic amylase from deep sea thermophilic Bacillus sp. WPD616. Enzyme
and Microbial Technology. 39: 805–810.
Mabrouk, S. B., Messaoud, E. B., Ayadi, D., Jemil, S., Roy, A., Mezghani, M. and
Bejar, S. (2007). Cloning and sequencing of an original gene encoding a
maltogenic amylase from Bacillus sp. US149 strain and characterization of the
recombinant activity. Molecular Biotechnology. 38: 211-219.
Machovič, M. and Janeček, Š. (2007). Amylolytic Enzymes: Types, Structures and
Specificities. In Polaina, J. and MacCabe, A. P. (Eds). Industrial Enzymes. (pp.
3-18). Slovakia: Springer Netherlands.
Matsuzaki, H., Yamane, K., Yamahuchi, K., Nagata, Y. and Maruo, B. (1974).
Hybrid α-amylases produced by transformants of Bacillus subtilis. I.
90
Purification and characterization of extracellular α-amylases produced by the
parental strains and transformants. Biochimica et Biophysica Acta (BBA) –
Protein Structure. 365(1): 235-247.
Mergulhão, F. J. and Gabriel A. M. (2005). Analysis of factors affecting the
periplasmic production of recombinant proteins in Escherichia coli. Journal of
Microbiology and Biotechnology. 17(8): 1236-1241.
Novagen. (2005). pET System Manual. (11th ed.). Germany: EMD Bioscience Inc.
Oh, K. W., Kim, M. J., Kim, H. Y., Kim, B. Y., Baik, M. Y., Auh, J. H. and Park, C.
S. (2005). Enzymatic characterization of a maltogenic amylase from
Lactobacillus gasseri ATCC 33323 expressed in Escherichia coli. FEMS
Microbiology Letters. 252:175–181.
Pandey, A., Nigam, P., Soccol, C. R., Singh, D. and Mohan, R. (2000). Advances in
microbial amylases. Biotechnology and Applied Biochemistry. 2: 135-152.
Pardi, Norlela (1999). Pengesanan Mikroorganisma Penghasil β-siklodekstrin
glukanotransferase (β-CGTase) Daripada Mikroorganisma Jenis Alkalopilus.
Universiti Teknologi Malaysia: Tesis Ijazah Sarjana Muda.
Park, K. H., Kim, M. J., Lee, H. S., Han, N. S., Kim, D. M. and Robyt, J. F. (1998).
Transglycosylation reactions of Bacillus stearothermophilus maltogenic
amylase with acarbose and various acceptors. Carbohydrate Research. 313:
235–246.
Park, K. H., Kim, T., Cheong, T. K., Kim, J. W., Oh, B. H. and Svensson, B. (2000).
Structure, specificity and function of cyclomaltodextrinase, a multispecific
enzyme of the α-amylase family. Biochimica et Biophysica Acta. 1478: 165–
185.
Purhagen, J. K., Sjöö, M. E. and Eliasson A. C. (2011). Starch affecting anti-staling
agents and their fuction in freestanding and pan-backed bread. Food
Hydrocolloids. 25(7): 1656-1666.
Robyt, J. F. (1995). Mechanisms in the glucansucrase synthesis of polysaccharides
and oligosaccharides from sucrose. Advances in Carbohydrates Chemistry and
Biochemistry. 51: 151–157.
Roh, H. J., Kang, S. C., Lee, H. S., Kim, D. K., Byun, S. B., Lee, S. J. and Park, K.
H. (2004). Transglycosylation of tagatose with maltotriose by Bacillus
stearothermophilus maltogenic amylase (BSMA). Tetrahedron: Asymmetry.
16(1): 77-82.
91
Sakai, S., Kubota, M., Yamamoto, K., Nakada, T., Torigoe, K., Ando, O., and
Sugimoto, T. (1987). Cloning of cyclodextrin glucanotransferase genes from
Bacillus stearothermophilus and Bacillus macerans. Journal of the Japanese
Society if Starch Science. 34: 140-147.
Si, J. Q. and Drost-lustenberger, C. (2001). Novamyl - A true anti-staling enzyme.
Madrid: The Meeting at IATA.
Sindhu, G. S., Sharma, P., Chakrabarti, T. and Gupta, J. K. (1997). Strain
improvement for the production of a thermostable α-amylase. Enzyme and
Microbial Technology. 24: 584-589.
Svensson, B., Jensen, M. T., Mori, H., Bak-Jensen, K. S., Bønsager, B., Nielsen, P.
K., Kramhøft, B., Prætorius-Ibba, M., Nøhr, J., Juge, N., Greffe, L.,
Williamson, G., and Driguez, H. (2002). Facinating facets of function and
structure of amylolytic enzymes of glycoside hydrolase family 13. Biologia. 57
(11): 5–19.
Takaku, H. (1988). Handbook of amylase and related enzymes. The Amylase
Research Society of Japan. (pp. 215-217). Oxford: Pergamon Press.
Takata, H., Kuriki, T., Okada, S., Takesada, Y., Iizuka, M., Minamiura, N. and
Imanaka, T. (1992). Action of neopullulanase. Journal of Biological
Chemistry. 267: 18447-18452.
Towbin, H., Staehelin, T. and Julian, G. (1979). Electrophoretic transfer of proteins
from polyacrylamide gels to nitrocellulose sheets: Procedure and some
applications. Proceedings of the National Academy of Science. 76(9): 4350-
4354.
Underkofler, L. A, Danault, L. J. and Hou, E. F. (1965). Enzymes in starch in dustry.
Die Starke. 17: 179-184.
Vandeputte, G. E., Vermeylen, R., Geeroms, J. and Delcour, J. A. (2003). Rice
starches. III. Structural aspects provide insight in amylopectin retrogradation
properties and gel texture. Journal of Cereal Science. 38: 61–68.
Vihinen, M., and Mäntsala, P. (1989). Microbial amylolytic enzymes. Critical
Reviews in Biochemistry and Molecular Biology. 24: 329–418.
Willey, J. M., Linda S., Christopher J. W., and Lansing M. P. (2008). Prescott,
Harley, and Klein's Microbiology. New York: McGraw-Hill Higher Education.
Whitehurst, R. J. and Law, B. A. (2002). Enzymes in starch modification. Enzymes in
Food Technology. 8: 200-228.
92
Wood, T. K. and Peretti, S. W. (1991). Effect of chemically-induced cloned-gene
expression on protein synthesis in E. coil. Biotechnology and Bioengineering.
38: 397-412.
Würsch, P. and Gumy, D. (1994). Inhibition of amylopectin retrogradation by partial
beta-amylolysis. Carbohydrate Research. 256: 129–137.
Yuuki, T., Nomura, T., Tezuka, H., Tsuboi, A., Yamagata, H., Tsukagoshi, N., and
Udaka, S. (1985). Complete nucleotide sequence of a gene coding for heat-
and pH-stable α-amylase of Bacillus licheniformis: Comparison of the amino
acid sequences of three bacterial liquefying α-amylases deduced from the DNA
sequences. The Journal of Biochemistry. 98: 1147-1156.
Zhao, H. L., Xue, C., Wang, Y., Duan, Q. F., Xiong, X.H., Yao, X. Q. and Liu, Z. M.
(2008). Disruption of Pichia pastoris PMR1 gene decreases its folding capacity
on human serum albumin and interferon-alpha2b fusion protein. Yeast. 25:
279–286.