1
Screening & Optimization Study on Fungal Proteases and their
Applications
Riya M. Bhanushali1, Hetal K. Panchal2
1,2 Dolat-Usha Institute of Applied Sciences and Dhiru-Sarla Institute of Management
& Commerce, Valsad, 396001, India
ABSTRACT
The present study focuses on production of extracellular proteases by a local fungal isolate
through solid state fermentation. All the fungal isolates were screened for their protease
producing ability & two isolates showing highest protease production were selected for further
studies. Optimization of different fermentative variables like carbon and nitrogen sources, pH,
temperature and various solid substrates was done to enhance enzyme production. OVAT
method was used to study optimization. Protein concentration and enzyme activity was
measured. The maximum protease production was found when the medium was supplemented
with corn seed meal as a solid substrate, glucose as a carbon source, peptone as a nitrogen
source. The optimum pH & temperature was 8 and 37°C respectively. The enzyme was further
purified by ammonium sulphate fractionation and dialysis at 60%, 70% & 100% saturation.
Where, 100% saturation showed best results. Then proteases was resolved by SDS-PAGE
analysis. Various applications like, Antioxidant activity, Milk Clotting Activity (MCA),
Efficiency to remove blood stains & feather degradation activity was successfully performed
using fungal protease. Proteins from germinating seeds of chick pea, mung bean, soybean and
cowpea were hydrolysed for the production of amino acids. Amino acids were recovered,
estimated & utilized for chelation of metalnutrients viz., Cu & Mn. The resultant chelates were
employed to detect with Fourier Transform Infra-Red Spectrophotometer (FTIR) analysis. The
resultant amino acids-metal nutrient chelates can be utilized as organic fertilizer.
Keywords: Fungal protease, Optimization, Antioxidant activity, Milk clotting activity, seed
proteins- amino acids- chelates, FTIR
1. INTRODUCTION
Proteases are among the oldest and most diverse families of enzymes known and are involved
in every aspects of organism’s function. Proteases are complex group of hydrolytic enzymes
which are responsible for the hydrolysis of protein molecules into small peptides & amino
acids. Because of their broad substrate specificity, proteases have a wide range of applications
such as in leather processing, detergent formulations, baking, brewing, meat tenderization,
peptide synthesis, cheese manufacturing, soya sauce production, protein hydrolysate,
pharmaceutical industry, waste treatment, silk industry, organic synthesis, recovery of silver
from waste photographic film, as well as analytical tools in basic research and have high
commercial value [1, 2, 3, 4]. They can be synthesized by plants, animals, and microorganisms
constituting around 60% of the worldwide enzyme market [5]. Microorganisms are the most
common sources of commercial enzymes due to their physiological and biochemical
properties, facile culture conditions and ease of cell manipulation [6]. Microbial proteases are
preferred to animal and plant sources because of various advantages. A variety of
microorganisms such as bacteria, fungi, yeast and actinomycetes are known to produce these
enzymes [7]. Among them fungi exhibit a wider variety of proteases than bacteria. Protease
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produced by SSF have greater economic feasibility. SSF is more advantageous as compared to
SmF because of low fermentation technology, low cost, higher yields-concentration of
enzymes and reduced waste output. In the present investigation, protease producing fungi were
isolated & screened. Various optimization parameters were studied to maximise the protease
production. And the ability of the selected isolate was tested by performing different activities.
2. MATERIALS AND METHODS
2.1 Collection & identification of Fungi
About Twenty-five different fungi were directly collected from the Microbiology Laboratory
of Dolat-Usha Institute of Applied Science and Dhiru-Sarla Institute of Management &
Commerce, Tithal Road, Valsad. The samples were inoculated onto sterile Sabouraud’s agar
plates (Peptone – 10gm, Glucose – 40gm, NaCl – 5gm, Agar – 24gm, D/W – 1000ml, pH –
5.2). The plates were incubated at room temperature for 6-7 days. After incubation, fungal
morphology were studied macroscopically by observing colony features (Colour and Texture)
and microscopically by staining with lacto phenol cotton blue and observe under compound
microscope for the conidia and arrangement of spores.
2.2 Screening by Plate Assay Method
Production of proteolytic enzymes by fungal isolates was detected by using the Plate Assay
Method [8], in which gelatin is act as the protein source of that medium. The fungal isolates
were spot inoculated on the medium supplemented with 1% gelatin (Peptone – 5gm, Beef
extract – 3gm, NaCl – 5gm, Agar – 24gm, D/W – 1000ml, pH – 6). After inoculation, plates
were incubated at Room temperature for 3-4 days. Following incubation, gelatin degradation
was observed as a clearing zone around the fungal colonies. The zone of gelatinolysis was
clearly seen when plates were flooded with aqueous solution of Frazier’s reagent (15gm HgCl2
mixed in 20ml 7M conc. HCl and 80ml D/W). Mercuric chloride solution react with gelatin to
produce a white precipitate which made the clearing zone visible. The zone indicates the
extracellular protease activity of the fungal strain. Enzyme activity was measured by the
following formula: EA = D – d. Where, D= diameter of colony plus clearing zone, d= diameter
of colony. Two most potent isolates were chosen for further production, optimization &
application studies.
2.3 Protease Production
The selected two fungal isolates were inoculated in sterile protease specific broth (Yeast extract
– 1gm, MgSO4 – 0.02gm, glucose – 2gm, K2HPO4 – 0.1gm, D/W – 100ml, pH – 5.0). Flasks
were inoculated & incubated at room temperature for 5-6 days on rotary shaker. At the end of
incubation, the contents of flasks were filtered through Whatmann filter paper No. 1 and then
filtrates were centrifuged at 8,000 rpm for 10 minutes.
Pellet were discarded after centrifugation and clear supernatant was used as a source of
protease. The supernatant was further used for subsequent studies [9].
2.4 Measurement of Protein and Enzyme activity
2.4.1 Protein estimation:
Protein estimation of crude enzyme extract was determined according to the Folin-Lowry’s
method.
2.4.2 Enzyme activity:
Protease activity in the crude enzyme extract was determined according to the method of
Carrie Cupp-Enyard by using casein as substrate. Two test tubes were taken and labelled as
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test (T) and blank (B). 5ml of 0.65% casein solution was added in test and blank, were placed
at 37°C for 30minutes to occur enzymatic reaction. The reaction was terminated by addition of
5% Trichloro acetic acid (TCA) solution in both the tubes. 1ml of enzyme solution was added
in test (T) tube only and it was allowed to stand for 15 minutes at room temperature. Then
solution from both the tubes were filtered by using Whatmann no. 1 filter paper.
2ml of filtrate from both the tubes were taken into two new tubes. 5 ml sodium carbonate was
added in both test tubes followed by addition of 1 ml of 2 fold diluted Folin Ciocalteu phenol
reagent. Then resulting solution were incubated in dark for 30 minutes at room temperature for
the development of blue colour. The absorbance of the blue colour compound was measured
at 660nm against a reagent using tyrosine standard [10].
2.5 Optimization of process parameters by One Variable At a Time (OVAT) method
2.5.1 Effect of various types of Carbon source: Various carbon sources were investigated
for their effect on enzyme production. The carbon sources tested were Glucose,
Maltose, Fructose & Sucrose [11].
2.5.2 Effect of various types of Nitrogen source: Protease production was investigated
using different nitrogen sources like Peptone, Yeast extract and Beef extract [12].
2.5.3 Effect of various pH: The effect of pH was studied at pH 5, 6, 7, & 8 [13].
2.5.4 Effect of various Temperature: The effect of different temperatures like- 4°C, Room
temperature and 37°C were studied [14].
2.6 Effect of various solid substrates (SSF):
Solid substrates like Cotton seed meal, Peanut seed meal, Corn seed meal and Soybean husk
were checked for the maximum production of protease as they are easily available at low cost
[15].
2.7 Extraction and Purification
The crude protease extract obtained by culturing fungal isolate in optimized protease
production medium was subjected to Ammonium sulphate fractionation (60%, 70%, & 100%).
The precipitates so obtained by centrifugation were dissolved 0.1M phosphate buffer (pH 6.7)
and dialyzed against the same buffer for overnight at room temperature. The samples were then
tested for protease activity & protein estimation. Then specific activity, fold purification and
% yield were calculated.
2.8 Electrophoresis SDS-PAGE analysis
The molecular weight of fungal proteases are generally in the range of 20 and 50 kDa. In order
to evaluate the proteolytic enzyme profile, crude enzymatic extracts corresponding to fungal
strains were analysed by polyacrylamide gel electrophoresis. Hence, SDS- PAGE analysis
allowed to distinguish a clear difference between protein patterns of the examined fungal
strains [16].
2.9 Applications of fungal protease
2.9.1 Antioxidant activity:
The reducing power of the fungal extract was determined by the method described by Oyaizu
(1986) with modifications [17]. Various concentration of fungal extract that is protease, were
mixed with 1 ml of 0.2M phosphate buffer (pH 6.6) and 1 ml of 1% potassium ferricyanide.
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The reaction mixture was incubated at 50°C for 20 minutes followed by addition of 1 ml of
10% trichloro acetic acid (TCA) and centrifuged at 2000 rpm for 10 minutes. An aliquot of
supernatant (1 ml) was mixed with 1 ml of deionized water and 250µl of 0.1% (v/v) ferric
chloride.
Absorbance was measured at wavelength 700nm against the diluent as blank. Higher
absorbance indicates greater reducing power. Butylated Hydroxy Toluene (BHT) was used as
the standard antioxidant.
2.9.2 Milk Clotting activity:
Milk clotting activity was measured using a modified method of Sousa and Malcata. The milk
substrate was prepared by dissolving 12gm of skimmed milk powder in 100cm3 CaCl2 solutions
(0.01 mol L-1). The pH value of the milk was adjusted to 6.0 with 1M HCl before use. The milk
substrate (2 cm3) was heated at 60°C, and thoroughly mixed with 2 cm3 of the enzyme solution.
The time for formation of fragments was measured with stopwatch. One unit of milk clotting
activity (MCA) is equal to the amount (mg) of enzymes required to coagulate 1cm3 of
reconstituted skimmed milk in 1 min at 60°C and pH 6.5. The MCA was calculated by using
Equation: MCA = 2400 / t × F. Where, t is the time for the formation of fragments (s), F is the
dilution coefficient [18].
2.9.3 Feather degradation activity:
Feathers were microscopically observed before the treatment. And then feather was subjected
to the crude enzyme extract. Which is incubated at 37°C, for 14 days. After incubation the
feather was microscopically examined for the feather degradation [19]. Distilled water blank
containing feather tube was used as control.
2.9.4 Silk degumming activity of the protease:
Degumming of silk was done by using commercial process [20]. In which enzyme bath was
prepared by adding protease enzyme 50 ml, 0.5 g sodium bicarbonate, 1 g non-ionic detergent
(Tween 80) with M: L ratio 1:20 & phosphate buffer (pH 7). 35.15 g of silk yarn washed with
soft water & then immersed into the prepared enzyme bath.
The temperature was maintained at 90°C for 70 minutes. Then the silk yarn was removed,
washed & dried. After drying, weight loss was compared to calculate the percentage of silk
degumming. And increased protein concentration in the solution of enzyme bath was indicated
through performing protein estimation by Folin Lowry’s method.
2.9.5 Removal of blood stain from the cotton cloth:
Fibrinolytic potential of the purified enzyme was determined by incubating the pieces of cotton
fabric impregnated with blood [21]. The dried blood stains were fixed with 2% (v/v)
formaldehyde for 30 min and the excess formaldehyde is removed by rinsing with water. The
cotton fabrics were then allowed to dry.
After drying, the cotton fabrics were incubated separately with 2ml of partially purified
protease along with detergent, 2ml of D/W with detergent and 2ml of distilled water at 37°C
for 1 hour. The cotton fabrics were then rinsed with water and checked for the removal of blood
stains after drying.
2.9.6 Production of amino acids-metal nutrient chelates:
2.9.6.1 Production of free amino acids- Fungal spore suspension was inoculated in modified
production media suggested by [22] (malt extract 1gL-1, glucose 6 gL-1, yeast extract 1 gL-1,
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peptone 2 gL-1, K2HPO4 0.5 gL-1, MgSO4.7H2O 0.5 gL-1, FeSO4. 7H2O 0.01 gL-1, pH-7.5) and
incubated on rotary shaker at 37°C for 9 days. After incubation the filtered broth was subjected
to 100% ammonium sulphate precipitation & dialysis. Then this dialyzed sample were used for
protease activity & also utilized for the digestion of seed proteins.
Each seed samples chick pea, mung bean, soybean and cowpea (10gm each) were germinated
with sterile D/W for 48 hours in incubator at constant temperature (37°C) and humidity (80%)
and extracted in distilled water.
The seeds were crushed together using blender and crushed material was adjusted to pH- 7.5
with 0.1N NaOH and subjected to fungal protease for the enzymatic digestion at 37°C for 4-5
days to recover maximum amount of amino acids. Then the digested material was treated with
95% ethanol for 30min and centrifuged at 10,000xg for 10 min and extracted with D/W [23].
Seed crush without digestion were used as control. Total amino acids were estimated from the
filtrate by Ninhydrin method and utilized for synthesis of metal-nutrient chelate.
2.9.6.2 Production & analysis of amino acids-metal nutrient chelate
The amino acids (Chelating solution) recovered from enzymatic digestion were utilized for
production of chelates [24]. One gram each of metal nutrients like, copper sulphate and
manganese sulphate was added separately to 100ml of chelating solution and incubated for 2
hour on rotary shaker at 37°C. After 2 hours of incubation it was employed to detect chelate
using FTIR spectrophotometer.
3. RESULTS AND DISCUSSION
3.1 Identification of fungi
Among most isolates the genera Aspergillus were dominant. The colony pigmentation was
white, yellow-brown to black shades of green, black grown evenly with dense & erect
conidiophores. Morphology of conidia was observed by Lacto-phenol cotton blue (LPCB)
mounting.
3.2 Screening by plate assay method
Screening of fungi for their protease activity was carried out by the hydrolysis of substrate
incorporating in the medium by plate assay method. After incubation period, enzyme activity
were detected by appearance of zones around the fungal colonies. Two fungal sp. (F 5 & F 15)
showed highest zones around the colony, were used for further study. Among fungal species
of certain genera, Aspergillus, Penicillium, Paecliomyces, Rhizopus and Rhizomucor are well
known producers of proteases [25, 26].
3.3 Measurement of Protein and Enzyme activity
By performing protein estimation and enzyme activity, Specific activity was calculated by
using formula: Specific activity = Enzyme activity/Protein concentration
From the above results it can be said that F 15 has high protease activity than F 5, but in case
of Specific activity F 5 has good results (Table 1).
Table 1. Protease activity of selected fungal isolates
Isolate No. Enzyme activity Protein conc. Specific activity
F 5 6.5 µg/min/ml 1.8 mg/ml 3.61 Unit/ml
F 15 6.7 µg/min/ml 1.9 mg/ml 3.52 Unit/ml
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3.4 Optimization by OVAT method
3.4.1 Effect of Carbon sources: Different carbon sources were investigated to get maximum
enzyme production & activity. And promising results were obtained when medium was
supplemented with Fructose and Glucose as a carbon source for F 5 & F15, respectively.
(Fig.1) Increased yield of alkaline protease production were reported by several other
researchers [21] using different sugars such as Dextrose, Sucrose, Lactose, Maltose.
3.4.2 Effect of Nitrogen sources: The growth of the microorganisms and enzyme production
requires nitrogen source. Maximum protease activity found when medium was supplemented
with peptone as Nitrogen source. (Fig. 2) Researchers [12] observed that yeast extract showed
promising results.
3.4.3 Effect of pH: pH of the fermentation medium is one of the most important factor affecting
enzyme production. In the present study a pH of 8.0 was found to be optimum for protease
production. (Fig. 3) An optimum pH of 8.0 and 7.0 was reported for protease produced by
Aspergillus flavus and Aspergillus niger respectively [13].
3.4.4 Effect of Temperature: Incubation temperature also plays an important role in enzyme
production and growth of microorganisms, which can be activated at one temperature and
inhibited at another. The optimum temperature for protease production was 37°C for both the
fungi. (Fig. 4) Similarly, [27] reported the optimum temperature for protease production by the
mesophilic fungi Synergistes species was at 35°C.
0
2
4
6
8
10
Glucose Fructose Maltose SucroseSpe
cifi
c ac
tivi
ty (
Un
it/m
l)
C-SOURCES
F 5 F 15
0
1
2
3
4
5
6
7
Peptone Yeat Extract BeefExtract
Spe
cifi
c ac
tivi
ty (
Un
it/m
l)
N-SOURCESF 5 F 15
0
0.5
1
1.5
2
2.5
3
3.5
4
pH 5 pH 6 pH 7 pH 8
Spe
cifi
c ac
tivi
ty (
Un
it/m
l)
pHF 5 F 15
00.5
11.5
22.5
33.5
44.5
4◦C R.T 37◦CSpe
cifi
c ac
tivi
ty (
Un
it/m
l)
TEMPERATURES
F 5 F 15
Figure.1 Effect of Carbon sources Figure. 2 Effect of Nitrogen sources
Figure. 3 Effect of various pH Figure. 4 Effect of Temperatures
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3.5 Enzyme production using Solid state fermentation: The selection of solid substrates
for enzyme production in SSF process depends upon several factors, mainly related to
cost and availability of the substrate. Out of four solid substrates corn seed meal gave
good results as compared to others. (Fig. 5) [28] got good results using wheat bran as
the solid substrate.
3.6 Extraction and Purification: For both the fungi maximum activity was found in 100%
saturation in Ammonium sulphate as well as in Dialysis (Table 2, 3). According to [29],
the highest protease activity was found with 60 to 80% ammonium sulphate saturation.
Table 2: Results of enzyme activity after Ammonium sulphate precipitation
Parameters
(After Ammonium
sulphate precipitation)
F 5 F 15
% Saturation % Saturation
60 % 70 % 100 % 60 % 70 % 100 %
Specific activity
(Unit/ml)
2.87 4.67 5.47 2.76 3.02 4.77
Fold purification 0.795 1.293 1.515 0.784 0.857 1.355
% Yield 32.9% 53.3% 68.6% 31.2% 40.8% 72.6%
Table 3: Results of enzyme activity after Dialysis
Parameters
(After Dialysis)
F 5 F 15
% Saturation % Saturation
60 % 70 % 100 % 60 % 70 % 100 %
Specific activity
(Unit/ml)
1.98 3.44 3.48 1.88 2.02 2.93
Fold purification 0.548 0.952 0.963 0.534 0.573 0.832
% Yield 19.8% 41.5% 42.3% 28.6% 31.3% 50.7%
0
2
4
6
8
10
Cotton Seed Corn Seed Soyabean Husk Peanut Seed
Spe
cifi
c ac
tivi
ty (
Un
it/m
l)
SubstratesF 5 F 15
Figure. 5 Enzyme production using various solid substrates
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3.7 Electrophoresis SDS-PAGE analysis:
3.8 Applications of fungal protease:
3.8.1 Antioxidant activity- From the Fig. 7, protease of F 15 has more antioxidant activity
than the protease of F 5. Darker the colour, higher is the reducing potential.
3.8.2 Milk clotting activity- Protease of F 5 has higher MCA activity than the protease of F
15. According to findings of [18], Ginger protease has maximum MCA that is about
179 Unit/cm3.
Table. 4 Results of Milk clotting activity
Fungal sample Time (in sec) MCA (Units/cm3)
F 5 360 sec 6.66 Unit/cm3
F 15 387 sec 6.20 Unit/cm3
0
0.1
0.2
0.3
0.4
0.5
Undil. 1:2 dil.
Ab
sorb
ance
(n
m)
Dilution
Antioxidant activity
BHT F 5 F 15
F 5 F 15
Figure. 6 Analysis of crude
enzyme by SDS-PAGE
The molecular weight of protease was determined
by comparing it with the standard protein marker
(6.5 kDa – 200 kDa). The molecular weight of
fungal proteases are generally in the range of 20
and 50 kDa [30].
The development of clear bands on the blue
background of the gel indicated the presence of
protease activity. SDS-PAGE was done from the
supernatant extracted from both the isolates F 5 &
F 15. Thicker bands in gel means there is more of
that particular size molecule in the sample.
From the Fig. 6 for F 5 isolate band was observed
near 40 kDa. Moreover F 15 also gave good
proteolytic activity as it shows bands near 44.3
kDa marker.
Figure. 7
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3.8.3 Feather degradation activity- Degradation rate was examined during 7 days with day
to day observation. From the Fig. 8 it has been said that, there was no degradation in
control tube.
3.8.4 Silk degumming activity- Degumming of silk by using fungal protease shows
difference in colour as well as weight loss. Weight loss was calculated in form of
percentage. And protein concentration was increased in enzyme bath. Which was
detected by Folin Lowry’s method. From the above results it can be said that protease
of F 5 has more efficiency to remove the protein (sericin) layer from the silk fibre.
Table. 5 Results of silk degumming
Fungal isolate Protein concentration Weight loss (in %)
F 5 2.49 mg/ml 5.14 %
F 15 2.45 mg/ml 2.13 %
3.8.5 Removal of blood stain from cotton cloth- Microbial proteases are widely used in
detergent industry. Proteases from both the fungal isolate F 5 and F 15 gave better
results of washing with/without detergent (Fig. 9).
Day: 1 Day: 7
Figure. 8 Microscopic examination of feather degradation
A
.
F
. E
.
D
.
C
.
B
.
Figure. 9
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3.8.6 Production of amino acids- metal nutrient chelates
Production of free amino acids
Results shows that amount of amino acids released from undigested seed crush and seed crush
digested with protease of F 5 and F 15 noted gradual increase in amino acid with increase in
incubation period. Amino acid estimation was performed by Ninhydrin method. L-lysine was
used as standard amino acid. Amino acids released from treated & untreated seed crush were
recorded at regular interval. The maximum release of amino acids 2.49 mg/ml was recorded at
96 h of incubation in F 5 and 1.79 mg/ml at 96 h of F 15.
Production and analysis of amino acid- metal nutrient chelate
The amino acids (chelating solution) recovered from enzymatic (F 5 & F 15) digestion were
utilized for the production of chelates. The metal nutrients like Cu2+ and Mn2+ chelated with
organic chelating agents were vary in colours. In present investigation, the detection of metal
nutrient chelate has been confined by FTIR spectrophotometer.
Figure. 9
A. Distilled water + Stained cloth(Control)
B. Distilled water + Stained cloth + Enzyme of F 5
C. Distilled water + Stained cloth + Enzyme of F 5 + Detergent
D. Distilled water + Stained cloth + Enzyme of F 15
E. Distilled water + Stained cloth + Detergent
F. Distilled water + Stained cloth + Enzyme of F 5 and F 15 +
Detergent
Figure. 10 (a)
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Figure. 10 (d)
Figure. 10 (c)
Figure. 10 (b)
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FTIR can be used to monitor the modification in the vibrational absorption bands of a ligand
due to metal-ligand complex formation. The simple stretching vibrations in the 1600 cm-1 to
3500 cm-1 region are the most predictable, and absorptions in this region are used to identify
Figure. 10 (e)
Figure. 10 (f)
Figure. 10
(a) FTIR spectra of chelating solution of F 5
(b) FTIR spectra of Amino acid- metal nutrient chelate [CuSO4]of F 5
(c) FTIR spectra of Amino acid- metal nutrient chelate [MnSO4] of F 5
(d) FTIR spectra of chelating solution of F 15
(e) FTIR spectra of Amino acid- metal nutrient chelate [CuSO4] of F 15
(f) FTIR spectra of Amino acid- metal nutrient chelate [MnSO4] of F 15
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functional groups in molecules. During chelation, binding of the carboxylic groups and primary
amine groups of amino acids to the metal nutrients is indicated by the broadening of peak and
changes of the peak position of hydroxyl groups.
In present investigation, detection of metal nutrient chelate has been confined to the higher
frequency region in which internal vibrations of the chelating solutions were observed.
4. CONCLUSION
Based on the results obtained in this study, various optimization conditions like carbon-
nitrogen source, pH, temperature and solid substrate for SSF were identified to get maximum
protease production. Enzyme purification and characterization had been performed. Various
applications like, Antioxidant activity, Milk Clotting Activity (MCA), feather degradation
activity, Efficiency to remove blood stains & Silk degumming activity was successfully
performed using fungal protease.
The amino acids produced from leguminous seeds can chelate metalnutrients. This amino acid-
metalnutrient chelates can be utilized as liquid organic fertilizer which can be useful to
overcome the metalnutrient deficiency in short term vegetable crops. Hence, these enzyme
have number of commercial applications at various places like: Dairy industry, Detergent
industry, Laundry, Poultry farm, Slaughter house, Textile industry, fertilizer manufacturing
companies, etc.
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Page No : 7338
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