Chapter Five: Enzyme Purification and Study of Enzyme Characteristics

CHAPTER FIVE:
ENZYME PURIFICATION AND STUDY
OF ENZYME CHARACTERISTICS
Chapter 5: Enzyme purification and study of enzyme characteristics
5.1 INTRODUCTION:
5.1.1 Keratinases
Proteases are enzymes that carry out proteolysis, which is hydrolysis of the
peptide linkages which connect amino acids (the building blocks of protein). Having
highly recognizable commercial applications, proteases occupy a crucial position in the
enzyme market. Depending upon their mode of action, proteases are classified into
different classes- Serine proteases, aspartic proteases, cysteine proteases and
metalloproteases. The sources of proteases range from prokaryotes to bacteria, archaea
and viruses to eukaryotes like plants and animals. Proteases are one of the most important
groups of industrial enzymes and account for nearly 60% of the total enzyme sale (Rao et
al., 1998).
Keratinases belong to the group of proteolytic enzymes that are capable of
specifically hydrolyzing keratin containing substrates. By virtue of this capability,
keratinases are gaining increasing importance in the commercial sector (Brandelli et al.,
2010). This is because keratin rich wastes such as feathers, horn, hair, nails, etc. are
generated in large amounts as by-products of agro-industrial processing and keratinases
produced by microorganisms enable the biodegradation of such wastes. Keratinases are
released as extracellular proteases by several microorganisms belonging to both bacteria
and fungi. Among bacteria, keratinase is produced majorly by Gram positive species,
namely Bacillus sp., Clostridium spp., etc. However, recent reports have also mentioned
of Gram negative bacteria producing keratinase, namely by Pseudomonas sp. and Proteus
sp. Among the fungi, keratinase is produced by Microsporum spp., Trychophyton spp.,
Aspergillus sp. etc. (Brandelli, 2008). Keratinases produced by fungi are also of medical
relevance. Bacterial keratinases have been purified and characterized and are being
studied further for their commercial significance.
5.1.2 Enzyme production (Rajni Hatti-Kaul, 2004):
Enzyme production using microorganisms can be carried out by fermentation
technology, using methods such as solid state fermentation and submerged fermentation.
Submerged fermentation involves cultivation of microorganisms in liquid nutrient
broth. This involves growing carefully selected microorganisms in closed vessels
containing the fermentation medium under highly aerobic conditions. While
School of Sciences, SVKM’S NMIMS (Deemed-to-be) University 156

microorganisms grow and metabolize the substrate, extracellular enzymes are released
into enzymes into solution. Solid-state fermentation (SSF) is another method used for
the production of enzymes. In solid-state fermentation microorganisms are cultivated a
solid substrate, such as grains, rice and wheat bran.
In laboratory, after an enzyme producing microorganism is selected, media
components and process parameters are optimized to achieve maximum enzyme
production by the selected isolate. The optimized parameters are then incorporated into
the production medium and the isolate is cultivated under the preferred process
parameters. The culture is then processed for recovery of enzyme and the enzyme is
processed for further studies.
5.1.3 Enzyme purification:
Enzyme purification has several purposes:
1. To increase catalytic activity of the enzyme, in order to produce an enzyme
preparation, that is of industrial significance. Even though the enzyme producing
isolate has undergone a genetic manipulation for elevated enzyme production,
purification step is required for a viable product.
2. To study the enzyme characteristics, namely the optimum pH, temperature, stability
at various conditions, Km, etc., without interference from other proteins.
3. To crystallize it, and to determine its structure using X-ray crystallography (David A.
Bender, 2006).
Enzyme purification involves several steps that eliminate other proteins and chemicals
that may interfere with the enzyme’s catalytic activity.
5.1.4 Methods/Stages of enzyme purification:
1. Salt precipitation (Salting out of proteins): This step involves precipitation of
proteins, thus eliminating residual nutrients of the medium and other released by-
products. Salt precipitation is usually carried out using ammonium sulfate.
Principle: Proteins are soluble in aqueous media because they have hydrophilic amino
acid side-chains which are provided by the basic amino acids, the acidic amino acids and
the neutral hydrophilic amino acids. A salt like ammonium sulfate interferes with these
interactions between amino acid side-chains and water, by reducing the available water
and will reduce the solubility of the protein. This enables protein-protein interactions

instead of protein-water interactions and the protein will come out of solution. Depending
upon the hydrophilicity of the protein, different proteins would separate at different
ammonium sulfate saturation levels. Higher the hydrophilicity, higher ammonium sulfate
concentration would be required to break the protein-water interactions to enable its
precipitation (Wingfield P, 2001; Burgess RR, 2009).
Ammonium sulfate precipitation is followed by dialysis, to remove the salt which
is bound to the protein. The protein/enzyme solution is placed in a bag of selectively
permeable membrane (e.g. gelatin/cellophane), immersed in a large volume of buffer,
under constant stirring at 4oC. The removal of ammonium salt is managed by the pore
size of the membrane that allows molecules of ammonium sulfate to cross, while
inhibiting the protein molecules to cross (Berg JM et al., Biochemistry, 5th edition, 2002).
2. Chromatographic methods: Depending upon the net charge of the

protein/enzyme of interest, the method to be employed can be either cation-exchange
chromatography or anion-exchange chromatography, which are differentiated on the
basis of the ion exchange resin.
i) Anion-exchange chromatography: The resin used is diethylaminoethyl-
cellulose (DEAE- Cellulose), which is a weak base. The cationic nature of the resin
enables anionic proteins to bind while allowing cationic proteins to pass through. The
binding of the protein to the resin depends upon the pI value of the protein and the pH of
the buffer solution in which the DEAE-Cellulose is equilibrated. The unbound proteins
can be collected in the unadsorbed fraction. The proteins which are bound can be eluted
using a salt gradient (i.e. NaCl linearly increasing from 0 mM to 200 mM). While elution
using a salt gradient, the positive ions of the salt will compete with those of the proteins,
for binding to the column, thus enabling the separation of protein (Berg JM et al.,
Biochemistry, 5th edition, 2002).
ii) Cation Exchange Chromatography: The resin used is CM-cellulose
(Carboxymethyl cellulose) is a weak acid which binds cations and allows anions to pass
(Berg JM et al., Biochemistry, 5th edition, 2002).

5.1.5 Study of Enzyme Characteristics (Nelson DL and Cox MM, 2004):

Enzymes have their specific characteristics i.e. assay requirements for maximum
activity, kinetics and also optimal requirements for stability and storage. The rate at
which an enzyme works is influenced by several factors:-
1. Concentration of Substrate (the more of them available, the quicker the enzyme
molecules collide and bind with them). The concentration of substrate is
designated as [S] and is expressed in units of molarity.
2. Reaction temperature: As the temperature rises, molecular motion increases and
hence collisions between enzyme and substrate speed up. But as enzymes are
proteins, there is an upper limit beyond which the enzyme becomes denatured and
ineffective.
3. Inhibitors: For example, competitive inhibitors bind to the same site as that of
the substrate, preventing the binding of the substrate. Non-competitive inhibitors
are molecules that bind to some other site, than that of the active site, affecting the
enzymes catalytic ability.
4. pH: The conformation of a protein is influenced by pH and as enzyme activity is
crucially dependent on its conformation, its activity is likewise affected.
The current chapter elucidates the process of purification of keratinase

enzyme from the crude cell free supernatant using ammonium sulfate precipitation
and dialysis, followed by Ion- Exchange Chromatography. This would be followed by
studying the key characteristics with respect to optimum pH and temperature for
enzyme assay, influence of chemicals on enzyme activity, molecular weight and
stability at various pH and temperature.

5.2 MATERIALS AND METHODS:

5.2.1 Enzyme production:
5.2.1.1 Preparation of production media: Optimized concentrations of media
components were incorporated into the production medium. The volume of the
production medium was raised up to 100 ml. The media optimized media
composition and incubation parameters for the respective feather degrading isolates
was as follows:
Table 5.1: Optimized media components and process parameters for the feather
degrading isolates
Isolate Isolate Isolate Isolate

B. sonorensis B. licheniformis B. subtilis
Components
NaCl (g/L) 5 5 5
K2HPO4 (g/L) 1 1 1
KH2PO4(g/L) 1 1 1
(NH)2SO4(g/L) 0.1 0.1 0.1
MgSO4(g/L) 0.2 0.2 0.2
Feathers(g/L) 7.5 7.5 10
Incubation temperature (oC) 35 30 35
Initial pH 7 8 7
The components were weighed accordingly and added into a 500 ml flask. They were
dissolved in minimal amounts of distilled water and then the volume was made up to
100 ml. The media was sterilized by autoclaving.
5.2.1.2 Inoculation and incubation: The isolates which were to be inoculated
were freshly grown on feather agar plates, from which a saline suspension was
prepared, turbidity of which was comparable with MacFarland’s opacity standard
number 2. This saline suspension was used to inoculate in the production medium. 1
ml of this suspension was used as inoculum, and it was aseptically added in to the
sterile medium. The inoculated medium was mixed and kept for incubation.
Incubations were carried out under shaker conditions at desired temperatures, i.e. using

Incubator-Shaker. The shaking was maintained at 120 rpm. Incubation was carried out
for five days. On the fifth day after inoculation, the supernatant was processed for
harvesting the enzyme.
5.2.2 Partial purification using ammonium sulfate precipitation (Jayaraman J,

2003):
5.2.2.1 Preparation of crude enzyme: After incubation, the crude enzyme was obtained
by centrifugation of the culture broth. Culture broth was aseptically dispensed into
sterile centrifuge tube. Centrifugation was carried out using a cold centrifuge, at
10,000 rpm for 20 minutes at 4oC. The supernatant was collected in a beaker and
it was proceeded for ammonium sulfate precipitation.
5.2.2.2 Ammonium Sulfate precipitation: Ammonium sulfate precipitation was carried

out at three saturation levels- 50%, 70% and 90%.
Ammonium sulfate precipitation chart: The following chart (table 5.2) was
referred, in which the values on the left hand side column indicate the initial
saturation level of the solution (in %). The values in the uppermost row indicate
the required saturation level (in %). For a required saturation level, first the initial
concentration of ammonium sulfate (in percent) is to be selected from the left side
column, and then the required final concentration is to be selected from the top
row. The intersection will give the exact amount of ammonium sulfate (in grams
at 25°C) to be added to one liter of solution to produce a desired change in the
present saturation of ammonium sulfate.

Table 5.2: Ammonium sulfate precipitation chart (Jayraman J, 2003)
Final concentration of Ammonium Sulfate (% saturation)
% 10 15 20 25 30 33 35 40 45 50 55 60 65 70 75 80 85 90 95 100
Grams of solid ammonium sulfate to be added to 1 liter of solution
0 56 84 114 144 176 196 209 243 277 313 351 390 430 472 516 561 610 662 713 767
10 28 57 86 118 137 190 183 216 251 288 326 365 406 449 494 540 592 640 694
Initial concentration of ammonium sulfate (% saturation)
15 28 57 88 107 120 153 185 220 256 294 333 373 415 459 506 556 605 657
20 29 59 78 91 123 155 189 225 262 300 340 382 424 471 520 569 619
25 30 49 61 93 125 158 193 230 267 307 348 390 436 485 533 583
30 19 30 62 94 127 162 198 235 273 314 356 401 449 496 546
33 12 43 74 107 142 177 214 252 292 333 378 426 472 522
35 31 63 94 129 164 200 238 278 319 364 411 457 506
40 31 63 97 132 168 205 245 285 328 375 420 469
45 32 65 99 134 171 210 250 293 339 383 431
50 33 66 101 137 176 214 256 302 345 392
55 33 67 103 141 179 220 264 307 353
60 34 69 105 143 183 227 269 314
65 34 70 107 147 190 232 275
70 35 72 110 153 194 237
75 36 74 115 155 198
80 38 77 117 157
85 39 77 118
90 38 77
95 39
The crude enzyme was first proceeded for precipitation at 50% saturation. For
this, the amount (grams) of ammonium sulfate to be added was calculated using
the ammonium sulfate precipitation chart. The calculation was as follows:
For 1000 ml, 50% saturation,
Ammonium sulfate required = 313 g
For 100 ml, Ammonium sulfate required = 31.3 g
31.3 g of ammonium sulfate was weighed.

The crude enzyme was placed in a beaker, which was in turn placed in an
acrylic container containing ice, so as to maintain cold conditions. The entire
assembly was placed on a magnetic stirrer. Addition of the salt was carried out
under constant stirring and cold conditions. The process of addition should be
carried out slowly, for up to one hour, in order to avoid frothing in the solution.
After the addition of salt was completed, stirring was continued for another 30
minutes.
The beaker containing the salt- saturated crude enzyme was then placed in
the refrigerator, overnight for the proteins to precipitate. The precipitated proteins
were recovered by cold centrifugation at 10000 rpm for 20 minutes. The proteins
were collected and suspended in 10mM phosphate buffer. The supernatant was
retained for the next level of ammonium sulfate saturation i.e. 70%.
The volume of the supernatant was measured and the difference in amount
of ammonium sulfate salt to be added was calculated. The addition process was
followed as mentioned above. The proteins were collected and suspended in
10mM phosphate buffer. The supernatant was subjected to next saturation level
i.e. 90%.
5.2.3 Dialysis of the salt-precipitated protein:

Preparation of phosphate buffer:
1M potassium phosphate buffer pH 7.0
Solution A- 1M KH2PO4: 68 grams in 500 ml distilled water
Solution B- 1 MK2HPO4: 87.09 grams in 500 ml distilled water
For 100 ml 1M potassium phosphate buffer (pH 7.0), 61.5 ml of solution B and
38.5 ml of solution A were mixed.
The buffer was diluted using distilled water, to achieve the desired molarity.
Dialysis Membrane- 110 (10 Mt) (Himedia Laboratories Pvt. Ltd.):
Av. Flat width- 32-34 mm
Av Diameter- 21.5 mm
Capacity approx. - 3.63 ml/cm

Preparation of the dialysis bag: A dialysis membrane was cut from the roll,
washed thoroughly in a beaker containing chilled distilled water. One end of this
membrane was sealed using a clamp. From the other end, the protein solution was
added using a pipette. After the addition is done, the open end was sealed using a
clamp, such that a little space was retained. This was done to avoid bursting of the
membrane due to excess flow of water inside the membrane.
Dialysis: The membrane was placed in a 1000 ml capacity glass beaker
containing 1000 ml of 10mM phosphate buffer, pH 7. This assembly was placed
inside the refrigerator. Gentle stirring was done using glass rod at regular
intervals. The buffer was changed every 2 hours. To ensure that the ammonium
salt is completely removed from the protein, 1 ml of the dialysis buffer was mixed
with 1% Barium Chloride solution. The absence of precipitate indicates that the
dialysis buffer is free from ammonium salt, and thus, desalting has successfully
occurred.
Assessing the enzyme activity and the protein content: The dialyzed protein
was assessed for its keratinase activity by performing the keratinase assay as
described previously, and the protein content was determined by Lowry assay.
5.2.4 Protein estimation by Folin-Lowry assay (Lowry et al., 1951) :

Reagents used & their preparation:
1. Solution A = 2 % Na2CO3 in 0.1N NaOH:
2 g of Na2CO3 was dissolved in 100 ml of 0.1 N NaOH which was prepared by
dissolving 0.4 g of NaOH in 100ml D/W.
2. Solution B = 1 ml of 1 % CuSO4 solution + 1 ml of 2 % Sodium Potassium
Tartarate:
 1 % CuSO4 solution
1 g of CuSO4 was dissolved in 100 ml D/W.
 2 % Sodium Potassium Tartarate
2 g of Sodium Potassium Tartarate was dissolved in 100 ml of D/W.
3. Solution C = 50 ml of Solution A + 1 ml of Solution B. This solution was
prepared fresh.

4. Folin- Ciocalteau phenol reagent (Sigma) was diluted 1:1, using distilled
water, freshly before use.
Lowry standard assay:
Lowry standard assay was performed using Bovine Serum Albumin (BSA) as a
standard; the concentration range was selected as 0 to 200 µg/ml, with an interval
of 20 µg/ml.
Preparation of Standard:
Concentration of BSA stock required: 1000 µg/ml
0.1 gram of BSA was weighed and taken into a conical flask. It was dissolved in
minimal amount of distilled water, and the volume was made up to 100 ml. In
order to ensure complete solubility, the flask was kept in an ultra-sound bath for
15 minutes.
Table 5.3: Dilution chart for protein estimation by Folin- Lowry method:
 Stock solution: Standard Bovine Serum Albumin (BSA)

 Diluent: Distilled water
 Concentration of Stock: 1000 µg/ml
 Range: 0-200 µg/ml
 Interval: 200 µg/ml
Tube Concentration of Volume of Volume of Total
Stock (µg/ml) stock (ml) diluent (ml) volume (ml)
Blank 0 0 1 1
1 20 0.02 0.98 1
2 40 0.04 0.96 1
3 60 0.06 0.94 1
4 80 0.08 0.92 1
5 100 0.1 0.90 1
6 120 0.12 0.88 1
7 140 0.14 0.86 1
8 160 0.16 0.84 1
9 180 0.18 0.82 1
10 200 0.2 0.80 1

Protocol for Lowry assay:

1. 1 ml of appropriately diluted sample was taken in a test tube.
2. To this, 0.5 ml of Solution C was added. The tube was mixed using vortex and
incubated for 15 minutes at room temperature.
3. After incubation, 5 ml of 1:1 diluted Folin-Ciocalteau phenol reagent was
added and mixed well by vortex, and incubation was carried out for 30
minutes at room temperature.
4. After incubation, absorbance was measured at 650 nm using Perkin-Elmer
UV- Visible Spectrophotometer.
5.2.5 Purification by Ion-Exchange Chromatography (Jayaraman, 2011):

Further purification was carried out by Ion-Exchange Chromatography using
DEAE- Cellulose (Diethyl amino ethyl cellulose) which is a weak anion
exchanger.
Reagents required and their preparation:
1. 1M NaCl containing 0.1N NaOH:
0.8 grams of NaOH was dissolved in 200 ml distilled water. To this, 11.688
grams of NaCl was added and mixed to dissolve.
2. 10mM phosphate buffer pH 7:
Phosphate buffer was prepared as mentioned in section 5.2.3 (dialysis of the
salt- precipitated protein).
3. 0.1 to 0.5N NaCl:
For 0.1M NaCl, 0.58 grams of NaCl was dissolved in 100ml distilled water.
For 0.2M NaCl. 1.16 grams of NaCl was dissolved in 100ml distilled water.
For 0.3 M NaCl, 1.74 grams of NaCl was dissolved in 100ml distilled water.
Method:
 Preparation of packing material:
10 grams of DEAE-cellulose was taken in a beaker. To this 200 ml of 1M
NaCl containing 0.1N NaOH, was added. Stirring was carried out vigorously

with a glass rod for a few minutes. The slurry was filtered through Whatmann
No. 1 paper. Washing was carried out till the filtrate became colorless. The
filter cake was extensively washed with distilled water till the pH of the
filtrate becomes neutral (pH 7.0).
The filter cake was transferred into a beaker and suspended in 200 ml of 0.1N
HCl and stirred well. In this step, the amino group of DEAE-cellulose gets
charged with proton. The excess acid is removed by washing with water as
mentioned before till the filtrate is of neutral pH. The material was then
suspended in 10 mM phosphate buffer pH 7.0.
 Column packing:
A column of size 10 cm x 1cm was mounted on a vertical stand. The outlet of
the column was packed with a little glass wool. The suspension of DEAE-
cellulose was poured into the column gently through the sides, avoiding
trapping of air bubbles. After the column is packed, equilibration was carried
out using 10mM phosphate buffer, pH 7.0.
 Loading and elution:
Without disturbing the top surface, protein sample was carefully added using a
micropipette. The sample was thus loaded and left undisturbed for 15 minutes,
to allow the keratinase enzyme to bind to the matrix. Then, the column was
washed with 10 mM potassium phosphate buffer at pH 7 and the protein was
eluted using 0.1 to 0.5 M NaCl at the flow rate 24 ml/h. The fractions
collected were assayed for enzyme activity and protein content. The fractions
were stored at -20 oC, and used as purified enzyme.
 Assessment of the protein content and enzyme activity:
The protein fractions collected by elution from DEAE- cellulose
chromatography were assessed for their keratinase activity and protein content
estimation.
5.2.6 Study of enzyme characteristics: Enzyme characteristics were studied in terms
of molecular weight, determination of optimum pH, temperature, substrate
concentration and effect of chemicals. The enzyme assay was performed under
varying conditions with respect to varying pH, Temperature, substrate

concentration and presence of chemicals. For assessing the stability of the

enzyme, the enzyme was incubated at different conditions and the residual
enzyme activity was determined.
Enzyme source: The purified enzyme obtained after DEAE-Cellulose
chromatography, was diluted to the ratio 1:5 times using distilled water. This
diluted enzyme was used for all the experiments of characterization.
5.2.6.1 Determination of molecular weight by Sodium Dodecyl Sulfate- Poly
Acrylamide Gel Electrophoresis (SDS-PAGE) (Laemlli, 1970):
Composition of reagents used:
1. SDS- PAGE Electrophoresis Buffer (Tank buffer), pH 8.3:
Composition:
25 mM Tris
250 mM glycine (electrophoresis grade)
0.1 % SDS
Preparation of Electrophoresis Buffer (5X):
5X stock:- 15.1 Tris base and 94 grams glycine was dissolved in 900 ml of de-
ionized water, then 50 ml of 10% (w/v) stock solution of electrophoresis grade
SDS was added and the volume was adjusted to 1000 ml with water. The
stock solution was stored at 4oC.
2. Preparation of Tris Buffer (buffering range 7.1 to 8.9):
1.5 M Tris Cl (100 ml): 18.165 grams Tris base was dissolved in 100 ml
distilled water. The pH was adjusted to 8.8 using HCl.
1.0 M Tris Cl (100 ml): 12.1 grams of Tris base was dissolved in 100 ml
distilled water. The pH was adjusted to 6.8 using HCl.
3. Preparation of 30% Acrylamide mix:
A stock solution containing 29% (w/v) acrylamide and 1% (w/v) N’ N’
methylene bisacrylamide was prepared in distilled, warm water (warm water
assists the dissolution of the bisacrylamide). This stock solution was stored at
4oC.
4. 10% stock solution of SDS:
Dissolve 0.1 gram SDS in 10 ml distilled water. It was stored below 25oC.

5. Ammonium Persulfate solution (10%):

0.1 grams of ammonium persulfate was dissolved in 10 ml distilled water. The
solution was freshly prepared before addition.
6. 10% Resolving gel:

Table 5.4: Composition of resolving gel
Solution Components Component Volume (ml)/30 ml
Distilled Water 11.9
30% acrylamide mix 10

1.5 M Tris (pH 8.8) 7.5
10% SDS 0.3
10% ammonium persulfate 0.3
TEMED 0.012
7. 5% Stacking gel:
Table 5.5: Composition of stacking gel
Solution Components Component volume (ml)/5ml
Distilled water 3.4
30% Acrylamide mix 0.83
1.0M Tris (pH 6.8) 0.63
10% SDS 0.05
10% ammonium persulfate 0.05
TEMED 0.005
8. Water saturated butanol solution:

100 ml n-Butanol was combined with 5ml distilled water and stored in a
plastic bottle, shaken well. The top layer was used for overlaying gels. It was
stored below 25oC.

9. 4 X SDS- PAGE loading buffer:

Composition:
40% Glycerol
250 mM Tris-Cl buffer, pH 6.8
8% SDS
0.04 % Bromophenol blue
5 % Beta- mercaptoethanol
Method:
1. SDS-PAGE was performed according to the protocol mentioned in Molecular
cloning - A laboratory manual by J. Sambrook, P.Maniatis, E.F. Fritsch; 2nd
edition.
2. The glass plates were assembled for casting slab gels.
3. The reagents were combined for two resolving gels, according to the
composition mentioned above.
4. Immediately after the addition of TEMED and ammonium persulfate, the gel
was pipetted into the casting tray, carefully, avoiding air bubbles.
5. The gel was overlayed with water saturated butanol (2mm height)
6. The polymerization was allowed to take place for 30 minutes at room
temperature
7. The top of the gel was drained and gently washed with distilled water. It was
pad dried with a tissue.
8. The stacking gel solution was prepared according to the composition
mentioned above. It was poured over the solidified resolving gel.
9. Immediately, a Teflon comb is inserted. It was allowed to polymerize for 30
minutes at room temperature.
10. The comb was carefully removed. The extraneous polymerized acrylamide
was removed from the top of the gel.
11. Tris-Glycine buffer was added to the top and bottom reservoirs.
12. The protein samples to be loaded were mixed with 4X electrophoresis buffer
(8 µl of the electrophoresis buffer + 32 µl of the sample) in a micro-tube

13. This sample solution was heated in a boiling water bath for 10 minutes. 40 µl
of the samples was then loaded into the wells with the help of a micro pipette.
In one of the wells, 4 µl of PAGERULERTM PLUS Prestained Protein Ladder
was added.
14. The protein samples were run at voltage of 200 volts, till the tracking dye
bromophenol blue, just reached the lower end of the gel.
15. After the gel was run completely, the buffer was decanted into a tray. The
assembly was dismantled, and the gel plate was soaked in the buffer
sufficiently to enable the separation of gel plates.
16. The left- hand side lower end of the gel was given a small cut. The gel was
removed from the gel plate and soaked into the buffer. It was proceeded for
silver staining.
Silver staining (Switzer et al., 1979):
Reagents required & preparation:
1. Fixing solution: 40% ethanol, 10% acetic acid, and 50% water
40% ethanol was measured into a measuring cylinder of 100 ml capacity, to
which 10% acetic acid was added. The volume was brought up to 100 ml with
distilled water
2. Sensitizing solution: 0.02% sodium thiosulfate
0.04 grams of Na2S2O3 was dissolved in 200 ml distilled water.
3. 0.1% Silver Nitrate solution:
0.2 grams AgNO3 was dissolved in 200 ml was dissolved in distilled water.
To this 40ul of 35% fromaldehyde was added just before use. This reagent
was chilled.
4. Developing solution: 3% sodium carbonate
7.5 grams Na2CO3 was dissolved in 250 ml distilled water, to which 0.05%
formaldehyde (125µl formaldehyde) was added just before use.
5. 5% acetic acid solution:
5 ml of glacial acetic acid solution was taken in a measuring cylinder, the
volume was made up to 100 ml using distilled water.

Method:
1. The gel was incubated in a fixing solution for 1 hour.
2. The gel was washed with distilled water, overnight with several changes of
distilled water.
3. The gel was sensitized using 0.2 % sodium thiosulfate, for 1 minute.
4. The gel was washed thrice with distilled water (20 seconds per wash).
5. The gel was incubated for 20 minutes in 4oC chilled silver staining solution.
This staining solution was added carefully from the corner of the tray.
6. The gel was washed in water thrice (20 seconds per wash).
7. The gel was then placed in a new staining tray and washed with distilled water
for 1 minute.
8. The gel was developed using developing solution. The developing process is
terminated just when the staining is enough, using 5% acetic acid solution.
9. The gel was washed with water for 20 seconds.
10. The gel was stored in 1% acetic acid at 4oC.
5.2.6.2 Determination of optimum pH for enzyme assay:
The enzyme- substrate reaction was carried out at varying pH values. The pH
values for the assay conditions were obtained by adjusting the pH of the substrate
using the appropriate buffers.
Preparation of buffers:
 For pH 5.5 to 6.5: Acetate buffer (pH 4.0, 50 mM)
Solution A: 11.55 ml glacial acetic acid/1000ml distilled water (0.2M glacial
acetic acid)
Solution B: 27.2 grams of sodium acetate/1000ml distilled water (0.2M sodium
acetate)
41 ml of solution A and 9ml of solution B were mixed, and diluted to 200 ml
using distilled water.
 For pH 7.0 to 8.5: Phosphate Buffer (pH 6.0, 50 mM)
Solution A- 1M KH2PO4: 68 grams in 500 ml distilled water
Solution B- 1 MK2HPO4: 87.09 grams in 500 ml distilled water

6.6 ml of solution B and 4.34 ml of solution A were mixed to obtain 50 mM

phosphate buffer, pH 6.
 For pH 9 to 9.5 : Glycine- NaOH buffer (pH 10, 50 mM)
Solution A: 0.2M Glycine: 15.01 grams glycine/1000ml distilled water
Solution B: 0.2M NaOH: 8 grams NaOH/1000ml distilled water
50 ml of solution A was mixed with 32 ml of solution A, and the volume was
made up to 200 ml.
Enzyme Assay: The purified enzyme was diluted (1:5) was incubated with the
substrate (at different pH values) at 50oC, for 10 minutes in a water bath. The
reaction was stopped by the addition of 20% Tricholoroacetic acid. The reaction
mixture was centrifuged at 10,000 rpm for 20 minutes. The supernatant was taken
for determining the absorbance at 280 nm, along with a substrate blank and
enzyme blank. The enzyme activity was calculated as mentioned previously.
5.2.6.3 Enzyme stability at varying pH:

The pH of the enzyme solution was adjusted at different pH values using the
above mentioned buffers. Thus, the enzyme was incubated at different pH values,
at room temperature for one hour. The enzyme assay was then performed to
determine the residual activity.
5.2.6.4 Determination of optimum temperature for enzyme assay:

Once the optimum pH was determined the enzyme-substrate reaction was carried
out at that optimum pH. The reaction mixture was incubated at varying
temperatures in a water bath (25 to 60oC) for 10 minutes. The reaction mixture
was centrifuged at 10,000 rpm for 20 minutes. The supernatant was taken for
determining the absorbance at 280 nm, along with a substrate blank and enzyme
blank. The enzyme activity was calculated as mentioned previously.
5.2.6.5 Enzyme stability at varying temperatures:

The enzyme solution was incubated at different temperatures (25 to 60oC) for one
hour after which the residual activity was determined.

5.2.6.6 Effect of varying substrate concentrations on enzyme activity:

Preparation of substrate: Keratin
Concentration of Keratin Stock solution: 500 µg/ml
Dilutions of the keratin solution were prepared using 50mM Tris.Cl buffer pH 8.0
as follows:
Table 5.6: Keratin dilution chart
Volume of stock Volume of diluent (ml) Concentration achieved
(ml) (µg/ml)
- 10
0
1 9 50
2 8 100
3.5 6.5
175
3.8 6.2
190
4.3 5.7
215
4.6 5.4 230
4.9 5.1
245
5.2 4.8
260
6 4
300
7 3 350
8 2
400
9 1
450
10 -
500
Enzyme assay: The enzyme was reacted with the varying concentrations of keratin
substrate as mentioned in the above table. The assay was carried out at the optimum pH
and temperatures, respectively. The absorbance was measured, and the enzyme activity
was determined.

5.2.6.7 Effect of chemicals on enzyme activity:

Preparation of metal solutions: All the metal salts were from SRL chemicals
LTD. The stock solutions of 100 mM of these metal salts were prepared. These
stock solutions were diluted appropriately to achieve three different molar
concentrations for each metal salt: 1mM, 5mM and 10mM concentrations as
follows:
1. Silver chloride (AgCl)
Molecular weight: 143.32 grams
For 100 mM, 1.43 grams was dissolved in 100 ml distilled water.
2. Calcium chloride (CaCl2):
3. Copper sulfate (CuSO4. 5H2O):
4. Ferric chloride (FeCl2):
5. Mercuric chloride (HgCl2)
6. Nickel chloride (NiCl2)
7. Magnesium sulfate (MgSO4)
8. Manganese chloride (MnCl2)

9. Barium chloride (BaCl2)

10. Zinc sulfate (ZnSO4)
11. PMSF (Phenyl Methyl Sulfonyl Flouride, C7H7FO2S)
For 100 mM, 1.74 grams was dissolved in 100 ml DMSO.
Enzyme assay: 0.5 ml of the enzyme and 0.5 ml of substrate were reacted at
optimum pH and temperature parameters for the respective enzymes. To the
enzyme-substrate reaction mixture, 1 ml of metal-ion solution was added, and the
incubation was carried out. For comparison, enzyme- substrate reaction was
carried out in the absence of any metal ion. The enzyme activity obtained at each
substrate concentration was determined and tabulated. From this data, a
Michaelis-Menton curve was plotted using the software GraphPad Prism 6, from
which the Vmax and Km values were determined for both the enzymes.
5.2.7 Studying feather degradation by combination of B. licheniformis and B.

subtilis:
Purpose of the experiment: On studying the feather degrading activity and the
characteristics of purified enzymes of B. licheniformis and B. subtilis, it was
determined that the keratinase of isolate B. licheniformis showed allosteric
properties and had a high Km value, whereas the keratinase of isolate B. subtilis
showed Michaelis-Menton type kinetics, bearing a lower Km value
comparatively. The current experiment was carried out to study the combined
feather degrading potential of both these isolates and compare with that of the
individual isolates.

Experiment design: In this experiment, the above two mentioned isolates were
cultivated in combination in MSM containing 1% feathers (table 5.7), and
incubation was carried out (at 33oC, shaker at 120 rpm) to allow feather
degradation to take place.
Simultaneously, individual isolates were also incubated at their respective
optimized conditions. The enzyme activity was assessed on the day of complete
feather degradation.
Table 5.7: Components of MSM for cultivation of combination of B.

licheniformis and B. subtilis
Components Quantity
NaCl (g/L) 5
K2HPO4 (g/L) 1
KH2PO4(g/L) 1
(NH)2SO4(g/L) 0.1
MgSO4(g/L) 0.2
Feathers(g/L) 10
Incubation temperature (oC) 33
Initial pH 7.5

5.3 RESULTS:
5.3.1 Protein Estimation by Folin-Lowry Method (Standard Assay):
The below table (Table 5.8) represents the absorbance values at 650 nm, obtained for the
standard concentrations of BSA (0-200 µg/ml). A standard graph (Figure 5.1) was
plotted, and the equation of the graph was used to calculate the protein concentrations of
the unknown samples for further experiments.
Table 5.8: Folin-Lowry Standard Assay for BSA
Concentration Reading 1 Reading 2 Reading 3 Average S.D
(µg/ml) (A650) (A650) (A650)
0 0 0 0 0.00 0.00
20 0.0606 0.0599 0.0624 0.06 0.00
40 0.1321 0.122 0.13 0.13 0.01
60 0.1925 0.1786 0.1856 0.19 0.01
80 0.2479 0.2326 0.2454 0.24 0.01
100 0.2899 0.2978 0.2905 0.29 0.00
120 0.3671 0.3241 0.3477 0.35 0.02
140 0.4325 0.4136 0.4079 0.42 0.01
160 0.4603 0.4763 0.475 0.47 0.01
180 0.5285 0.4804 0.5081 0.51 0.02
200 0.545 0.5213 0.5718 0.55 0.03
Figure 5.1: Standard graph of Folin-Lowry assay

5.3.1 Partial Purification and Dialysis:

An increase in specific activity was obtained after treating the crude enzyme for ammonium sulfate precipitation and
partial purification. The below table indicates the difference in specific activities observed at various saturation levels of
ammonium sulfate precipitation, while also comparing with that of the crude enzyme. The table 5.9 indicates that, at 90%
saturation level, maximum specific activity was obtained for isolates B. sonorensis and B. licheniformis, while for B. subtilis,
maximum specific activity was obtained at 50% saturation levels.
Table 5.9: Enzyme activity and Specific activity at different saturation levels of ammonium sulfate precipitation
Isolates Isolate B. sonorensis Isolate B. licheniformis Isolate B. subtilis

Enzyme Specific Enzyme Specific Enzyme Specific
Activity Activity Activity Activity Activity Activity
(U/ml/min) (U/mg) (U/ml/min) (U/mg) (U/ml/min) (U/mg)
Crude Enzyme 20.07+0.09 38.98+0.18 14.34+0.11 46.42+0.05 16.93+0.80 51.36+3.06
50% Saturation 84.51+1.14 123.27+1.67 88.86+1.41 112.64+1.79 143.99+1.04 189.17+0.48
70% Saturation 57.87+1.11 95.23+1.84 104.47+1.95 120.70+2.25 92.94+1.36 83.27+0.92
90% Saturation 92.05+1.7 119.07+2.54 126+1.65 259.19+2.83 63.17+0.50 104.11+1.08
(*Results are expressed as mean + S.D., n=3)

The results indicated that keratinases of B.licnehiformis and B. subtilis had higher specific activities as compared to that
of B. sonorensis. Thus, enzymes of B. licheniformis and B. subtilis were proceeded for further purification by Ion-exchange
chromatography.

5.3.2 DEAE- Cellulose Chromatography:

Purification by DEAE- Cellulose Chromatography resulted in further increase in enzyme
activity. The graph (figure 5.2 A) below represents the enzyme activity and the specific
activity for isolate B. licheniformis. On determining the Enzyme activity and the protein
content of each fraction, it was observed that the 14th and 15th fraction showed the
maximum enzyme activity, they were pooled and selected for studying enzyme
characteristics.
Figure 5.2 A: Elution profile of protein sample of B. licheniformis
40 180
Fraction 14: 222.28
Enzyme Activity units/ml/min
35 160
Fraction 15: 236.57
Protein Content (µg/ml)

30 140
120
25
100
20 Enzyme
80 Activity
15
60 Protein
10 40 content
5 20
0 0
0 10 20 30 40 50
Fraction Number
The graph (figure 5.2 B) below represents the enzyme activity and the specific activity
for isolate B. subtilis. On determining the Enzyme activity and the protein content of each
fraction, it was observed that the 11th and 12th fraction showed the maximum enzyme
activity, and they were pooled for studying enzyme characteristics.
Figure 5.2 B: Elution profile of protein fractions of B. subtilis
50 140
Enzyme Activity Units/ml/min
45 Fraction 11: 366.46

120
Protein content (µg/ml)
40 Fraction 12: 363.68

35 100
30 80
25 Enzyme
20 60 activity
15 40 Protein
10 Content
20
5
0 0
0 5 10 15 20 25
Fraction Number

Table 5.10 A and B are the purification tables, representing the enzyme activity and the specific activity at each stage of
purification for both isolates.
The enzyme was 4.93 times purified for isolate B. licheniformis, which resulted in increased specific activity. The enzyme of
B. subtilis was 7.11 times purified as indicated in the purification table.
Table 5.10 A: Purification table for the enzyme of isolate B. licheniformis
Stages of purification Total Total Enzyme Total Protein Specific Purification %Yield
volume (ml) Activity (U) (mg) Activity (U/mg) Fold
Crude 100 1434.4 30.9 46.42 1 100

(NH4)SO4Precipitation 10 1260 6.01 209.65 4.51 87.84
DEAE- Cellulose 6 211.47 0.924 228.86 4.93 14.74
Table 5.10 B: Purification table for the enzyme of isolate B. subtilis
Stages of purification Total Total Enzyme Total Protein Specific Purification %Yield
volume (ml) Activity (U) (mg) Activity (U/mg) Fold
Crude 100 1693.4 32.96 51.37 1 100
(NH4)SO4Precipitation 10 1432.62 7.58 189.23 3.68 84.60
DEAE- Cellulose 6 247.02 0.676 365.41 7.11 19.09

5.3.3 Study of Enzyme Characteristics:
5.3.3.1 Determination of Molecular Weight: SDS-PAGE chromatography:
The below images (Figures 5.3 A and B) represents the progress of enzyme
purification (from crude enzyme to purified enzyme) in terms of SDS-PAGE
chromatography for the keratinases of B. licheniformis and B. subtilis. The molecular
weight of the purified keratinase was determined to be 25 kDa for isolate B.
licheniformis and 45 kDa for B. subtilis, which indicated that the kerarinases were
different for both the isolates.
1 2 3 4
250
130
100
70
55
35
25 25 kDa
15
Fig 5.3 A: Electrophoresis pattern of active fraction obtained from DEAE-Cellulose

chromatography of protein sample of Isolate B. licheniformis (lane 4), compared
with crude protein extract (lane 2), ammonium sulfate fraction (lane 3) and column
fraction subjected (lane 4)

1 2 3 4
250
130
100
70
55 45
kDa
35
15
10
Fig 5.3 B: Electrophoresis pattern of active fraction obtained from DEAE-Cellulose

chromatography of protein sample of Isolate B. subtilis (lane 4), compared with
crude protein extract (lane 2), ammonium sulfate fractions (lane 3) and pre-stained
PAGE ruler (lane 1)

5.3.3.2 Effect of pH on enzyme activity:

5.3.3.2.1 Optimum pH for enzyme activity:
The below table indicates the enzyme activities obtained at various pH values for isolates
B. licheniformis and B. subtilis. On assessing the enzyme activity at different pH values,
it was observed that the enzyme of isolate B. licheniformis was best active within the pH
range of 7.5 to 8.0. The graph (Figure 5.4 A) represents the enzyme activities observed at
different pH values for B. licheniformis. For B. subtilis, maximum enzyme activity was
obtained at pH 8.0. The below graph (Figure 5.4 B) represents the enzyme activities
obtained at pH 8.0.
Table 5.11: Effect of pH on Figure 5.4 A: Effect of pH on enzyme

Enzyme activity of purified activity of B. licheniformis.
enzyme of isolates B.
100
licheniformis and B. subtilis
Enzyme units/ml/min
pH Enzyme Enzyme 80
Activity B. Activity B. 60
licheniformis subtilis 40
(U/ml/min) (U/ml/min)
20
5.0 8.746+2.01 5.48+0.42
0
4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10
5.5 22.76+0.18 20.93+0.58 pH values
6.0 52.5+2.70 31.54+1.04 Figure 5.4 B: Effect of pH on enzyme

6.5 60.14+3.72 59.34+3.30 activity of B. subtilis.
7.0 60.3+2.98 87.53+3.04

140
7.5 73.04+1.87 87.82+2.39 120
Enzyme Units/ml/min
100
8.0 86.20+1.73 127.03+2.2
80
8.5 28.21+2.74 70.65+2.28 60
9.0 33.15+3.72 39.23+3.64 40

20
9.5 28.17+1.49 24.56+3.06 0
4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10
pH values
*Results are represented as Mean + S.D., where n=3. P values<0.05 were considered
significant.

5.3.3.2.2: Enzyme stability at varied pH:

Table 5.12 represents the residual enzyme activities of the purified enzymes of isolates B.
licheniformis and B. subtilis, obtained after incubating the enzymes at various pH.
The enzyme of isolate B. licheniformis was stable in the pH range of 7.5 to 8.0, as
represented in the below graph (figure 5.5 A). The enzyme of isolate B. subtilis was
stable within the pH range of 7.0 to 8.0, as represented in the below graph (Figure 5.5 B).
The enzymes were capable of retaining up to 41% and 48% activity at pH 9, and upto
45% activity at pH 6.0, after one hour of incubation.
Table 5.12: Enzyme stability at
Figure 5.5 A: Stability at varied pH, B.
varied pH of purified enzymes of
licheniformis.
B. licheniformis and B. subtilis
pH Residual Residual
120
Enzyme Enzyme 100
Relative Activity (%)
Activity (%), Activity (%), 80

B. B. subtilis 60
licheniformis 40
5.0 13.19+ 0.91 25.87+ 0.83 20
5.5 14.35+0.51 29.37+0.21 0

4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10
6.0 45.11+1.31 44.55+1.10 pH values
6.5 75.60+2.61 63.73+1.20 Figure 5.5 B: stability at varied pH, B.

7.0 88.60+1.54 94.11+1.72 subtilis.
7.5 96.96+1.52 98.57+2.32 120

100
8.0 100 100
Relative Enzyme Activity (%)
80
8.5 53.94+0.45 81.95+0.99
60
9.0 41.01+0.75 48.02+0.25 40
9.5 46.35+1.21 35.57+2.83 20
0
4.5 5.5 6.5 7.5 8.5 9.5 10.5
pH values
significant.

5.3.3.3 Effect of temperature on enzyme activity:

5.3.3.3.1 Determination of optimum temperature for enzyme activity
Table 5.13 represents the enzyme activities obtained by carrying out the enzyme assay at
varied temperatures ranging from 30 to 60 oC. On assessing the enzyme activities at
various temperatures, increased enzyme activity was observed in the range of 40 to 50oC,
for isolate B. licheniformis, whereas for B. subtilis, it was 45 to 50oC. The below graphs
(Figure 5.6 A and B) represent the enzyme activities observed at various temperatures for
the purified enzyme of both the isolates.
Table 5.13: Effect of temperature on Figure 5.6 A: Effect of temperature on

enzyme activity of isolate B. licheniformis.
enzyme activity of purified enzymes
of B. licheniformis and B. subtilis.
Temperatur Enzyme Enzyme
125
o
e ( C) Activity of B. Activity B.
Enzyme Units/ml/min
105
licheniformis subtilis
85
(U/ml/min) (U/ml/min)
65
30 53.17+3.51 17.42+2.84
45
33 49.98+3.21 18.34+1.6
25
35 65.58+1.47 55.45+2.99 25 30 35 40 45 50 55 60 65
Temperature (degree Celcius)
37 76.72+3.34 60.73+0.8
Figure 5.6 B: Effect of temperature on
40 99.2+2.35 75.07+1.53 enzyme activity of isolate B. subtilis.
45 109.99+1.65 93.48+3.81
120
50 102.78+1.86 100.42+3.4
Enzyme Units/ml/min
100
55 70.58+1.46 72.82+1.07 80
60 52.43+1.35 66.2+1.36 60
40
20
0
25 35 45 55 65
Temperature (degree Celcius)
significant.

5.3.3.3.2 Thermostability of the enzymes:

Table 5.14 represents the residual enzyme activities of the purified enzymes of isolates B.
licheniformis and B. subtilis after incubating the enzymes at various temperatures for one
hour. The below graphs (figure 5.7 A and B) represent the stability of the enzyme at
different temperatures. The enzyme was found to be stable within the temperature range
of 30 to 50oC.
Table 5.14: Thermostability of Figure 5.7 A: Thermostability of enzyme of

purified enzymes of B. licheniformis isolate B. licheniformis.
and B. subtilis at varied temperatures

Temperature Residual Residual 120
(oC) 100
Residual Activity (%)
Activity (%), Activity
B. (%), 80
licheniformis B. subtilis 60
40
30 78.21+0.28 86.31+0.13
20
35 80.22+0.32 89.01+0.31 0
30 40 50 60 70
40 100 89.08+0.04 Temperature (degree C)
45 98.85+0.20 100 Figure 5.7 B: Thermostability of enzyme of

isolate B. subtilis.
50 92.88+2.08 94.55+0.16
120
55 92.2+0.16 53.97+0.25
100
Residual Activity (%)
60 29.7+0.23 34.56+1.60
80
60
40
20
0
30 35 40 45 50 55 60 65
Temperature (Degree C)
significant.

5.3.3.4 Effect of substrate concentration on enzyme activity:

The enzyme activity was assessed by incubating the enzyme with different concentrations
of substrate at optimum pH and temperature. Table 5.15 represents the represents the
enzyme activities at different substrate concentrations for isolate B. licheniformis.
Figures 5.8 A and B show the Allosteric plot and Lineweaver Burk plot of the data along
with the Vmax and the Km values. Vmax was determined to be 157.9 U/ml/min and Km
was determined to be 139.6 µg/ml.
Table 5.15: Enzyme Activity at Figure 5.8 A: Allosteric plot of

different substrate concentrations purified enzyme of B. licheniformis
for B. licheniformis
Substrate Enzyme Activity
Concentration (Units/ml/min)
(µg/ml)
0 0+0
50 2.00+0.12
100 65.41+0.30
175 98.36+1.21
190 111.44+2.08
215 105.62+0.97
Figure 5.7 B: Lineweaver Burk plot of
230 104.80+1.21 purified enzyme of isolate B.
licheniformis
245 107.46+0.27
260 108.32+1.12
300 104.99+3.004
350 104.56+1.054
400 107.66+0.103
450 107.10+0.47
significant.

Table 5.16 shows the enzyme activities at different substrate concentrations for purified
enzyme of isolate B. subtilis. Figures 5.9 A and B represent the Michaelis-Menton curve
and the Lineweaver Burk plot of the same, along with the Vmax and the Km values.
Vmax was determined to be 148.2 Units/ml/min and Km was determined to be
78.51µg/ml.
Table 5.16: Enzyme Activity at Figure 5.9 A: Michaelis -Menton plot

different substrate concentrations of purified enzyme of B. subtilis
for isolate B. subtilis
Substrate Enzyme Activity
Concentration (Units/ml/min)
(µg/ml)
0 0+0
50 37.55+0.041
100 79.16+0.81
175 106.48+0.64
190 120.05+1.11
215 120.24+0.09 Figure 5.9 B: Lineweaver Burk plot
230 118.36+2.47 of purified enzyme of B. subtilis
245 116.85+0.68
260 117.60+2.31
300 117.45+1.12
350 117.20+2.01
400 116.66+1.71
450 117.27+0.36
500 118.07+0.38
significant.

5.3.3.5 Effect of chemicals on enzyme activity

Metal ions such as Ag, Ca, Cu, Fe, Hg, Ni, Mg, Mn, Ba, Zn and Phenyl Methyl
Sulfonyl Flouride (PMSF) were assessed for their effect on enzyme activity. Table
5.17 represents the Inhibition/activation of enzyme of isolate B. licheniformis,
expressed as Relative Activity (%). Figure 5.10 A shows the graphical representation
of the same. The enzyme was activated by Mg ion.
Table 5.17 A: Effect of Metal ions on Enzyme Activity of B. licheniformis
Metal Relative Activity (%) at Relative Activity (%) at Relative Activity (%)
ion conc 1 mM conc 5 mM at conc 5 mM
None 100 100 100
Ag 48.73 40.66 30.33
Ca 59.14 53.56 42.77
Cu 26.29 24.25 17.76
Fe 30.52 22.40 14.11
Hg 36.28 24.22 17.63
Ni 57.52 42.37 36.74
Mg 94.46 100.83 102.96
Mn 30.12 39.14 30.14
Ba 38.77 31.64 27.56
Zn 33.62 27.85 17.84
PMSF 21.41 17.29 14.51
Figure 5.10 A: Effect of Metal ions on Enzyme Activity of B. licheniformis
120
100
80
Conc 1 mM
60
Conc 5 mM
40
Conc 10 mM
20
0
None Ag Ca Cu Fe Hg Ni Mg Mn Ba Zn PMSF
Metal ions
significant.

Table 5.17 B represents the inhibition/activation of enzyme of isolate B. subtilis in terms

of relative activity (%). Figure 5.10 B is a graphical representation of the same. The
enzyme was activated by Mg and Ca, and inhibited by other chemicals.
Table 5.17 B: Effect of metal ions on Enzyme Activity of B. subtilis
Relative Activity (%) Relative Activity (%) Relative Activity (%)

Metal ion at conc. 1mM at conc. 5 mM at conc. 5 mM
None 100 100 100
Ag 22.24 11.74 9.45
Ca 82.63 94.82 106.90
Cu 66.72 69.42 65.19
Fe 39.99 35.18 30.25
Hg 34.71 27.64 24.66
Ni 31.61 22.76 17.82
Mg 98.35 106.83 114.72
Mn 60.94 47.70 43.33
Ba 48.51 38.07 32.82
Zn 39.09 32.99 19.80
PMSF 14.86 14.1 12.09
Figure 5.10 B: Effect of metal ions on Enzyme Activity of B. subtilis

120
100
80
Conc 1 mM
60
Conc 5 mM
40
Conc 10 mM
20
0
none Ag Ca Cu Fe Hg Ni Mg Mn Ba Zn PMSF
Metal ions
significant.

5.3.4. Studying feather degradation by combination of B. licheniformis and B.

subtilis:
On studying combined feather degrading activity of B. licheniformis and B.
subtilis, it resulted in faster feather degradation as compared to the individual isolates.
While individual cultures took about 5-7 days for complete degradation, the combination
carried out complete degradation by 3 days. Also, combination of the two isolates had
increased enzyme activity than the individual isolates.
Figure 5.11 shows the comparative feather degradation by the combination
culture and individual isolates. Table 5.18 represents the enzyme activities of the
combination culture and individual isolates.
Figure 5.11: Comparative feather degrading activity of A) Isolate B. licheniformis B)

combination culture and C) Isolate B. subtilis on the third day after inoculation.
Complete degradation of 1% feathers was achieved on the third day after
inoculation for the combination culture, whereas, for the individual cultures, it was
achieved in 5-6 days.
A B C

Table 5.18: Comparative enzyme activities of individual isolates v/s combination

culture of B. licheniformis and B. subtilis.
Culture Average Enzyme Activity

(on day 3) (Units/ml/min)
B. licheniformis 14.97 + 0.37
B. subtilis 16.18 + 0.87
Combination of B. licheniformis 33.68 + 0.95

and B. subtilis
significant.

5.4 DISCUSSION:
5.4.1 Enzyme Purification:
Purification of the crude keratinase contained in the cell free supernatant of the
culture medium by ammonium sulfate precipitation and ion-exchange chromatography
resulted in increase in enzyme activity, which reflected in purification fold of 4.93 and
7.11 times, respectively for isolates B. licheniformis and B. subtilis. Purification steps
therefore resulted in elimination of interfering materials present in the crude cell- free
extract thereby resulting in increased enzyme activity. A higher level of purification can
be achieved by Gel- filtration chromatography (Suntornsuk W et al., 2005).
The keratinase from B. licheniformis was determined to be a 25 kDa monomer,
whereas, the keratinase from B. subtilis was determined to be a 45 kDa monomer.
Overall, the current findings are in agreement with the previous reports about bacterial
keratinases having molecular weight ranging from 18 kDa to 240 kDa (Adriano
Brandelli, 2008). However, the results differ from earlier reports about certain
keratinases; for example, the serine protease B. licheniformis PWD- 1 has a molecular
weight of 33 kDa. (Lin et al., 1992). Also, another study on B. subtilis, has reported a 25
kDa keratinase (Suh and Lee, 2001). This indicates that the purified monomeric
keratinases from both the isolates in the current study would be different from the
previously reported keratinases of the same species.
5.4.2 pH and temperature optima:

Most bacterial keratinases are active over a pH range of 7.0 to 10 (Lin et al.,
1992; Nam et al., 2002; Gessesse et al., 2003; Bernal et al., 2006; Tatineni et al., 2008;
Cai et al., 2008; Brandelli, 2008; Ionata et al., 2008; Prakash et al., 2010). Certain
keratinases are active at alkaline pH, for example keratinase of Nocardiopsis spp., and
that of B. halodurans (Takami et al., 1999, Mitsuiki et al., 2004), while keratinase from
B. subtilis has maximum catalytic potential at of pH 6.0. In the current study, the
enzymes display optimum catalytic activity at pH 8.0. Keratinases are stable over a pH
range of 7.0- 10.0., except for keratinase of Nocardiopsis spp., which is stable over a
remarkable pH range i.e. pH 1.5- 12.0. The keratinases isolated from B. licheniformis
and B. subtilis, show maximum stability in the pH range of 7 to 8, for one hour, by

retaining more than 80% catalytic activity. This data is in accordance with previous
studies on keratinases belonging to Bacillus spp., having the similar pH range for stability
(Brandelli, 2008, Łaba and Rodziewicz, 2009).
Most keratinases possess optimum activity within the temperature range of 30 to
70oC (De Toni et al., 2002; Cai et al., 2008; Brandelli, 2008; Rajput et al., 2010), with
the exception of keratinase isolated from Fervidobacterium islandicum AW-1, which
shows maximum keratinolytic activity at 100oC (Nam et al., 2002). Moreover,
keratinases from most bacteria belonging to the Bacillus spp. have an optimum catalytic
temperature above 45oC (Cai et al., 2008, Lin et al., 1992, Gessesse et al., 2003). In the
current study, the keratinases had temperature optima of 45 to 50oC. The keratinase of
isolate B. licheniformis retained maximum activity within 30 to 55oC, and the keratinase
of B. subtilis could retain activity up to 86% within 30 to 50oC. It was observed that the
keratinase of B. licheniformis showed a remarkable stability by retaining upto 92.2%
activity at 55oC, whereas the keratinase of B. subtilis showed stability upto 50oC by
retaining 94.55% activity, beyond which, the activity retained was 53.9%.
5.4.3 Effect of Substrate Concentration:

On studying the enzyme activity response of keratinases to varied concentrations
of keratin substrate, it was observed that the activity response of keratinase of isolate
Bacillus licheniformis was sigmoidal, i.e. low level of substrate concentrations were not
sufficient for activating the enzyme, thus resulting in low levels of keratinase activity. In
other words, higher concentration of substrate was required for raising the enzyme
activity. The enzyme activities obtained at varied substrate concentrations represented a
sigmoidal curve, typical of an allosteric enzyme. Also, the enzyme had a high Km value.
(Lehninger Nelson and Cox, 2004). In case of B. subtilis, the activity response was a
Michaelis-Menton Curve, which is indicated by a steep increase in enzyme activity at
lower substrate concentrations, thus characterized by a low Km.
5.4.4 Effect of Chemicals:

In the study of assessing the effect on metal ions on enzyme activity, it was found
that divalent metal ions Calcium and Magnesium had a stimulatory effect on the enzyme

activity. Also manganese had a marginal stimulatory effect at lower concentrations, while
other metal ions had an inhibitory effect. These findings are in accordance with previous
reports which have indicated that heavy metal ions such as Cu2+ (Nam et al., 2002; Riffel
et al., 2003; Thys et al., 2004), Hg2+ (Riffel et al., 2003; Thys et al., 2004) and Zn2+
(Thys et al., 2004) have inhibitory effects on keratinolytic activity. There are also reports
about stimulation of keratinase by Ca2+, Mg2+ and Mn2+ (Nam et al., 2002; Riffel et al.,
2003), which support the current study. Both keratinases, being inhibited by PMSF,
indicated that they belonged to the serine protease group. It has been reported that
keratinases from Gram positive bacteria are serine proteases, whereas those from the
Gram negative bacteria are metalloproteases (Brandelli, 2008).
5.4.5 Feather degrading activity of combination of B. licheniformis and B. subtilis:

The combination of isolates B. licheniformis and B. subtilis resulted in rapid and
efficient degradation of feathers. The combination of the two isolates, results in the
complementation of two kinetically different keratinases: allosteric type from B. subtilis
and Michaelis-Menton type from B. subtilis. This resulted in a faster utilization of
substrate, resulting in rapid breakdown of feathers, bringing about a complete
degradation of feathers within 3 days of inoculation, Thus, the mixed culture of B.
licheniformis and B. subtilis, provides an efficient alternative to bring about microbial
degradation of feathers.

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Chapter Five: Enzyme Purification and Study of Enzyme Characteristics

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CHAPTER FIVE:

ENZYME PURIFICATION AND STUDY

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2. Chromatographic methods: Depending upon the net charge of the

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5.1.5 Study of Enzyme Characteristics (Nelson DL and Cox MM, 2004):

The current chapter elucidates the process of purification of keratinase

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5.2 MATERIALS AND METHODS:

Isolate Isolate Isolate Isolate

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5.2.2 Partial purification using ammonium sulfate precipitation (Jayaraman J,

5.2.2.2 Ammonium Sulfate precipitation: Ammonium sulfate precipitation was carried

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Table 5.2: Ammonium sulfate precipitation chart (Jayraman J, 2003)

Final concentration of Ammonium Sulfate (% saturation)

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5.2.3 Dialysis of the salt-precipitated protein:

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5.2.4 Protein estimation by Folin-Lowry assay (Lowry et al., 1951) :

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 Stock solution: Standard Bovine Serum Albumin (BSA)

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Protocol for Lowry assay:

5.2.5 Purification by Ion-Exchange Chromatography (Jayaraman, 2011):

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concentration and presence of chemicals. For assessing the stability of the

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5. Ammonium Persulfate solution (10%):

6. 10% Resolving gel:

30% acrylamide mix 10

10% SDS 0.3

10% ammonium persulfate 0.3

8. Water saturated butanol solution:

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9. 4 X SDS- PAGE loading buffer:

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6.6 ml of solution B and 4.34 ml of solution A were mixed to obtain 50 mM

5.2.6.3 Enzyme stability at varying pH:

5.2.6.4 Determination of optimum temperature for enzyme assay:

5.2.6.5 Enzyme stability at varying temperatures:

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5.2.6.6 Effect of varying substrate concentrations on enzyme activity:

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5.2.6.7 Effect of chemicals on enzyme activity:

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9. Barium chloride (BaCl2)

5.2.7 Studying feather degradation by combination of B. licheniformis and B.

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Table 5.7: Components of MSM for cultivation of combination of B.

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Figure 5.1: Standard graph of Folin-Lowry assay

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5.3.1 Partial Purification and Dialysis:

Isolates Isolate B. sonorensis Isolate B. licheniformis Isolate B. subtilis