CHAPTER 4

RESULTS AND DISCUSSION

In this study we report the isolation of three mesophilic bacteria that produce proteolytic

and keratinolytic enzymes, which can efficiently degrade chicken and pigeon feather. The three

isolate SN1, SN2, SN3 was identified as a strain of Bacillus megateruim, Bacillus thuringienesis

and Bacillus pumilis respectively based on Morphological, Cultural and Biochemical

characteristics and chromogenic method by growth on Hicrome bacillus agar. Earlier studies

from our lab involving screening of micro-organism from same soil sample of dumping site of

Ghazipur poultry processing plant, we have reported isolation of Pseudomonas thermaerum

GW1, GenBank accession GU95151, this bacteria showed proteolytic activity but not

keratinolytic activity [48].

117

4.1 Protease and Keratinase isolated from microbial sources from soil

collected from feather dumping site at Ghazipur poultry waste site,

Ghaziabad

118

4.1.1 Screening of keratinolytic bacteria

It was found that the enriched feather degrading culture contained micro-organism exhibited

keratinolytic activity. The feathers were fully degraded within 120 hrs of incubation with the

microbes from selected soil (Fig. 4.1). Three bacterial strains that visually degraded feather were

isolated when allowed to grow on medium containing feather meal powder as sole carbon and

nitrogen source. The strain SN1, SN2, SN3 were selected as they produced clear zones on

incubation at 30ºC for 72hrs suggesting the presence of keratinolytic activity (Fig. 4.2).

119

A B

Figure 4.1: Degradation of chicken feathers by the bacterial strain isolated from soil of Ghazipur poultry dumping

site, India, in submerged cultivation at 30°C. (A) Feather control without the bacterial strain, (B) feather after 120

hrs of incubation with the bacterial strain showed complete degradation.

120

SN1

SN2

SN3

Figure 4.2: Production of clear zones in feather meal (10g/L) agar plates by keratinolytic bacteria. Three strains

were identified Bacillus sp. SN1, Bacillus sp. SN2, and Bacillus sp. SN3 that produced clear zone. Microbial

culture was spreaded on feather meal agar plate and incubated at 30°C for 72 hr.

121

4.1.2 Isolation, identification and characterization of keratinolytic strains

The identification of the keratinolytic bacteria was based on cell morphology, colony

morphology and several biochemical tests (Table 4.1a). Isolates SN1, SN2, SN3 were

determined to be Gram-positive, sporulating, motile bacillus. The isolate SN1, SN2 formed

yellow colored colonies and SN3 showed white colored colony on feather meal agar plate. These

results suggested that these three strains belong to genus Bacillus (Table 4.1b, 4.1c). On the basis

of morphological characteristic and cultural characteristic of Hicrome Bacillus agar and was

identified (Table 4.2). They (SN1, SN2, and SN3) were further identified to be as sample B.

megaterium, B. thuringenesis, B. pumilis respectively (Fig. 4.3). These strains degraded the

chicken feathers and pigeon feathers completely. Their cultural characteristic in the Hicrome

bacillus agar is given in table 4.2 with B. megaterium SN1 showing yellowish green, irregular

colonies, B. thuringenesis SN2 showed blue circular colonies, B. pumilis SN3 showed green, flat,

circular shiny colonies.

122

Figure 4.3: Growth of bacterial strain SN1, SN2, SN3 on chromogenic differentiation agar Hicrome bacillus agar

from Himedia M1651. Yellowish green colonies represent B. megaterium SN1; Blue colonies represent

B. thuringenesis SN2; Green colored colonies represent B. pumilis SN3.

123

Table 4.1a: Results of morphological, physiological, Cultural Characteristic of three isolated bacterial strain SN1,

SN2, SN3 were conducted.

Observations Details of experiment

B. megaterium SN1 B. thuringenesis SN2 B. pumilis SN3

Shape of Bacteria Rod Short rod Rod

Endospore formation + + +

Motility Motile Highly motile Motile

Gram character + + +

Anaerobic growth _ _ _

Colony Characteristics

Growth Rapid Rapid Rapid

Shape Circular Irregular Circular

Surface Smooth shiny Smooth Smooth shiny

Margin Entire Entire Entire

Color Cream White Cream

Elevation Convex Flat Convex

Consistency Buttery Viscous Buttery

Opacity Opaque Opaque Opaque

124

Table 4.1b: Table depicts the Biochemical characteristic of three isolated bacterial strain SN1, SN2, SN3 were

conducted. Collectively these characteristics indicated that the isolates were of genus Bacillus.

Biochemical Characterstics B. megaterium SN1 B. thuringenesis SN2 B. pumilis SN3 Glucose -/- A/- -/-

Lactose -/- A/- -/-

Mannitol -/- A/- -/-

Indole production _ _ _

Methyl red reaction _ + +

Voges-proskaure reaction _ _ _

Citrate utilization _ _ _

Catalase + + +

Gelatinase _ + +

Caesinase + + +

Amylase _ _ _

Cellulase _ _ _

Deaminase _ _ _

Symbol: +: Positive; -: Negative; A/- :Acid/No gas; -/- :No acid/No gas

Table 4.1c: Table depicts that growth on Triple sugar iron (TSI) agar. Bacillus sp. SN1, Bacillus sp. SN3 showed

alkaline reaction on slant and acidic reaction on butt. Bacillus sp. SN2 showed acidic reaction both on slant and

butt.

Bacterial Isolates Color and reaction of slant

Color and reaction of

butt

Gas production

Hydrogen sulphide

production

B. megaterium SN1 Red, Alkaline Yellow, Acidic _ _

B. thuringenesis SN2 Yellow, Acidic Yellow, Acidic _ _

B. pumilis SN3 Red, Alkaline Yellow, Acidic _ _

125

Table 4.2: Cultural characteristics on Hicrome bacillus agar

Colony Characteristics B. megaterium SN1 B. thuringenesis SN2 B. pumilis SN3

Growth Luxuriant Luxuriant Luxuriant

Shape Irregular, large Circular, Small Circular, Small

Surface Smooth Smooth Smooth shiny

Margin Irregular Entire Entire

Color Yellowish green Blue Green

Elevation Convex Flat Flat

Opacity Opaque Opaque Opaque

Morphological characteristic and cultural characteristic of the three isolates on Hicrome Bacillus

agar Himedia M1651. Strains SN1, SN2, SN3 were identified as B. megaterium, B.

thuringenesis, B. pumilis respectively. (Fig 4.3)

126

4.1.3 Factors affecting growth and enzyme production

Bacterial growth and enzyme production of keratinase and protease by the microbes of soil

sample was monitored during growth in enrichment media - Horikoshi media, Feather meal

media1, and feather meal media 2. Growth determination of three bacteria with was done taking

absorbance at 600nm of bacterial growth media at regular intervals from 0 hrs -144 hrs (Fig. 4.4)

log phase of growth is from 24hrs till 48 hrs for all the strains and in stationary phase till 120 hrs.

127

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 24 48 72 96 120 144Time (hr)

Abs

orba

nce

at 6

00nm

Bacillus SN1

Bacillus SN2

Bacillus SN3

Bacilluslicheniformis

Figure 4.4: Growth determination of bacterial strains B. megaterium SN1, B. thuringenesis SN2, B. pumilis

SN3, Standard strain B. licheniformis 1483 (Collected from MTCC was used for comparative study) were done

taking absorbance at 600nm of bacterial growth media. It is observed that Log phase is from 24 hours till 48

hours and stationary phase is till 120 hours for all the strains.

128

4.1.4 Selection of media

The maximum protease activity of mixed microbial culture was seen at 120 hrs (5th day) of

incubation of in feather meal media 2 (Fig 4.5) and the maximum keratinase activity of mixed

microbial culture was seen on the 144 hrs (6th day) of incubation in feather meal media 2 (Fig.

4.6). Relatively the microbial culture could produce 2.4 times more protease in feather meal

media 2 on incubation and 2.53 times more keratinase in Feather meal media 2 than in peptone

broth (Table 4.3 & 4.4). Comparison was made standard strain of B. licheniformis (MTCC

1483).

Ammonium sulphate fraction of the crude cell free supernatant which was seeded with the soil

inoculum showed 1.5 fold purification for keratinase and 1.3 fold purification for Proteases.

Microbes were isolated from a poultry processing plant that showed keratinolytic activity and

ability to degrade chicken and pigeon feathers.

129

0

10

20

30

40

50

60

1 2 3 4 5 6 7 8

Time (Days)

Prot

ease

act

ivity

(U/m

l)

Horikoshi mediaFeather M eal media 1Feather M eal media 2

Figure 4.5: Protease activity produced by feather degrading bacterial strains of feather dumping site, activity was measured by a modified method of Tsuchida et al., 1986 by using casein as substrate. One protease unit is defined as the amount of enzyme that releases 1 µmol of tyrosine per ml per minute under the above assay conditions. The

specific activity is expressed in the units of enzyme activity per milligram of protein. In different culture media Horikoshi media, Feather meal media 1, Feather meal media 2. Maximum protease units 54 U/ml were seen in

feather meal media 2.

130

Table 4.3: Purification table that compares the yield, specific activity of protease from micro-organisms grown in

Peptone broth and the enriched media (Horikoshi media, Feather meal media 1 and Feather meal media 2) Enzyme

assay was determined as mentioned in materials and methods. The Standard strain B. licheniformis (Crude and 0-

80% Ammonium sulphate) was kept as control

Purification step Protein (mg)

Total activity(Uª)

Specific activity (U/mg)

Yield (%)

Purification (Fold)

Peptone broth 138.75 1095250 7893.69 100 1

Horikoshi media (0-80% A.S.) 217.5 1194075 5503 1.090 .697

Feather meal media 1 (0-80% A.S.) 63 51770 1297 0.0746 0.1644

Feather meal media 2 (0-80% A.S.) 279.5 2850750 10199 2.6028 1.292

Crude (B.lic) 137.95 782177.5 5670.05 100 1

B.lic (0-80% A.S.) 176.25 509362 2890.81 0.6512 0.5098

131

0

0.2

0.4

0.6

0.8

1

1.2

1 2 3 4 5 6 7 8

Time (days)

Ker

atin

ase

activ

ity (U

/ml)

Horikoshi media

Feather M eal media 1

Feather M eal media 2

Figure 4.6: Keratinase activity produced by feather degrading bacterial strains dumping site, activity was measured

by a modified method of Cheng et.al., 1995 by using keratin as a substrate. One unit (U) of enzyme activity was the

amount of enzyme that caused a change of absorbance of 0.01 at 440 nm in 20 min at 45ºC, in different culture

media Horikoshi media, Feather meal media 1, Feather meal media 2. Maximum activity 1.2 U/ml was produced in

feather meal media 2.

132

Table 4.4: Purification table that compares the yield, specific activity of keratinase from micro-organisms grown in

Peptone broth and the enriched media (Horikoshi media, Feather meal media 1 and Feather meal media 2) The

Standard strain B. licheniformis (Crude and 0-80% Ammonium sulphate) was kept as control. Enzyme assay was

determined as mentioned in materials and methods

Purification step Protein (mg)

Total activity (Uª)

Specific activity (U/mg)

Yield (%)

Purification (Fold)

Peptone broth 138.75 11,500 82.88 100 1

Horikoshi media (0-80% A.S.) 217.5 23,900 110.138 207.82 1.328

Feather meal media 1 (0-80% A.S.) 63 4,650 73.80 40.80 0.890

Feather meal media 2 (0-80% A.S.) 279.5 33,900 121.28 294.78 1.463

Crude (B.lic ) 137.95 9,050 65.61 100 1

B.lic (0-80% A.S.) 176.25 18,500 104.96 204.41 1.596

133

4.1.5 Hydrolysis of protein substrates

Hydrolysis of protein substrates (Keratin, Casein, Gelatin, Bovine Serum Albumin- 2mg/ml) by

cell free supernatant collected after 120 hrs of incubation with bacterial stains B. megaterium

SN1, B. thuringenesis SN2, B. pumilis SN3, Standard strain B. licheniformis 1483 (collected

from MTCC was used for comparative study). All strains preferred keratin and casein as

preferred substrate. Gelatin was least preferred source as substrate. Maximum utilization of

keratin and casein was by strain SN2 as seen by the absorbance. (Fig. 4.7)

134

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

B.SN1 B.SN2 B.SN3 B.lic.Bacterial Isolates

abso

rban

ce a

t 660

nm

KeratinCaseinGelatinBSA

Figure 4.7: Hydrolysis of protein substrates (Keratin,Casein,Gelatin,Bovine Serum Albumin 2mg/ml ) by cell

free supernatant collected after 120 hrs of bacterial growth {strains identified were B. megaterium SN1, B.

thuringenesis SN2, B. pumilis SN3, Standard strain of B. licheniformis 1483 (collected from MTCC was used

for comparative study)}.All strains preferred Keratin and casein as preferred substrate Gelatin was least

preferred source as substrate . Maximum utilization of keratin and casein was by strain SN2 as seen by the

absorbance.

135

4.2 Milk clotting activity (MCA) by isolated bacterial strains

136

4.2.1 Protease and Keratinase activity

Protease and keratinase activity of all three isolated bacterial strains and standard Bacillus

licheniformis strain was checked at regular interval. Maximum protease activity of B.

megaterium SN1, B. pumilis SN3 was found at 96hr and B. thuringenesis SN2 at 72 hr then

Bacillus licheniformis at 144 hr (Fig. 4.8). Whereas maximum keratinase activity of B. pumilis

SN3, Bacillus licheniformis was found at 72 hr, B. thuringenesis SN2 at 96 hr, B. megaterium

SN1 at 120 hr (Fig. 4.9).

137

0

10

20

30

40

50

60

70

24 48 72 96 120 144 168Time (Hr)

Prot

ease

act

ivity

(U/m

l)

Isolate SN1 Isolate SN2Isolate SN3B.lic.

Figure 4.8: Protease activity of all three isolated bacterial strains and standard Bacillus licheniformis strain

was checked at regular interval

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

24 48 72 96 120 144Time (Hr)

Ker

atin

ase

activ

ity (U

/ml)

Isolate SN1Isolate SN2Isolate SN3B.lic.

Figure 4.9: Keratinase activity of all three isolated bacterial strains and standard Bacillus licheniformis

strain was checked at regular interval

138

4.2.2 Milk clotting activity

The result in Table 4.5 summarizes all bacterial isolate and their ammonium sulphate fractions

have caesinolytic activity. Milk clotting activity was seen in crude and ammonium sulphate

fraction of B. megaterium SN1, B. thuringenesis SN2, B. pumilis SN3 except crude and

ammonium sulphate fraction of standard strain Bacillus licheniformis (Fig. 4.10). Interestingly

highest ratio of milk clotting activity (with CaCl2 and MnSO4) to caesinolytic activity in B.

megaterium SN1 (30-60% ammonium sulphate fraction) followed by B. megaterium SN1 (0-30%

ammonium sulphate fraction), B. megaterium SN1 (60-90% ammonium sulphate fraction), B.

pumilis SN3 (0-80% ammonium sulphate fraction), B. thuringenesis SN2 (0-80% ammonium

sulphate fraction). 30-60% ammonium sulphate fraction of B. megaterium SN1 showed highest

milk clotting activity while 0-80% ammonium sulphate fraction of B. pumilis SN3 showed

highest caesinolytic activity. It is not clear whether the coagulation of skimmed milk by bacterial

source of enzyme is due to single action of enzyme or the synergistic action of many enzymes.

But these bacterial strains showed promising proteolytic activity.

139

Table 4.5: Milk clotting activity, Caesinolytic activity, Ratio of milk clotting units to caesinolytic activity of

bacterial strain. 0.5 ml of tested materials was added to a test-tube containing 5ml of reconstituted skim milk

solution (10 g dry skim milk/100 ml, 10mM CaCl2 and 10mM MnSO4) preincubated at 35ºC for 5min. The mixture

was mixed well and the clotting time T (s)

Milk clotting activity(SU/ml)

Ratio (SU / OD 660nm)

Bacterial strains CaCl2 MnSO4

Caesinolytic

activity (OD 660nm) CaCl2 MnSO4

B. licheniformis (Crude) ND ND 0.474 0 0

B. licheniformis (0-80%) ND ND 0.426 0 0

B. megaterium SN1 (Crude) 1.429 2.5 0.250 5.716 10

B. megaterium SN1 (0-30%) 10 12.5 0.115 86.96 108.69

B. megaterium SN1 (30-60%) 50 100 0.192 260.4 520.84

B. megaterium SN1 (60-90%) 9.09 11.1 0.132 68.87 84.09

B. thuringenesis SN2 (Crude) 0.83 1.11 0.458 1.81 2.42

B. thuringenesis SN2 (0-80%) 1.11 8.33 0.816 1.36 10.20

B. pumilis SN3 (Crude) 1.25 1.25 0.435 2.87 2.87

B. pumilis SN3 (0-80%) 1.66 14.28 0.644 2.58 22.17

140

Figure 4.10: Milk coagulation: B. megaterium SN1, B. thuringenesis SN2, B. pumilis SN3 strain showed milk

clotting activity. C- Control; 1- Crude enzyme of isolate SN1; 2- 0-30% A. S. sample of isolate SN1; 3- 30-60% A. S. sample of isolate SN1; 4- 60-90% A. S. sample of isolate SN1; 5- Crude enzyme of isolate SN2; 6- 0-80% A. S.

sample of isolate SN3; 7- Crude enzyme of isolate SN3; 8- 0-80% A. S. sample of isolate SN3.

Figure 4.11: Milk coagulation: 30-60%, 60-90% A. S. fraction of B. megaterium SN1, crude and 0-80% A. S.

fraction of B. thuringensis SN2 showing floating pellet. C- Control; 1- Crude enzyme of isolate SN1; 2- 0-30% A. S. sample of isolate SN1; 3- 30-60% A. S. sample of isolate SN1; 4- 60-90% A. S. sample of isolate SN1; 5- Crude enzyme of isolate SN2; 6- 0-80% A. S. sample of isolate SN3; 7- Crude enzyme of isolate SN3; 8- 0-80% A. S.

sample of isolate SN3.

141

4.2.3 Antimicrobial activity

A visually clear zone around enzyme disk indicates the presence of antibacterial components

against bacterial strain in the enzyme preparation (Table 4.6). B. thuringenesis SN2 showed

antimicrobial activity against Micrococcus luteus and Bacillus subtilis, Bacillus

amyloliquifaceance, Escherichia coli.

This is very important from enzymological view point with respect to characterization, enzyme

catalysis, kinetic study of enzyme etc. These Bacillus sp. presented in this study is useful in

industry regarding food (Dairy Industry).

Table 4.6: Inhibition (mm) of bacterial growth

B. licheniformis B. megaterium SN1

B. thuringenesis SN2

B. pumilis SN3 Reference Strain

Te TS Te TS Te TS Te TS Micrococcus luteus

(MTCC 106) 18 ND 17 ND 20 12 ND ND

Bacillus subtilis (MTCC 1789) 20 ND 20 17 25 10 20 16

Bacillus amyloliquifaceance

(MTCC 1270) 15 10 30 16 20 10 23 ND

Pseudomonas fluroscence

(MTCC 2421) 25 ND ND ND 15 ND 25 22

Escherichia coli (MTCC 1695) 25 13 20 12 20 15 10 ND

Te: Standard antibiotic Tetracycline; TS: Test samples; Values given in mm; ND: Not determined

142

4.3 Production and Purification of Protease and Keratinase enzyme from

Bacillus megateruim SN1

143

4.3.1 Degradation of feather by Bacillus megateruim SN1

In this study an extracellular protease and keratinase was purified and characterized from

Bacillus megaterium SN1 that was isolated from soil of Poultry waste site in Ghazipur. This is

potential strain degrading feather within 72hrs (fig. 4.12). This is a first report that shows

production extracellular acidic proteases and keratinase. The extra cellular protease and

keratinase was harvested by centrifugation (10,000g, 20 min) of the feather meal media 2 that

was maintained at 30°C, 160 rpm and 72 hrs of incubation with 10% feather.

144

BA

Figure 4.12: Degradation of pigeon feathers by the Bacillus megaterium SN1 isolated from soil of Ghazipur poultry

dumping site, India, in submerged cultivation at 30°C. (A) Feather control without the bacterial strain, (B) feather

after 72 hrs of incubation with the bacterial strain showed complete degradation.

145

4.3.2 Bacterial growth determination and enzyme production

The isolated colony was grown in media and growth was determined by taking absorbance at

600nm. Protease as well as keratinase level was checked for 7 days after regular interval see Fig.

4.13, 4.14. Protease and keratinase activity was maximum during log phase of growth.

146

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

1 2 3 4 5 6 7Days

O.D

. at 6

00nm

0

10

20

30

40

50

60

Prot

ease

act

ivity

(U/m

l)

Growth determinat ion

Protease act ivity

Figure 4.13: Growth determination and protease activity of isolate SN1 Figure shows that maximum amount of

protease activity 54 units/ml is produced during log phase of growth of Bacillus megaterium. There is drop in the

protease activity in later days of growth (stationary phase).

147

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

1 2 3 4 5 6 7Days

O.D

. at 6

00nm

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Kera

tinas

e ac

tivity

U/m

l

growth determinat ion

Kerat inase act ivity

Figure 4.14: Growth determination and keratinase activity of isolate SN1 Figure shows that maximum amount of

keratinase 0.7 units/ml is produced when the Bacillus megaterium is during stationary phase of growth (5 days of

incubation) and that there is drop in the keratinase activity in later days of growth.

148

4.3.3 Effects of different parameters on enzyme activity

Various substrates like (chicken feather, pigeon feather, hair and nail) were evaluated for the

production of enzyme (Fig. 4.15). Isolated strain B. megaterium SN1 grew in four nutrient

sources and produced protease and keratinase. The maximum yield of protease and keratinase

was seen in basal media supplemented with pigeon feather as compared to other substrate like

chicken feathers, hair and nail. Also complete degradation of the pigeon feather and chicken

feather was seen.

149

0

20

40

60

80

100

120

Chickenfeather

Pigeonfeather

Hair Nail

Diff. Substrate

Rel

ativ

e ac

tivity

(%)

Keratinase activity

Protease activity

Figure 4.15: Graph depicts the relative activity of proteases and keratinases in presence of different proteinaceous

substrates. Maximum activity of enzyme was detected in pigeon feathers which were treated as 100% activity.

150

4.3.4 Purification of protease and keratinase

The extracellular protease produced by B.megaterium SN1 was purified in two steps by

30-60% ammonium sulphate precipitation followed by strong anion exchange chromatography

on 25 Q sepharose. The recovered active fraction from 30-60% ammonium sulphate of culture

broth was loaded on the 25 Q sepharose column. The bound protease was eluted with 0.2 and

0.4 M NaCl (in 10 mM Tris–HCl buffer, pH 8.0). The fraction showing the presence of

caseinolytic activity was pooled (Fig. 4.16). We report 29.28 fold purification of protease with

295.98 units/mg specific activity. The results of purification of protease from B. megaterium

SN1 are summarized in Table 4.7. Similarly the fractions showing the presence of keratinase

were pooled and 655.64 fold purification of keratinase was achieved with specific activity of

544.69 U/mg protein and 12.4% recovery see Table 4.8. This keratinolytic active fraction was

further separated on SDS polyacrylamide gel electrophoresis for molecular weight determination

and also zymography studies

Many authors have suggested various strategies in purification of keratinases like Correa

et al 2010 [384] reported that the Amazonian bacterium Bacillus sp. P7 produced extracellular

keratinase that was partially purified by 60% ammonium sulphate precipitation, gel filtration

Sephadex G-200, and ion-exchange chromatography SP-Sepharose, DEAE-Sepharose FF,

resulting in a purification factor of 29.8-fold and a yield of 27%. Zhang et al 2009 [385] reported

that a new alkaline keratinase extracted from Bacillus sp. 50-3 by ammonium sulfate

precipitation, DEAE-Sephadex-A50 column and 17.7-fold purification with a yield of 46.5% was

obtained.

Our purification process includes just three steps where we have identified the

concentration of ammonium sulphate that is needed for precipitation of enzyme. In contrast,

some purification protocols described for Bacillus and other microorganisms keratinases

often involve more than two steps that normally contain two kinds of chromatography

procedures [361, 362, 327, 358]. In this study, the purified enzyme comes from a feather

meal medium containing just feather keratin protein; thus, keratinase could be purified

through a relatively simple procedure. On the other hand, the 0–30% ammonium sulfate

precipitation may have removed many unwanted proteins. Hence, this approach to the

151

purification of keratinase from Bacillus megaterium SN1 expended less time and energy, further

increasing its biotechnological industrial potential.

152

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 2021222324252627282930

Fractions

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

ProteaseactivityKeratinaseactivity

Figure 4.16: Activity profile of protease and keratinase isolated from B. megaterium SN 1 by 25 Q sepharose ion

exchange chromatography. The two peaks for protease and keratinase could be separated fraction 2-6 showed the

presence of keratinase activity and protease activity was seen in fraction 19-21.

153

Table 4.7: Summary of purification steps of protease from B. megaterium SN1

Purification Step

Total enzyme

activity (U)

Total protein

(mg)

Specific activity (U/mg)

Purification fold

Recovery (%)

Crude enzyme 6212.516 613.887 10.11 1.0 100

(0-30%) (NH4)2SO4

ppt, dialyzed 1196.188 334.601 4.468 0.45 19.25

(30-60%) (NH4)2SO4

ppt, dialyzed 3864.54 637.161 7.581 0.75 62.21

(60-90%) (NH4)2SO4

ppt, dialyzed 2720.96 288.052 11.807 1.16 43.80

25 Q sepharose 95.90 0.324 295.98 29.28 8.02

154

Table 4.8: Summary of purification steps of keratinase from B. megaterium SN 1

Purification Step

Total enzyme

activity (U)

Total protein

(mg)

Specific activity (U/mg)

Purification fold

Recovery (%)

Crude enzyme 510 613.887 0.84 1 100

(0-30%) (NH4)2SO4

ppt, dialyzed 192 334.601 0.58 0.69 37.65

(30-60%) (NH4)2SO4

ppt, dialyzed 312 637.161 0.49 0.59 61.18

(60-90%) (NH4)2SO4

ppt, dialyzed 72 288.052 0.25 0.31 14.12

25 Q sepharose 97.5 0.179 544.69 655.64 19.12

155

4.3.5 SDS-PAGE and Zymogram analysis

The 25 Q sepharose fraction was analysed on SDS-PAGE (10%), showed presence of single

band indicating a homogeneous preparation. The enzyme has a molecular weight of 30 kDa.

Zymogram activity staining also revealed one clear zone of proteolytic activity against the blue

background for purified sample at corresponding positions in SDS-PAGE (Fig. 4.17, Lane 5). It

is reported in literature that molecular weight of major keratinases, vary from 20 to 50 kDa.

keratinase from B. licheniformis PWD-1 showed MW 33 kDa [324]; B. subtilis KS-1 at MW

25.4 kDa [363]; B. Pseudofirmus FA30-10 ,MW 27.5 kDa [363]; S. Pactum DSM40530 at MW

30 kDa [33], X. maltophilia POA-1 at MW 36 kDa [365], Vibrio sp. kr2 at MW 30 kDa [366],

Chryseobacterium sp. kr6 at MW 64 kDa [362], Microbacterium sp. kr10 at MW 42 kDa [363];

S. albidoflavus K1-02 at MW 18 kDa [367].

156

M 1 2 3 4 5

200 150 120 100

80 70 60 50 40 30 25 20 15 10

Figure 4.17: Silver staining of purified enzyme of Bacillus megaterium SN1 on 10% SDS PAGE stained showed molecular weight of 30 kDa. Lane: M, Broad range molecular weight marker; Lane 1: Crude enzyme; Lane 2: 30-60% Ammonium suphate sample; Zymogram- Lane 3: Zymo Crude enzyme; Lane 4: Zymo 30-60% Ammonium

sulphate sample; Lane 5: 25 Q sepharose purified fraction showing molecular weight of 30kDa

157

4.3.6 pH Optimum

Activity of the enzyme was determined at different pH ranging from 2.0-11.0. The maximum pH

recorded was 3.0 for protease and keratinase activity. Protease and keratinase activity was found

to be stable in the acidic range starting from the pH 3-7 (Fig 4.18). Maximum activity in the

acidic range suggests a positive biotechnological potential in the food and found that feather

protein can be metabolized which is used as animal feed protein [47, 369, 370]. Additionally, the

relative enzyme activity was higher than 40% even at neutral and some alkaline conditions,

indicating the potential versatility of such enzyme preparations for diverse applications. Several

authors have reported that microbial keratinases typically have temperature optima in the range

of 30–80 °C, and optimum pH in the neutral to alkaline range [371]. For instance, keratinase

from B. subtilis MTCC (9102) presented maximum activity at 40 °C and pH 6.0 [325]; optimal

activity of B. subtilis KS-1 keratinase was observed at 60 °C and pH 7.0 [69]; keratinase from

Streptomyces sp. S.K1-02 showed optimal activity at 70 °C and pH 10.0 [372]; and

Microbacterium sp. kr10 keratinase exhibited maximal activity at 50 °C and pH 7.5 [363].

158

0

20

40

60

80

100

120

pH

Rel

ativ

e ac

tivity

(%)

Pro tease activity

Kerat inase act ivity

2 3 4 5 6 7 8 9 10 11

Figure 4.18: Effect of pH on caesinolytic activity and keratinolytic activity of enzyme from Bacillus megaterium

SN1, pH optima was measured by incubating the enzyme with the substrate at different pH values. The maximum

protease and keratinase activity obtained at pH 3.0 and it was considered as 100% activity.

159

4.3.7 Temperature Optimum

The thermal stability of the enzyme was also tested at different temperatures 20°C, 30°C, 40°C,

50°C, 60°C, 70°C and 80°C (Fig. 4.19). The optimum temperature recorded was at 60°C for

protease and 70°C for keratinase activity beyond which there was a rapid decline. The protease

and keratinase activity was found to be stable in the temperature range from 40°C to 80°C and

50°C to 70°C respectively.

160

0

20

40

60

80

100

120

10 20 30 40 50 60 70 80Temprature (ºC)

Rel

ativ

e ac

tivity

(%)

protease activity

keratinase activity

Figure 4.19: Effect of temperature on caesinolytic activity and keratinolytic activity from Bacillus megaterium SN1.

The relative activity was defined as the percentage of activity detected with respect to the maximum caesinolytic

activity and keratinolytic activity. The maximum protease and keratinase activity obtained at temperature 60°C and

70°C was considered as 100% activity respectively.

161

4.3.8 Effect of Metal Ions

Interestingly Mn2+, Co2+ (10mM) strongly activated protease activity by 2.1 fold, 1.28 fold

respectively, whereas Mn2+, Co2+ and Mg2+ strongly activated keratinase activity by 1.17, 1.15

and 1.12 fold respectively by acting as salt or ion bridges that stabilize the enzyme in its active

conformation and might protect the enzyme against thermal denaturation [325, 373]. While Hg2+

and Ba2+ strongly inhibited protease activity and Hg2+ and Fe2+strongly inhibited keratinase

activity. Interestingly presence of Mn2+ activated protease and keratinase activity by 2.1 and 1.17

fold respectively (Fig. 4.20). These results suggest that concerned metal ions apparently played a

vital role in maintaining the active conformation of the enzyme at high temperatures. This is

possible because of the activation by the metal ions.

Hg+2 is recognized as an oxidant agent of thiol groups, and the enzyme inhibition by this ion

could suggest the presence of important -SH groups (such as free cysteine) at or near the active

site [271, 328]. However, Hg2+ might also react with tryptophan residues and carboxyl groups in

amino acids of the enzyme [374].

162

0

50

100

150

200

250

Ni Ca Mg Co Cu Mn Hg Zn Fe Ba

Metal ion

Rel

ativ

e ac

tivity

(%)

Protease activity

Keratinase activity

Figure 4.20: Effect of metal ion on protease activity and keratinase activity from Bacillus megaterium SN1. The

relative activity was defined as the percentage of activity detected with respect to protease activity and keratinase

activity without metal ions considered as 100%. The maximum protease activity and keratinase activity obtained

with Mn2+ was observed. Barium could inhibit protease activity.

163

4.4 Production and Purification of Protease and Keratinase enzyme from

Bacillus thuringenesis SN2

164

4.4.1 Degradation of feather by Bacillus thuringenesis SN2

In this study an extracellular protease and keratinase was purified and characterized from

Bacillus thuringenesis SN2 that was isolated from soil of Poultry waste site in Ghazipur. This

strain degraded feather within 96 hrs (fig. 4.21). The extra cellular protease and keratinase was

harvested by centrifugation (10,000g, 20 min) of the feather meal media 2 that was maintained at

30°C, 160 rpm and 96 hrs of incubation with 10% feather.

165

A B

Figure 4.21: Degradation of pigeon feathers by the Bacillus thuringenesis SN2 isolated from soil of Ghazipur

poultry dumping site, India, in submerged cultivation at 30°C. (A) Feather control without the bacterial strain, (B)

feather after 72 hrs of incubation with the bacterial strain showed complete degradation.

166

4.4.2 Purification of protease and keratinase enzyme

The extracellular protease and keratinase was produced by B. thuringenesis SN2 was partially

purified by 0-80% ammonium sulphate precipitation. We report that there were 2.92 fold

increases in protease activity with 54.76 % recovery and keratinase activity increases with 1.8

fold and 41.68 % recovery. (Table 4.9, 4.10)

Table 4.9: Summary of purification steps of protease activity from B. thuringenesis SN2

Purification Step

Total enzyme activity

(U)

Total protein

(mg)

Specific activity (U/mg)

Purification fold

Recovery (%)

Crude enzyme 4713.1 66.66 70.71 1 100

(0-80%) (NH4)2SO4 ppt,

dialyzed 2581.96 12.5 206.56 2.92 54.76

Table 4.10: Summary of purification steps of keratinase from B. thuringenesis SN2

Purification Step

Total enzyme activity

(U)

Total protein

(mg)

Specific activity (U/mg)

Purification fold

Recovery (%)

Crude enzyme 249 66.66 4.6 1 100

(0-80%) (NH4)2SO4 ppt,

dialyzed 103.84 12.5 8.3 1.8 41.68

167

4.4.3 Bacterial growth determination and enzyme production

The isolated colony was grown in media and growth detected by taking the absorbance at 600nm

and protease as well as keratinase level was checked for 7 days after regular interval see Fig.

4.22, 4.23. Protease and keratinase activity was maximum during stationary phase of growth.

168

SN2

0

0.05

0.1

0.15

0.2

0.25

1 2 3 4 5 6 7Days

O.D

. at 6

00nm

0

5

10

15

20

25

30

35

Prot

ease

act

ivity

(U

/ml)

Growth determinat ionProtease act ivity

Figure 4.22: Growth determination and protease activity of B. thuringenesis SN2 Figure shows that maximum amount of protease activity 30 units/ml is produced during log phase of growth. There is drop in the protease

activity in later days of growth (stationary phase).

SN2

0

0.05

0.1

0.15

0.2

0.25

1 2 3 4 5 6 7Days

O.D

. at 6

00nm

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

Ker

atin

ase

activ

ity U

/ml

growth determinat ion

Kerat inase act ivity

Figure 4.23: Growth determination and keratinase activity of B. thuringenesis SN2 Figure shows that maximum amount of keratinase 0.32 units/ml is produced when the Bacillus thuringnesis is during stationary phase of growth

(4 days of incubation) and that there is drop in the keratinase activity in later days of growth.

169

4.4.4 Caesin Zymography

Activity staining on gel was confirmed by casein zymography, which was performed in

polyacrylamide slab gels containing SDS and casein (0.12% w/v) as co-polymerized substrate.

Caesinolytic bands were seen to have molecular weight 80 kDa, 60 kDa and 40 kDa (fig. 4.24)

170

Figure 4.24: Activity staining on gel was confirmed by casein zymography, which was performed in

polyacrylamide slab gels containing SDS and casein (0.12% w/v) as co-polymerized substrate. Lane: 1, molecular

weight marker; Lane: 2, Crude enzyme; Lane: 3, 0-80% Ammonium sulphate sample. Caesinolytic bands were seen

to have molecular weight 80kDa, 60kDa and 40kDa.

171

4.4.5 pH Optimum

Activity of the enzyme was determined at different pH ranging from 3.0-12.0. The optimum pH

recorded was 5.0 for caesinolytic activity and 3.0 for keratinolytic activity. caesinolytic activity

and keratinolytic activity was found to be stable in the acidic range starting from the pH 3-9 (Fig

4.25).

172

SN2

0

20

40

60

80

100

120

2 3 4 5 6 7 8 9 10 11 12pH

Rel

ativ

e ac

tivity

(%)

protease act ivity

Kerat inase act ivity

Figure 4.25: Effect of pH on the activity of caesinolytic activity and keratinolytic activity from B. thuringenesis.

SN2. pH optima was measured by incubating the enzyme with the substrate at different pH values. The maximum

caesinolytic activity and keratinolytic activity obtained at pH 5.0 and 3.0 was considered as 100% activity

respectively.

173

4.4.6 Temperature Optimum

The effect of temperature of the enzyme was also tested at different temperatures 10°C, 20°C,

30°C, 40°C, 50°C, 60°C, 70°C and 80°C (Fig. 4.26). The optimum temperature recorded was at

40°C for caesinolytic activity and 50°C for keratinolytic activity. The Caesinolytic activity and

keratinolytic activity was found to be stable in the temperature range from 30°C - 60°C.

174

SN2

0

20

40

60

80

100

120

10 20 30 40 50 60 70 80

Temprature (°C)

Rel

ativ

e ac

tivity

(%)

Protease act ivityKerat inase act ivity

Figure 4.26: Effect of temperature on caesinolytic activity and keratinolytic activity from B. thuringenesis SN2. The

relative activity was defined as the percentage of activity detected with respect to the maximum caesinolytic activity

and keratinolytic activity. The maximum caesinolytic activity and keratinolytic activity obtained at temperature

40°C and 50°C was considered as 100% activity respectively.

175

4.4.7 Effect of Metal ons

Interestingly Mn2+ and Co2+ strongly activated caesinolytic activity by 3.74 and 1.43 fold. Ba2+,

Mg2+, Ca2+ and Mn2+ strongly activated keratinolytic activity by 1.9, 1.52, 1.35 and 1.32 fold (Fig.

4.27). Whereas caesinolytic activity was strongly inhibited by Ba2+ and keratinolytic activity was

strongly inhibited by Fe2+ and Hg2+ (10mM). These results suggest that concerned metal ions

apparently played a vital role in maintaining the active conformation of the enzyme. This is

possible because of the activation by the metal ions. They are to be utilized for the application

purpose, these specific metal ion can be added either for enhancing or inhibiting the activity.

176

SN2

0

50

100

150

200

250

300

350

400

Ni Ca Mg Co Cu Mn Hg Zn Fe BaMetal ion

Rel

ativ

e ac

tivity

(%)

Caesinolytic activity

Keratinolytic activity

Figure 4.27: Effect of metal ion on caesinolytic activity and keratinolytic activity from B. thuringenesis SN2. The

relative activity was defined as the percentage of activity detected with respect to caesinolytic activity and

keratinolytic activity without metal ions considered as 100%. The maximum caesinolytic activity and keratinolytic

activity obtained with Mn2+ and Ba2+ was observed respectively.

177

4.5 Optimization of protease and keratinase production by Resilient Back

Propagation (RPROP) from Bacillus megaterium SN1

178

Experiments were conducted according to the various combinations with different rang

and code of variables as mentioned in Table 4.11 at constant pH, Temperature and RPM. From

the results it is evident that presence of yeast extract and feather is significant in terms of

caesinolytic enzyme production. The combination where NaCl was 0.5gm, Yeast extract 0.13 gm

and Feather was 15g (-1, 1, 1) yielded the maximum amount of caesinolytic enzyme (24.292

U/mg protein). Lowest value (7.8721 U/mg) is reported when the combination is NaCl was 1gm,

Yeast 0.1 gm and Feather was 5g (1, -1, -1).

In terms of keratinolytic enzyme production, it is evident that presence of feather is

significant. The combination where NaCl was 0.5gm, Yeast extract 0.1 gm and Feather was 10g

(-1,-1, 0) yielded the maximum amount of keratinolytic enzyme (17.2314 U/mg protein). Lowest

value (10.5634 U/mg) is reported when the combination is NaCl was 1.5gm, Yeast 0.2 gm and

Feather was 5g (1,0,-1).

Generally artificial neural network offer the advantage of being fact accurate and

valuable for prediction or approximation when numerical and mathematical models are not

adequate. ANN model fits the data very well with our experimentally measured value as shown

in Table 4.13 and 4.15.

Artificial neural network is a powerful generalization model used for prediction of enzyme

activity when the culture parameters are changed. Taking into consideration the result from these

analyses, it was concluded that ANN provides a desirable description of the experimental data

because of its generality and simplicity. A one hidden layer (6 neurons) RPROP network with

tangent-sigmoid transfer function in hidden layer and purelin in output layer was found to be an

optimum topology, which predicted the specific activity for keratinase with order of error 1.88 e-18.

Table 4.11: Range and code of the process variable

Variable Actual Code Actual Code Actual Code

NaCl 0.5g -1 1 g 0 1.5g +1

Yeast Extract 0.1g -1 0.2g 0 0.3g +1

Feather 5g -1 10g 0 15g +1

179

Table 4.12: Input –target pairs of network and corresponding predicted values of specific

activity of protease produced by Bacillus megaterium SN1

Input to network S. No.

NaCl Yeast extract Feather

corresponding target Specific activity

prediction by RPROP

Specific activity

1 0 0 0 22.42199 22.42 2 0 0 1 24.07511 24.0779 3 0 1 0 22.95631 21.8528 4 0 1 1 20.92159 21.8623 5 1 0 0 20.30444 20.0973 6 1 0 1 20.48604 21.5615 8 1 1 1 22.55164 21.8427 9 0 0 -1 16.60956 16.6297

13 0 -1 0 19.77484 19.5913 17 -1 0 0 14.59169 21.5527 21 0 -1 -1 15.13072 15.2343 27 -1 -1 -1 20.72026 20.7177

Table 4.13: Specific activity of protease enzyme predicted by trained network for various

combination of NaCl, yeast extract, feather

S. No. NaCl Yeast extract Feather Prediction of Specific activity

7 1 1 0 21.7199 12 1 1 -1 20.9523 14 0 -1 1 21.944 15 1 -1 0 15.302 16 1 -1 1 20.8603 18 -1 0 1 22.895 19 -1 1 0 20.46 20 -1 1 1 24.292 22 1 -1 -1 7.8721 23 -1 -1 0 21.410 24 -1 -1 1 21.52 25 -1 0 -1 21.619 26 -1 1 -1 17.7225

180

Table 4.14: Input –target pairs of network and corresponding predicted values of specific

activity of keratinase activity produced by Bacillus megaterium SN1

Input to the network S. No.

NaCl Yeast

extract Feather

Corresponding target Specific activity

Prediction by RPROP Specific

activity

1 0 0 0 15.01413 15.014 2 0 0 1 12.75981 12.759 3 0 1 0 12.59792 12.597 4 0 1 1 14.94694 14.946 5 1 0 0 12.71873 12.718 6 1 0 1 13.59126 13.591 8 1 1 1 17.01308 17.013 9 0 0 -1 15.22162 15.091

13 0 -1 0 16.77419 16.774 17 -1 0 0 14.59169 14.591 21 0 -1 -1 13.35282 13.352 27 -1 -1 -1 16.54015 16.54

Table 4.15: Specific activity of keratinase enzyme predicted by trained network for various

combination of NaCl, yeast extract, feather

S. No. NaCl Yeast extract Feather Prediction of specific activity

7 1 1 0 14.5562 10 0 1 -1 12.9543 11 1 0 -1 10.5634 12 1 1 -1 11.8972 14 0 -1 1 14.9405 15 1 -1 0 13.9996 16 1 -1 1 12.9222 18 -1 0 1 14.3316 19 -1 1 0 14.9583 20 -1 1 1 15.2118 22 1 -1 -1 12.5605 23 -1 -1 0 17.2314 24 -1 -1 1 14.5032 25 -1 0 -1 17.0882 26 -1 1 -1 15.5543

181