Chapter 8shodhganga.inflibnet.ac.in/bitstream/10603/34582/13/13_chapter8.pdf · Anjali Bose, Shabnam Pathan, Khyati Pathak and Haresh Keharia (2012). Keratinolytic ... Profile of

Chapter 8 Keratinolytic protease production by Bacillus amyloliquefaciens 6B using feather meal as substrate and application of feather hydrolysate as biocontrol (antifungal) as well as organic nitrogen input for agricultural soil This part has been communicated as: Anjali Bose, Shabnam Pathan, Khyati Pathak and Haresh Keharia (2012). Keratinolytic protease production by Bacillus amyloliquefaciens 6B using feather meal as substrate and application of feather hydrolysate as soil input. Waste & Biomass Valorization.

Feather as substrate for keratinase production Chapter 8

156

8.1. Introduction

Low commercial value keratinous wastes of animal origin such as nails, hair,

feathers, horns, hoofs, beak etc., which are generated in huge amount from poultry and

cattle slaughter houses, are hard-to-degrade. Considering the fact that feathers constitute

nearly 8.5% of the total weight of a chicken, the amount of solid waste generated

annually in India from approximately 823.5 million chickens, with an average

individual body weight of 2 kg, would be approximately 140 million kg (FAO 2002).

Currently, the waste feathers are either disposed by dumping or incineration which can

lead to serious health hazards and environmental pollution (Deydier et al 2005).

Alternately, they are transformed into animal feed supplement by harsh hydrothermal

and chemical treatment that result in loss of nutritionally essential amino acids, such as

methionine, lysine, and tryptophan, and formation of non-nutritive amino acids, such as

lysinoalanine and lanthionine (Dalev et al 1997). Consequently, microbial degradation

of such wastes along with production of commercially important enzymes (especially

keratinase) and other value-added products such as nutrient-rich animal feed or fertilizer

(Onifade et al 1998, Brandelli 2008, Jeong et al 2010, Lateef et al 2010) is desirable.

Contemporary to this problem, is the development of new fertilizers using natural

materials. In the same context, feather hydrolysate resulting from the bioconversion of

keratinous wastes being rich in nitrogen and inexpensive could be used as a potential

fertilizer for soil amendment (Gupta and Ramnani 2006), serving the dual purpose of

improving plant growth and stimulating microbial activity in soil (Vasileva-Tonkova et

al 2009). On the other hand, inadequate effectiveness of chemical control and

increasing restrictions on the use of fungicide/ pesticides have given increasing impetus

to biological control as an alternative tool for disease management in agriculture

(Raaijmakers et al 2002). Hence, microorganisms capable of degrading keratinous

waste as well as exhibiting antagonism against soil borne plant pathogens hold

prospects of dual benefit, waste utilization along with production of biocontrol

metabolites. Most of investigations reported in literature on keratinolytic

microorganisms have been directed toward keratinase production for increasing the

digestibility of feathers as an animal feed (Onifade et al 1998, Odetallah et al 2003,

Grazziotin et al 2006, Fakhfakh et al 2011). However, there is only one report on the

isolation and characterization of keratinolytic Stenotrophomonas maltophilia R13 with


157

plant growth-promoting activity (Jeong et al 2010). To the best of our knowledge, there

is no report on keratinolytic Bacillus sp. exhibiting biocontrol activity.

From this perspective, the poultry soil isolate Bacillus amyloliquefaciens 6B

(BA6B) was investigated for: its ability to grow on and efficiently degrade whole

feathers, production of keratinolytic protease and antifungal metabolites. Additionally,

the enzyme and the antifungal metabolites produced by BA6B were characterized.

Further, the fermentation broth with the bacterial cells and hydrolyzed feather was

investigated as nitrogenous soil input.

8.2. Materials and Methods

8.2.1. Materials

Bushnell and Haas Medium (BHM), Luria Bertani Broth, gelatin, yeast extract

and bovine serum albumin (BSA, protein standard) were obtained from Hi-Media,

India. Hammerstein casein was procured from Sisco Research Laboratories, India.

Chicken feathers were procured from a local poultry (Karamsad, Anand, Gujarat, India),

washed with tap water and air dried. The feathers were then ground to get feather meal

which was added to basal salt medium as a substrate for enzyme production. All other

solvents and chemicals used during the experiment were of analytical grade.

Phytopathogenic fungi namely, Aspergillus niger MTCC 16404, Aspergillus fumigatus

MTCC 343, Aspergillus parasiticus NCIM 898, Trichosporon sp. NCIM 1110,

Fusarium oxysporum NCIM 1072 were obtained from National Collection of Industrial

Microorganisms (NCIM), National Chemical Laboratory, Pune, India and Microbial

Type Culture Collection (MTCC), Institute of Microbial Technology, Chandigarh,

India.

8.2.2. Isolation and identification of keratin hydrolysing bacterium

Feather degrading bacteria were isolated upon enrichment of the poultry soil

(Karamsad, Gujarat) with whole feathers for 15 days. Upon incubation a 10% (w/v) soil

suspension was prepared in sterile distilled water and 100 µL aliquots of serially diluted

soil suspension were spread on 1% casein agar plates and incubated at 37°C for 48 to 72

h. Individual colonies exhibiting clear zones around them resulting from casein

hydrolysis were picked and purified by repeated sub-culturing on Luria Bertani agar

plate. The pure cultures were further screened for their ability to degrade feather on


158

feather meal agar (BHM supplemented with 1% w/v feather meal; FMA) plates. The

bacterial isolates exhibiting feather degradation on FMA plates were then investigated

for native feather degradation. The isolate exhibiting the highest keratinolytic activity

was then selected for further studies.

The identity of the selected bacterial isolate designated as 6B was determined on

the basis of 16S rRNA gene sequence analysis. The genomic DNA was extracted as

described by Ausubel et al (1997) and the 16S rRNA gene was amplified and sequenced

as described in Chapter 2. Further, the sequence has been submitted to Genbank at

NCBI.

8.2.3. Organic solvent tolerance of bacterial isolate 6B

The bacterium 6B was spot inoculated on casein agar plates using sterile tooth

picks upon which each plate was flooded with 7 mL of different solvents (iso-octane, n-

heptane, n-hexane, cyclohexane, xylene, toluene, benzene, chloroform, ethyl acetate and

methanol) and incubated at 37°C in air tight canister in order to prevent the evaporation

of solvent from the plates. Upon incubation, plates were observed for growth as well as

clear zone around the colony in the presence of solvents.

The stability of keratinolytic protease in organic solvents was investigated by

mixing equal volume of solvent and culture supernatant (crude enzyme) in screw cap

test tubes and incubated in shaking water bath (50 rpm) at 37 C. After 24 h of

incubation residual protease activity was assayed in the aqueous phase.

8.2.4. Profile of keratinolytic protease production by bacterial isolate 6B

The 500 mL Erlenmeyer flasks containing 200 mL feather meal broth (BHM

amended with 0.2% w/v feather meal; FMB) was inoculated with an overnight grown

culture of 6B to obtain an initial culture density (O.D.600nm) of 0.05 and incubated on

orbital shaker (150 rpm) at 37 C. The 2 mL aliquots were withdrawn after 3, 6, 9, 12,

24, 36 and 48 h of incubation, and monitored for growth and protease activity.

8.2.5. Medium optimization for keratinolytic protease production by bacterial isolate

6B

Various sugars (fructose, galactose, glucose, lactose, maltose, mannitol, sucrose

and xylose; 0.5% w/v) were supplemented individually to FMB, in order to investigate


159

their effect as co-carbon sources on the enzyme production by 6B. The influence of

varying concentrations (0.1% to 2%, w/v) of feather meal on the keratinolytic enzyme

production was also investigated.

8.2.6. Concentration of enzyme

The fermentation broth was harvested after 12 h of incubation and subjected to

centrifugation at 4°C and 12,310 ×g for 15 min. The supernatant was collected and

subjected to 40-80% ammonium sulphate saturation and kept overnight at 20°C to allow

protein precipitation. The precipitates were then harvested by centrifugation at 4°C and

27,540 ×g for 20 min. The pellet thus obtained was resuspended in minimum volume of

50 mM Tris-Cl buffer (pH 8.0) to allow solubilization of proteins and dialyzed against

same buffer overnight at 4°C.

The dialyzed enzyme preparation was then analyzed by Native and SDS-

polyacrylamide gel (10%, w/v) electrophoresis according to the method of Laemmli

(1970). One part of gel was subjected to activity staining in order to visualize protease

band and the other part was subjected to silver staining (Sambrook and Russell 2001) in

order to visualize protein bands. For activity staining, the native gel was transferred

onto a casein agar slab (1% casein and 1% (w/v) agar in 50 mM Tris-Cl buffer pH, 8.0)

and incubated at 37°C till transparent bands of protease activity appeared due to casein

hydrolysis in the agar slab. In case of SDS-PAGE, after electrophoresis, SDS was

removed by washing the gel for 15 min with 2.5% Triton X-100 and then twice for 10

min each with distilled water. Then, the gel was subjected to activity staining as

described above. Relative molecular mass (Mr) was estimated by comparison with

molecular mass standards (3.5 to 97.4 kDa) (Bangalore Genie, Bangalore, India).

8.2.7. Characterization of crude keratinolytic protease from isolate 6B

The keratinolytic protease activity was determined at different temperatures viz.,

30, 40, 50, 60, 70, 80°C in presence of 50 mM Tris-Cl buffer, pH 8.0. The

thermostability of enzyme was determined by pre-incubating the enzyme at 50 and

60°C in shaking water bath for varying time intervals upto 120 min and then assayed for

residual activity at 50ºC, which was expressed as percentage of initial activity.

The activity of keratinolytic protease was monitored over a pH range of 5.0 to

11.0 at 37°C. Sodium acetate buffer (50 mM) for pH 5.0-5.5, sodium phosphate buffer


160

(50 mM) for pH in range of 6.0 to 7.5, Tris-Cl buffer (50 mM) for pH 8.0 to 8.5 and

glycine-NaOH (50 mM) for pH in range of 9.0 to 11.0 were used to adjust the pH of the

reaction mixture. The pH stability of the enzyme was estimated by pre-incubating the

enzyme in buffers of various pH for 1 h and then assayed for protease activity at

optimum pH.

To determine the effect of various additives viz., metal ions (Ca2+

, Cu2+

, Fe2+

,

Mg2+

, Mn2+

, Hg2+

, Zn2+

; 5 mM), enzyme inhibitors (EDTA and PMSF; 5 mM),

surfactants (SDS, CTAB, Triton X-100, Tween 80; 1% w/v), and bleach-oxidant (H2O2;

1% v/v); the enzyme was pre-incubated with these additives for 1 h at 37°C and then

assayed using casein as a substrate. The enzyme activity was expressed as % activity

relative to control (without any of the aforementioned additives) which was considered

as 100%.

8.2.8. In vitro screening for antifungal activity

A co-culture technique (Trivedi et al 2008) was used to test the ability of

bacterium 6B to inhibit the growth of several phytopathogenic fungi (A. niger MTCC

16404, A. parasiticus NCIM 898, A. fumigatus MTCC 343, Trichosporon sp. NCIM

1110 and F. oxysporum NCIM 1072). The bacterium was spot inoculated on one side of

PDA plate and incubated at 37 C for 48 h after which each fungal strain was inoculated

individually, next to the bacterial culture and the plates were incubated at 27°C up to 5 d

for monitoring antifungal activity.

The antifungal activity of the culture supernatant harvested from stationary-phase

culture of isolate 6B was bioassayed against the above mentioned fungi using agar well

diffusion method. Briefly, the culture supernatant (100 µL) was inoculated in the center

of PDA plates which had been inoculated previously with test fungus. After the plates

were incubated at 28°C for 5 d, the inhibition of fungal growth was observed around the

well .The control plate was prepared by growing each test fungus with uninoculated

feather medium.

Further the antifungal metabolites from culture broth were precipitated by

lowering the pH of cell free supernatant upto 2.0 using (6M) HCl. The precipitate thus

obtained was solubilized in methanol, which was then analyzed by ESI-MS. Mass

spectra was recorded in positive ion mode using Esquire 3000 plus mass spectrometer

(Bruker Daltonics, Germany) consisting of two octopole followed by an ion trap.


161

Helium was used as collision gas for collision induced dissociation (CID) experiment

and the data obtained was processed using Esquire data analysis software, version 3.1.

8.2.9. Use of the feather hydrolysate as a bioactive agricultural nitrogen input (30 d

plant growth assay).

Pot study was carried out in triplicates to compare the ability of feather

hydrolysate and the reference commercial fertilizer as agriculture input and its effect on

the growth of Vigna radiate var. meha (mung). The seeds (5 seeds/test pot) were

planted in 6 liters plastic pots with 4 kg garden soil. The feather hydrolysate

(concentrated five times using rotary evaporator at 60°C; 153 mL of feather

hydrolysates/kg soil) and the fertilizer (625 mg of fertilizer/kg soil) were diluted with

tap water and poured onto the top of the soil to obtain the desired concentration of

soluble nitrogen (100 mg of N/kg soil). All the pots were watered regularly with tap

water to replace the evaporation loss and after 30 days of sowing, all plants were

carefully uprooted and washed. Various growth parameters such as plant height, plant

fresh weight, root length and root fresh weight were recorded.

8.2.10. Analytical procedures: enzyme activity

The enzyme activity was determined as per the modified method of Khardenavis

et al (2009). In brief, 2 mL of reaction mixture containing 1 mL of 1% (w/v) casein

(acc. to Hammerstein) and 1 mL of appropriately diluted enzyme in 50 mM Tris-Cl

buffer (pH 8.0) was incubated for 20 min at 50°C. The reaction was stopped by

precipitation of the residual substrate with 2 mL 10% (w/v) trichloroacetic acid. A

control was run in parallel in which the enzyme was added after addition of

trichloroacetic acid. The reaction mixture was then centrifuged at 13,201 ×g for 10 min

and supernatant was assayed for tyrosine by reading its absorbance was read at 280 nm

(absorption maxima for tyrosine) against the assay blank, using UV-Visible

spectrophotometer (Thermo Electron corporation, HEλIOS α). The standard curve of

tyrosine was prepared by reading absorbance of different dilutions of 500 µg

tyrosine/mL stock. One unit of protease activity was defined as the amount of enzyme

required to liberate 1 μg of tyrosine/min.mL under specified assay conditions. The total

protein was estimated according to the method of Lowry et al (1951) using bovine

serum albumin (fraction V) as standard.


161.1

Figure: Proteolytic activity of isolates 3B, 6B, 1B and 2A on (a) casein agar plate, (b) gelatin agar plate.

Figure: Initial screening for feather degradation by isolates 3B, 6B, 2A and 1B


162

8.1. Results and Discussion

8.1.1. Isolation and identification of keratin hydrolysing bacterium

Thirteen proteolytic bacterial strains isolated from the poultry farm soil sample

were screened for feather degradation on FMA plates. The growth of bacterial isolates

on FMA indicated their ability to utilize feather keratin as a sole carbon source for

growth. Amongst all, four isolates designated as 1B, 2A, 3B and 6B were selected on

the basis of their growth on FMA and were then screened for their ability to degrade

native feather. Among the four isolates, 6B exhibited complete degradation of intact

feather within 24 h of incubation at 37°C (Fig. 8.1).

Figure 8.1 Feather degradation by isolate designated 6B

The isolate 6B was found to be a Gram positive, spore-forming bacillus. The

nucleotide sequence of 16S rRNA gene of this isolate exhibited 100% sequence

homology with the partial sequence of Bacillus amyloliquefaciens DJFZ40 (GU568197)

16S rRNA gene. The 1,354 bp sequence of isolate 6B (Appendix 3) identified as B.

amyloliquefaciens has been submitted to NCBI GenBank (JQ904625).

8.1.2. Organic solvent tolerance of B. amyloliquefaciens 6B (BA6B)

The solvent tolerance of BA6B was determined by its ability to grow on casein

agar plates flooded with different solvents (iso-octane log Pow = 4.5, n-heptane log Pow

= 4.39, n-hexane log Pow = 3.86, cyclohexane log Pow = 3.2, xylene log Pow = 3.1,

toluene log Pow = 2.64, benzene log Pow = 2.13, chloroform log Pow = 2.0, ethyl acetate

Pow = 0.68 and methanol log Pow = -0.764) during incubation. The BA6B exhibited

growth as well as protease production in presence of all the solvents except ethyl acetate


163

and methanol, with maximum zone of hydrolysis on plates flooded with iso-octane

(Table 8.1).

Table 8.1: Organic solvent tolerance of B. amyloliquefaciens 6B and its protease.

Organic solvents log Pow Zone/Growth ratio Relative enzyme activity (%)

None (Control) 4.3 100

iso-Octane 4.5 4.5 99

n-Heptane 4.39 4.1 94

n-Hexane 3.86 3.6 85

Cyclohexane 3.2 2.6 82

Xylene 3.1 2.8 70

Toluene 2.64 2.4 46

Benzene 2.13 1.6 39

Chloroform 2.0 1.4 22

Ethyl acetate 0.68 ND 7

Methanol -0.76 ND 4

In general, organic solvents with a log Pow between 1.5 and 4.0 are extremely

toxic to microorganisms and other living cells because they partition preferentially in

the cytoplasmic membrane, disorganizing its structure and impairing vital functions

(Inoue and Horikoshi 1991). However, solvent toxicity not only depends on the

inherent toxicity of the solvent but also on the intrinsic tolerance of the bacterial species

and strains (Sikkema et al 1995). There exits few protease producing Bacillus sp. that

can grow in presence of toxic organic solvents such as DMSO, methanol, toluene,

xylene, cyclohexane, hexane and iso-octane (Gorbel et al 2003, Sareen and Mishra

2008, Fang et al 2009, Li et al 2009). The solvent tolerance in such microorganisms has

been attributed to the alteration in cell morphology, composition of the membrane fatty

acids and/or presence of active efflux pumps which maintain minimal sub toxic

concentration of organic solvents within cells (Torres et al 2011).

The stability of crude enzyme from BA6B was determined in presence of all the

above organic solvents at 50% (v/v) concentration upto 24 h (Table 8.1). The enzyme

was found stable in presence of non-polar (high log Pow value) solvents, retaining more

than 80% activity, whereas, the enzyme activity was significantly inhibited in presence

of solvents with lower log Pow value such as chloroform, ethyl acetate and methanol.

Low log Pow value corresponds to greater polarity and toxicity of the solvent to the

enzyme (Inoue and Horikoshi 1991). Similar inactivation of keratinase from B. subtilis

KD-N2 in presence of polar solvents was reported by Cai et al (2008). In contradiction

to our observations, keratinase from Kocuria rosea (Bernal et al 2006),


164

Fervidobacterium islandicum (Nam et al 2002) and Chryseobacterium sp. kr6 (Riffle et

al 2003) have been reported to be stable in presence of even polar organic solvents.

8.1.3. Production profile of the extracellular keratinolytic protease by BA6B in

batch culture

The isolate BA6B was able to produce keratinolytic protease in medium

containing feather meal as sole carbon source. It was observed that the enzyme

production by BA6B was growth associated and attained maxima (236.46 ± 12.5 U/mL)

at the end of exponential growth phase (Fig. 8.2). The production of similar growth-

associated keratinases has been reported by Brandelli and Riffel (2005) as well as Park

and Son (2009). Daroit et al (2011) also demonstrated growth associated keratinase

production in optimized medium by an Amazonian Bacillus sp. P45.

Figure 8.2 Time course of keratinolytic protease production by

Bacillus amyloliquefaciens 6B (Data are means of three replicates,

error bars indicate S.E.)

The keratinolytic protease production by BA6B was higher in comparison to those

reported for Serratia sp. HPC1383 (210 U/mL) (Khardenavis et al 2009) and B.

licheniformis RG1 (161 U/mL) (Ramnani and Gupta 2004).

Furthermore, keratinolytic protease production by BA6B was accompanied by rise

in media pH from 7.2 to 8.4. It has been proposed that microbial degradation of

keratinous substrate is a complex process and involves three steps, i.e. deamination,

sulphitolysis and proteolysis. Such change in pH due to deamination of peptides and

amino acids originating from keratin degradation, allows the substrate to swell and

create an environment required for sulphitolysis and proteolysis (Kunert 2000). Any


165

further increase in pH was limited by the volatility of ammonia, which escapes from

culture broth in gaseous form. Similar increase in medium pH from neutral to alkaline,

have been reported during keratinase production by several other feather-degrading

bacteria (Park and Son 2009, Lateef et al 2010) and fungi (Kaul and Sumbali 1999).

8.1.4. Optimization of media components and culture conditions for BA6B

keratinolytic protease production

The isolate BA6B produced maximum keratinolytic protease (380.85 U/mL) in

presence of xylose as co-substrate followed by lactose (286.16 U/mL) (Fig. 8.3).

Contrary to most reports (Cai and Zheng 2009, Park and Son 2009, Jeong et al 2010),

presence of mannitol, fructose, glucose, maltose and sucrose as co-carbon source in

medium resulted in reduced enzyme production by BA6B. Such suppression in enzyme

production has been attributed to catabolite repression, a phenomenon observed for

biosynthesis of bacterial proteases including keratinases (Cheng et al 1995, Hossain et

al 2007, Wang and Shih 1999).

Figure 8.3 Effect of co-carbon source on keratinolytic protease production by B.

amyloliquefaciens 6B. (Data are means of three replicates, error bars indicate S.E.)

In subsequent experiment, the effect of varying concentrations of feather meal on

keratinolytic protease production by BA6B was evaluated. The increase in BA6B

protease production was observed with increase in feather meal concentration up to

0.5% (w/v) in fermentation medium and further increase in feather meal up to 1% (w/v)

had no influence on protease production (Fig. 8.4). However, further increase in feather

meal concentration above 1% (w/v) in medium resulted in lower protease production.

Cheng et al (1995) reported highest yield of B. licheniformis PWD-1 keratinase in 1%


166

(w/v) feather meal medium. The higher feather meal concentrations (2%, w/v and

above) has been shown to increase medium viscosity, thereby affecting mass transfer

and causing oxygen limitation for the growing culture, ultimately resulting in lower

yields of keratinase (Park and Son 2009).

Figure 8.4 Effect of feather meal concentration on

keratinolytic protease production by B.

amyloliquefaciens 6B. (Data are means of three replicates,

error bars indicate S.E.)

8.1.5. Characterization of BA6B keratinolytic protease

Fig. 8.5 Profile of keratinolytic protease from BA6B resolved by 10% (a)

Native and (b) SDS polyacrylamide gel electrophoresis. (L1, L5: silver

stained crude enzyme; L3, L7: zymogram of crude enzyme; L2, L6: silver

stained concentrated crude enzyme, L4, L8: Zymogram of concentrate crude

enzyme; M: molecular weight markers)

The extracellular keratinolytic protease from cell free supernatant of feather meal

broth was concentrated upto 24-fold by employing 40 to 80% ammonium sulphate


167

precipitation. The molecular weight of BA6B protease was found to be ~58 kDa based

on its electrophoretic mobility in SDS-PAGE (Fig. 8.5). The molecular weight of

keratinases from other Bacillus sp. has been reported in the range of 25 to 69 kDa (Lin

et al 1992, Kojima et al 2006, Balaji et al 2008, Cai et al 2008). In contrast, Lysobacter

NCIMB 9497 and Kocuria rosea have been reported to produce higher molecular mass

keratinases (Allpress et al 2002, Bernal et al 2006).

Figure 8.6 Effect of temperature on (a) activity and (b) stability of B. amyloliquefaciens 6B

keratinolytic protease (Data are means of three replicates, error bars indicate S.E.)

The BA6B keratinolytic protease exhibited maximum activity at 50°C (Fig. 8.6a),

which is similar to reported for keratinase activity from other Bacillus sp. (Lateef et al.

2010; Daroit et al. 2011). The half life of the enzyme was found to be 44 and 19 min at

50 and 60°C, respectively (Fig. 8.6b). Although the BA6B protease has lower thermal

stability but its efficient feather degrading property suggests its potential application in

feather waste management.

The BA6B protease (Fig. 8.7a) exhibited maximum activity at pH 8.0 which is

similar to the previous reports from several other keratinolytic microorganisms (Riffel

et al 2003, Cai et al 2008). Generally, bacterial keratinases are reported to exhibit

neutral (Lateef et al 2010, Daroit et al 2011) or alkaline pH optima (Bernal et al 2006,

Prakash et al 2010, Xie et al 2010). Studies on pH stability revealed that the enzyme

was stable in the pH range of 8.0 to 11.0 retaining more than 85% activity after 1 h (Fig.

8.7b).


168

Figure 8.7 Effect of pH on (a) activity and (b) stability of B. amyloliquefaciens 6B keratinolytic

protease (Data are means of three replicates, error bars indicate S.E.)

The values of Km and Vmax of BA6B keratinolytic protease for caesin hydrolysis

were found to be 1.14 mg/mL and 1.43 mg/mL.min, respectively (Fig. 8.8).

Figure 8.8 (a) Substrate saturation curve and (b) Double reciprocal Lineweaver-Burk plot

for B. amyloliquefaciens 6B keratinolytic protease

Amongst different metal ions, Ca2+

, Zn2+

and Mn2+

were found to stimulate

enzyme activity while transition metal ions such as Cu2+

and Fe2+

strongly suppressed

the enzyme activity (Table 8.2). Such stimulatory effect of divalent metal ions on

enzyme activity has been attributed to structural alterations rather than catalytic effect

(Glusker et al 1999). Several keratinases have been reported to exhibit higher activity in

presence of Ca2+

and Mn2+

ions (Nam et al 2002, Riffel et al 2003, Corrêa et al 2010).

However, in present study Zn2+

was found to have stimulatory effect, which is contrary


169

to the observations reported by Khardenavis et al (2009), Corrêa et al (2010) and Daroit

et al (2011).

Table 8.2 Effects of various metal ions, inhibitors, surfactants and bleach oxidant on B.

amyloliquefaciens 6B protease activity

Additives (concentration) Residual enzyme activity (%)

Control 100

Metal ions (5mM) Ca2+ 130.94

Cu2+ 34.52

Fe2+ 17.04

Hg2+ 105.82

Mg2+ 99.94

Mn2+ 195.96

Zn2+ 167.24

Inhibitors (5 mM) EDTA 106.27

PMSF 1.25

Surfactants (1%; w/v) Triton X- 100 110.12

Tween 80 91.53

SDS 82.35

CTAB 21.47

Oxidising agent (1%, w/v) H2O2 94.66

Among the inhibitors tested, PMSF completely inhibited the enzyme activity

(Table 8.2), suggesting it to be a serine hydrolase. Usually, EDTA has negative effects

on keratinase activity, but we observed that the keratinolytic protease from 6B strain

was slightly simulated by EDTA (5mM), similar to the keratinase from

Fervidobacterium islandicum AW-1 (Nam et al 2002) and B. sublitis KD-N2 (Cai et al

2008).

Furthermore, the enzyme remained quiet stable in presence of non-ionic (Triton

X-100 and Tween 80) and anionic surfactant (SDS), whereas CTAB (cationic

surfactant) exerted inhibitory effect (Table 8.2). Similar stability of B. halodurans

PPKS-2 keratinase in presence of non-ionic and anionic surfactant has been reported

(Prakash et al 2010). In general as per the literature, the proteases belonging to Bacillus

genus are unstable against the bleech-oxidants (Anwar and Saleemuddin 2000).

However, BA6B protease was found to be stable in presence of H2O2. Several

researchers have engineered proteases for making them surfactant and bleach-stable

(Tuschiya et al 1992, Wolff et al 1996), whereas the BA6B protease exhibited inherent

stability towards surfactants and bleach-oxidant, which makes it suitable for use in the

detergent industry.


170

8.1.6. In-vitro screening for antifungal activity

The genus Bacillus includes species considered particularly promising as

biocontrol agents against plant pathogens (Ongena and Jacques 2008). Thus, the

bacterium BA6B was investigated for its biocontrol potential.

Figure 8.9 Antifungal activity of Bacillus amyloliquefaciens 6B against (a) Aspergillus parasiticus

NCIM 898, (b) Aspergillus niger MTCC 16404, (c) Aspergillus fumigatus MTCC 343, (d) Fusarium

oxysporum NCIM 1072, (d) Trichosporon sp. NCIM 1110 and (e) Chrysosporium indicum.

Table 8.3: Antifungal activity of Bacillus amyloliquefaciens 6B culture supernatant against fungal

phytopathogens

Test organism Inhibition Zone (mm)

Control (uninoculated broth) -

Aspergilus niger MTCC 16404 14

Aspergilus parasiticus NCIM 898 19

Aspergilllus fumigates MTCC 343 12

Fusarium oxysporum NCIM 1072 21

Trichosporon sp. NCIM 1110 17

In dual culture assays, the bacterium BA6B was found to inhibit all the test fungi

(A. niger MTCC 16404, A. fumigatus MTCC 343, A. parasiticus NCIM 898,

Trichosporon sp. NCIM 1110, F. oxysporum NCIM 1072) on PDA plates at 25°C (Fig.

8.9). Interestingly, BA6B grown in feather meal medium also inhibited mycelial growth

of all the fungi tested (Table 8.3) with maximum antagonism against F. oxysporum

(1072).


171

Figure 8.10 (a) ESI-MS spectrum of crude methanolic extract of B. amyloliquefaciens 6B, (b) ESI-

MS/MS spectrum of [M+Na]+ precursor ion at m/z 1058.6 and (c) sequence deduced on the basis of

fragmentation pattern of [M+Na]+ precursor ion.


172

Furthermore, to identify and characterize the bioactive compounds from BA6B,

the crude extract of the fermentation broth was subjected to ESI-mass spectrometry. The

peaks in ESI-MS spectra (Fig. 8.10a) at m/z, 1030.5, 1044.5, 1058.6 and 1072.6 differ in

mass by 14 Da and may be assigned as sodium adducts of C13-, C14-, C15- and C16-

fatty acid chain length variants of a same surfactin group, respectively (Vater et al.

2002). The peak at m/z 1058.6 was further subjected to MS2 analysis and on the basis of

fragment ions obtained, its sequence was deduced (Fig. 8.10b, c) The fragment ion at

m/z 1030.5 with a mass difference of 28 Da from the parent ion (m/z 1058.6) revealed

the carbonyl group coupled cleavage of lactone bond between fatty acid and C-terminal

end of peptide leading to the linearization of surfactin cyclic lipopeptide ring (Cavelier

et al 1999). The peak at m/z 707.2 in MS2 spectrum of m/z 1058.6 could be assigned as

sodiated form of internal fragment ion with molecular mass of 684 Da. The protonated

from of this fragment ion (m/z 685) has been reported to be ubiquitously detected in

MS2 spectra of surfactins [M+H]+ and is considered as characteristic marker ion for

identification of surfactins (Hue et al 2001). The sequence derived from the

fragmentation of m/z 1058.6 is: β-hydroxy fatty acid-Glu1-Leu

2-Leu

3-Val

4-Asp

5-Leu

6-

Leu7

(Fig. 8.10c). Similarly the peak at m/z, 1036.5 and 1075.4 could be assigned as

protonated [M+H]+ form and potassium adduct [M+K]

+of C15-surfactin with 1035 Da,

respectively. The m/z 1080.5 may be attributed to sodium adduct of C15-Iturin A

isoform (Pathak 2011).

Surfactin and iturin are the most commonly found antifungal lipopeptide

belonging to Bacillus genus (Ongena and Jacques 2008). Both the lipopeptides are

synthesized in a nonribosomal manner by large multienzyme complexes, exhibiting a

wide antimicrobial spectrum and exceptional surfactant activities (Ongena and Jacques

2008). They share a common cyclic structure consisting of either a β-amino (for iturin)

or β-hydroxy (for surfactin) fatty acid integrated into a peptide moiety. In addition,

surfactins show a strong synergistic action in combination with iturin (Maget-Dana et al

1992). It has also been reported that the lipopeptides also play a role to enhance

bacterial colonization on plant tissues and to induce plant resistance against pathogens

(Ongena and Jacques, 2008). Consequently, a great deal of interest has centered on the

use of such bioactive compounds in agriculture for eco-friendly and sustainable

suppression of crop diseases (Ongena and Jacques, 2008). Thus, the feather


173

hydrolysates resulting from the microbial conversion of feather keratin by BA6B may

represent a promising biocontrol agent against fungal phytopathogens.

8.1.7. Application of feather hydrolysate as nitrogenous soil input

The total nitrogen content of the amino acid rich feather hydrolysates resulting

from the conversion of feather keratin by BA6B was found to be 0.131 g/l.

Accordingly, the feather digest was concentrated five times and evaluated for its use as

organic nitrogen input on the growth of V. radiate var. meha (mung). It was observed

that the added feather hydrolysate exerted a beneficial effect on the germination and the

growth of mung bean. Plant height, plant fresh weight, root length and root fresh weight

was increased by 36%, 58%, 42%, 39%, respectively in the soil treated with feather

hydrolysate over the untreated soil (Table 8.4). Further in comparison to the reference

fertilizer in term of plant height and root length (Table 8.4), feather digest exhibited the

same effect as that of the reference fertilizer. Similar effect of feather hydrolysate as

nitrogen input has been reported by Kim et al (2005) for the growth of carrot and

Chinese cabbage. Cao et al (2012) evaluated the feather fermentation broth as a leaf

fertilizer and found 66 to 82% increase in pakchoi plant growth. Thus, the feather

hydrolysate obtained upon decomposition of feathers by BA6B, holds potential to be

applied as agriculture input (source of nitrogen and antifungal metabolites).

Table 8.4 Effect of feather hydrolysate on growth of Vigna radiate var. meha (mung) as compared

to the reference fertilizer.

Treatments Plant height

(cm)

Plant fresh weight

(g)

Root length

(cm)

Root fresh weight

(g)

None (Control) 26.4 ± 0.94 8.7 ± 0.98 15.7 ± 0.41 4.6 ± 0.64

Feather

hydrolysate 35.93 ± 0.69 13.8 ± 0.69 22.3 ± 0.86 6.4 ± 0.94

Fertilizer 37.32 ± 0.93 15.4 ± 0.52 23.8 ± 0.94 6.9 ± 0.78 Each value represents means ± S.E. of 15 replicates per treatment

8.2. Conclusion

In the present study, Bacillus amyloliquefaciens 6B isolated from feather dump

soil sample was found to efficiently degrade native feather within 24 h of incubation.

The rapid feather degrading ability of the bacterium makes this strain a potential tool for

the development of processes suitable for conversion of feather to value added products

such as feed or fertilizer. The keratinolytic protease production by B. amyloliquefaciens

6B was optimized using one-factor-at-a-time approach which resulted in 2.6-fold

increase in enzyme yield. The enzyme produced was characterized and used for the


174

preparation of feather hydrolysate, which was evaluated as nitrogenous soil input for

plant growth promotion. In addition, the strain was found to produce antifungal

metabolites. Thus, the feather hydrolysate obtained upon degradation of feather by

BA6B bacterial culture, may be useful as nitrogenous input for agricultural soils as well

as biocontrol agent against fungal phytopathogens.


175

References

Allpress JD, Mountain G, Gowland PC (2002) Production, purification and

characterization of an extracellular keratinase from Lysobacter NCIMB 9497. Lett

Appl Microbiol 34: 337-342.

Anwar A, Saleemuddin M (2000) Alkaline protease from Spilosoma obliqua: potential

application in bioformulations. Biotechnol Appl Biochem 31: 85-89.

Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JA, Smith JG, Struhl DJ

(1997) Current Protocols in Molecular Biology Unit (24). New York: John Wiley

& Sons.

Balaji S, Senthil Kumar M, Karthikeyan R, Kumar R, Kirubanandan S, Sridhar R,

Sehgal PK (2008) Purification and characterization of an extracellular keratinase

from a hornmeal-degrading Bacillus subtilis MTCC 9102. World J Microbiol

Biotechnol 24: 2741-2745.

Bernal C, Cairo J, Coello N (2006) Purification and characterization of a novel

exocellular keratinase from Kocuria rosea. Enzyme Microb Technol 38: 49-54.

Brandelli A, Riffel A (2005) Production of an extracellular keratinase from

Chryseobacterium sp. growing on raw feathers. Electron J Biotechnol 8: 35-42.

Brandelli A (2008) Bacterial keratinases: Useful enzymes for bioprocessing

agroindustrial wastes and beyond. Food Bioprocess Technol 1: 105-116.

Cai CG, Zheng XD (2009) Medium optimization for keratinase production in hair

substrate by a new Bacillus subtilis KD-N2 using response surface methodology. J

Ind Microbiol Biotechnol 36: 875-883.

Cai CG, Lou BG, Zheng XD (2008) Purification and characterization of keratinase from

a new Bacillus subtilis strain. J Zhejiang Univ Sci B 9: 60-67.

Cao Z-J, Lu D, Luo L-S, Deng Y-X, Bian Y-G, Zhang X-Q, Zhou M-H (2012)

Composition analysis and application of degradation products of whole feathers

through a large scale of fermentation. Environ Sci Pollut Res 19: 2690-2696.

Cavelier F, Enjalbal JM, Roque M, Sanchez P, Aubagnac J-L (1999) Comparison of

collisionally dissociation mass spectra for the identification of cyclopeptides and

cyclodepsipeptides. Rapid Commun Mass Spectrom 13: 880-885.

Cheng SW, Hu HM, Shen SW, Takagi H, Asano M, Tsai YC (1995) Production and

characterization of keratinase of a feather-degrading Bacillus licheniformis PWD-

1. Biosci Biotechnol Biochem 59: 2239-2243.


176

Corrêa APF, Dariot DJ, Brandelli A (2010) Characterization of a keratinase produced

by Bacillus sp. P7 isolated from an Amazonian environment. Int Biodeterior

Biodegrad 64: 1-6.

Dalev P, Ivanov I, Liubomirova A (1997) Enzymatic modification of feather keratin

hydrolyzates with lysine aimed at increasing the biological value. J Sci Food

Agric 73: 242-244.

Daroit DJ, Corrêa APF, Brandelli A (2011) Production of keratinolytic proteases

through bioconversion of feather meal by the Amazonian bacterium Bacillus sp.

P45. Int Biodeterior Biodegrad 65: 45-51.

Deydier E, Guilet R, Sarda S, Sharrock P (2005) Physical and chemical characterization

of crude meat and bone meal combustion residue: waste or raw material? J Hazard

Mater 121: 141-148.

Fang Y, Liu S, Wang S, Lv M (2009) Isolation and screening of a novel extracellular

organic solvent-stable protease producer. Biochem Eng J 43: 212-215.

Fakhfakh N, Ktari N, Haddar A, Mnif IH, Dahmen I, Nasri M (2011) Total

solubilisation of the chicken feathers by fermentation with a keratinolytic

bacterium, Bacillus pumilus A1, and the production of protein hydrolysate with

high antioxidative activity. Process Biochem 46: 1731-1737.

FAO (2002) http://www.fao.org/docrep/004/AD452E/ad452e30.htm (retrieved on

28/05/2012)

Ghorbel B, Sellami-Kamoun A, Nasri M (2003) Stability studies of protease from

Bacillus cereus BG1. Enzyme Microb Technol 32: 513-518.

Glusker JP, Katz AK, Bock CW (1999) Metal ions in biological systems. The Rigaku J

16: 8-16.

Grazziotin A, Pimentel FA, de Jong EV, Brandelli A (2006) Nutritional improvement of

feather protein by treatment with microbial keratinase. Anim Feed Sci Technol

126: 135-144.

Gupta R, Ramnani P (2006) Microbial keratinase and their prospective application: an

overview. Appl Microbiol Biotechnol 70: 21-33.

Hossain MS, Azad AK, Abu Sayem SM, Mostafa G, Hoq MM (2007) Production and

partial characterization of feather-degrading keratinolytic serine protease from

Bacillus licheniformis MZK-3. J Biol Sci 7: 599-606.


177

Hue N, Serani L, Laprevote O (2001) Structural investigation of cyclic peptidolipids

from Bacillus subtilis by high energy tandem mass spectrometry. Rapid Commun

Mass Spectrom 15: 203-209.

Inoue A, Horikoshi K (1991) Estimation of solvent-tolerance of bacteria by the solvent

parameter logP. Journal Ferment Bioeng 71: 194-196.

Jeong J-H, Lee, O-M, Jeon Y-D, Kim J-D, Lee N-R, Lee C-Y, Son H-J (2010)

Production of keratinolytic enzyme by a newly isolated feather-degrading

Stenotrophomonas maltophilia that produces plant growth-promoting activity.

Process Biochem 45: 1738-1745.

Kaul S, Sumbali G (1999) Production of extracellular keratinases by keratinolytic

fungal species inhabiting feathers of living poultry birds (Gallus domesticus): a

comparison. Mycopathologia 146: 19-24.

Khardenavis AA, Kapley A, Purohit HJ (2009) Processing of poultry feathers by

alkaline keratin hydrolyzing enzyme from Serratia sp. HPC1383. Waste Manage

29: 1409-1415.

Kim JM, Choi YM, Suh HJ (2005) Preparation of feather digests as fertilizer with B.

pumilus KHS-1. J Microbiol Biotechnol 15: 472-476.

Kojima M, Kanai M, Tominaga M, Kitazume S, Inoue A, Horikoshi K (2006) Isolation

and characterization of a feather degrading enzyme from Bacillus pseudofirmus

FA30-01. Extremophiles 10: 229-235.

Kunert J (2000) Physiology of keratinophylic fungi. In: Kushwaha RKS, Guarro J

(Eds.) Biology of dermatophytes and other keratinophylic fungi. Bilbao: Rev

Iberoamericana de Micología, pp. 77-85.

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of

bacteriophage T4. Nature 227: 680-685.

Lateef A, Oloke JK, Gueguim Kana EB, Sobowale BO, Ajao SO, Bello BY (2010)

Keratinolytic activities of a new feather-degrading isolate of Bacillus cereus LAU

08 isolated from Nigerian soil. Int Biodeterior Biodegrad 64: 162-165.

Li S, He B, Bai Z, Ouyang P (2009) A novel organic solvent-stable alkaline protease

from organic solvent-tolerant Bacillus licheniformis YP1A. J Mol Catal B 56: 85-

88.

http://www.sciencedirect.com/science/journal/09648305


178

Lin X, Lee CG, Casale ES, Shih JCH (1992) Purification and characterization of a

keratinase from a feather degrading Bacillus licheniformis strain. Appl Environ

Microbiol 58: 3271-3275.

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the

Folin phenol reagent. The J Biol Chem 193: 265-275.

Maget-Dana R, Thimon L, Peypoux F, Ptak M (1992) Surfactin/iturin A interactions

may explain the synergistic effect of surfactin on the biological properties of iturin

A. Biochimie 74: 1047-1051.

Nam GW, Lee DW, Lee HS, Lee NJ, Kim BC, Choe EA, Hwang JK, Suhartono MT,

Pyun YR (2002) Native-feather degradation by Fervidobacterium islandicum

AW-1, a newly isolated keratinase-producing thermophilic anaerobe. Arch

Microbiol 178: 538-547.

Odetallah NH,Wang JJ, Garlich JD, Shih JCH (2003) Keratinase in starter diets

improves growth of broiler chicks. Poult Sci 82: 664-670.

Ongena M, Jacques P (2008) Bacillus lipopeptides: versatile weapons for plant disease

biocontrol. Trends Microbiol 16: 115-125.

Onifade AA, Al-Sane NA, Al-Musallam AA, Al-Zarban S (1998) A review: potentials

for biotechnological applications of keratin-degrading microorganisms and their

enzymes for nutritional improvement of feathers and other keratins as livestock

feed resources. Biores Technol 66: 1-11.

Park GT, Son HJ (2009) Keratinolytic activity of Bacillus megaterium F7-1, a feather

degrading mesophilic bacterium. Microbiol Res 164: 478-485.

Pathak KV (2011) Purification and characterization of antifungal compounds produced

by Banyan endophytic Bacilli. Ph. D. Thesis, Sardar Patel University, Vallabh

Vidynagar, India.

Prakash P, Jayalakshmi SK, Sreeramulu K (2010) Production of keratinases by free and

immobilized cells of Bacillus halodurans strain PPKS-2: partial charcaterization

and its application in feather degradation and dehairing of the goat skin. Appl

Biochem Biotechnol 160: 1909-1920.

Raaijmakers JM, Vlami M, Souza JT (2002) Antibiotic production by bacterial

biocontrol agents. Antonie Leeuwenhoek 81: 537-547.


179

Ramnani P, Gupta G (2004) Optimization of medium composition for keratinase

production on feather by Bacillus licheniformis RG1 using statistical methods

involving response surface methodology. Biotechnol Appl Biochem 40: 191-196.

Riffel A, Lucas FS, Heeb P, Brandelli A (2003) Characterization of a new keratinolytic

bacterium that completely degrades native feather keratin. Arch Microbiol 179:

258-265.

Sambrook J, Russell DW (2001) Molecular Cloning: A Laboratory Manual (3rd ed),

New York: Cold Spring Harbor Laboratory Press.

Sareen R, Mishra P (2008) Purification and characterization of organic solvent stable

protease from Bacillus licheniformis RSP-09-37. Appl Microbiol Biotechnol 79:

399-405.

Sikkema J, de Bont JAM, Poolman B (1995) Mechanisms of membrane toxicity of

hydrocarbons. Microbiol Rev 59: 201-222.

Torres S, Pandey A, Castro GR (2011) Organic solvent adaptation of Gram positive

bacteria: applications and biotechnological potentials. Biotechnol Adv 29: 442-52.

Trivedi P, Pandy A, Palni LM (2008) In vitro evaluation of antagonistic properties of

Pseudomonas corrugata. Microbiol Res 163: 329-336.

Tuschiya K, Nakamura Y, Sakashita H, Kimura T (1992) Purification and

characterization of a thermostable alkaline protease from alkalophilic

Thermoactinomyces sp. HS682. Biosci Biotechnol Biochem 56: 246-250.

Vasileva-Tonkova E, Gousterova A, Neshev G (2009) Ecologically safe method for

improved feather wastes biodegradation. Int Biodeterior Biodegrad 63: 1008-

1012.

Vater J, Kablitz B, Wilde C, Franke P, Mehta N, Cameotra SS (2002) Matrix-assisted

Laser Desorption Ionization-Time of Flight mass spectrometry of lipopeptide

biosurfactants in whole cells and culture filtrates of Bacillus subtilis C-1 isolated

from petroleum sludge. Appl Environ Microbiol 68: 6210-6219.

Wang JJ, Shih JCH (1999) Fermentation production of keratinase from Bacillus

licheniformis PWD-1 and a recombinant B. subtilis FDB-29. J Ind Microbiol

Biotechnol 2: 608-616.

Wolff AM, Showell MS, Venegas MG, Barnett BL, Wertz WC (1996) Laundry

performance of subtilisin proteases. In: Bott R, Betzel C (Eds.) Subtilisin

enzymes: practical protein engineering. New York: Plenum Press, pp. 113-120.

http://www.sciencedirect.com/science/journal/09648305


180

Xie F, Chao Y, Yang X, Yang J, Xue Z, Luo Y, Qian S (2010) Purification and

characterization of four keratinases produced by Streptomyces sp. strain 16 in

native human foot skin medium. Biores Technol 101: 344-350.

Documents

Chapter 8shodhganga.inflibnet.ac.in/bitstream/10603/34582/13/13_chapter8.pdf · Anjali Bose, Shabnam Pathan, Khyati Pathak and Haresh Keharia (2012). Keratinolytic ... Profile of