De-hairing protease production by an isolated Bacillus cereus strain AT under solid-state fermentation using cow dung: Biosynthesis and properties

ORIGINAL ARTICLE

De-hairing protease production by an isolated Bacillus cereus strain AT under solid-state fermentation using cow dung: Biosynthesis and properties

Ponnuswamy Vijayaraghavan

^a,*

, Sophia Lazarus

^b

, Samuel Gnana Prakash Vincent

^a

aInternational Centre for Nanobiotechnology, Centre for Marine Science and Technology, Manonmaniam Sundaranar University, Rajakkamangalam 629 502, Kanyakumari District, Tamil Nadu, India

bDepartment of Biotechnology, Holycross College, Nagercoil, Kanyakumari District, India

Received 5 February 2013; revised 16 April 2013; accepted 19 April 2013 Available online 6 May 2013

KEYWORDS Cow dung;

Solid-state fermentation;

Bacillus cereusstrain AT;

Alkaline protease;

De-hairing

Abstract Agro-industrial residues and cow dung were used as the substrate for the production of alkaline protease byBacillus cereusstrain AT. The bacterial strainBacillus cereusstrain AT pro- duced a high level of protease using cow dung substrate (4813 ± 62 U g ¹). Physiological fermen- tation factors such as the incubation time (72 h), the pH (9), the moisture content (120%), and the inoculum level (6%) played a vital role in the enzyme bioprocess. The enzyme production improved with the supplementation of maltose and yeast extract as carbon and nitrogen sources, respectively.

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and zymogram analysis of the purified protease indicated an estimated molecular mass of 46 kDa. The protease enzyme was stable over a temperature range of 40–50C and pH 6–9, with maximum activity at 50C and pH 8. Among the divalent ions tested, Ca²⁺, Na⁺and Mg²⁺showed activities of 107 ± 0.7%, 103.5 ± 1.3%, and 104.6 ± 0.9, respectively. The enzyme showed stability in the presence of surfactants such as sodium dodecyl sulfate and on various commercially available detergents. The crude enzyme effec- tively de-haired goat hides within 18 h of incubation at 30C. The enzymatic properties of this pro- tease suggest its suitable application as an additive in detergent formulation and also in leather processing. Based on the laboratory results, the use of cow dung for producing and extracting

* Corresponding author. Tel.: +91 09486007351.

E-mail address:venzymes@gmail.com(P. Vijayaraghavan).

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enzyme is not cumbersome and is easy to scale up. Considering its cheap cost and availability, cow dung is an ideal substrate for enzyme bioprocess in an industrial point of view.

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1. Introduction

Proteases constitute one of the commercially important groups of extracellular microbial enzymes widely used in several indus- trial sectors such as detergent, food, pharmaceutical, chemical, leather and silk, apart from waste treatment. In recent years, the use of alkaline proteases in a variety of industrial pro- cesses like detergents, food, leather and silk has increased remarkably (Gessesse, 1997;Kembhavi et al., 1993). Till date, even though a wide range of micro-organisms are known to produce alkaline proteases, a large proportion of commer- cially available alkaline proteases are now being produced from Bacillus strains because of its ability to secrete large amounts of alkaline proteases having significant proteolytic activity and stability at considerably high pH and tempera- tures (Jacobs, 1995; Yang et al., 2000). These strains can be cultivated under extreme temperatures and pH conditions to give rise to products that are, in turn, stable over a wide range of harsh environments (Han and Damodaran, 1997).

Microbial proteases, especially fromBacillussp. are the most widely exploited industrial enzymes with major applications in detergent formulations (Johnvesly and Naik, 2001). But, alkaline proteases that find application for industrial pur- poses face some limitations, such as, lack of enzyme activity and stability towards sodium dodecyl sulfate (SDS) and H₂O₂ which have been the common ingredients in modern-bleach based detergent formulations (Joo et al., 2003). As a result, the demand for highly active preparations of proteolytic en- zymes with appropriate specificity and stability over a wide range of pH, temperature and retention of activity in the presence of ions and organic solvents continues to stimulate the search for new enzyme sources (Rajkumar et al., 2011).

The use of enzyme based products is currently being explored in many areas of leather making process, with increasing importance in the dehairing process, thus eliminating the use of hazardous sodium sulfide (Thanikaivel- ann et al., 2004). Due to the increasing demand of enzyme in the leather industry, there arises a need for new proteases (Shrinivas and Naik, 2011).

From an industrial point of view, it is estimated that around 30–40% of the production cost of enzymes is ac- counted for the cost of the growth medium (Joo et al., 2003).

Solid-state fermentation (SSF) processes are therefore, of spe- cial economic interest for countries having abundant biomass and agro-industrial residues, as they can be used as cheap raw materials (Tunga et al., 2001). Apart from these agro- industrial residues, in recent years, more attention has also been paid in utilizing the solid waste substrates such as: feather meal and corn steep liquor (De Azeredo et al., 2006); protein- aceous shrimp shell powder (Wang et al., 2008); proteinaceous tannery solid waste (Ravindran et al., 2011); sardinelle (Sardi- nella aurita) powder (Sellami-Kamoun et al., 2011) and shrimp shell waste (Ghorbel-Bellaaj et al., 2012) for the production of proteases. Considering the cost of the fermentation medium for enzyme production, an attempt was made to use cheap

and easily available cow dung as an effective substrate along with other agro-industrial residues. Reports on SSF of cow dung for the production of alkaline protease using Bacillus sp. are limited (Vijayaraghavan et al., 2012). Hence, the pres- ent investigation aimed to exploit cow dung that is cheap and globally available for alkaline protease production. This paper also reports some properties of the purified alkaline pro- tease and its potential application in the detergent- and leather processing industries.

2. Materials and methods

2.1. Bacterial isolation, media, and culture conditions

The bacterium,Bacillus cereusstrain AT was isolated from fer- mented rice and was cultivated aerobically at 37C in the med- ium for protease production which contained (g L ¹): peptic digest of animal tissue 5, beef extract 1.5, yeast extract 1.5, so- dium chloride 5, skim milk 10, pH 8. Morphological, physio- logical, and biochemical characteristics of the potent isolate B. cereus strain AT was studied as described by Holt et al.

(1994). The 16S rDNA gene was sequenced after genomic DNA extraction and PCR amplification as described elsewhere (Riffel et al., 2003).

2.2. Protease assay

The protease activity was determined as described byKumar et al. (1999) with little modification. The assay mixture con- tained 1 mL of casein solution (1%, w/v in 0.05 mol L ¹ tris–HCl buffer, pH 8) and 0.1 mL of enzyme. Following incu- bation for 30 min at 37C, 4 mL of trichloroacetic acid (10%) was added and mixed. This mixture was filtered through What- man 1 and the optical density was measured at 280 nm against the blank. One unit of enzyme activity is defined as the amount of the enzyme required to liberate 1lg of tyrosine min ¹ at 37C under assay conditions. The total protein content was estimated as per the method ofBradford (1976), using Bovine Serum Albumin as standard.

2.3. Inoculum

Inoculum was prepared by growingB. cereusstrain AT on a medium that contained (g L ¹): peptic digest of animal tissue 5, beef extract 1.5, yeast extract 1.5, and sodium chloride 5 (pH 8). This was sterilized at 121C for 20 min and cooled.

After that, a loopful culture ofB. cereusstrain AT was inocu- lated into this nutrient medium with rotary shaking at 150 rpm at 37C for 24 h and was stored at 2–8C until further use.

2.4. Solid state fermentation (SSF)

Apple peels, banana peels, cow dung, paddy straw and wheat bran were used as the substrate for the production of alkaline

protease. All substrates were dried (sun drying) for several days and further dried at 60C for 1 h. SSF was carried out separately in a 250 mL Erlenmeyer flask containing 10 g of cow dung and other substrates were moistened with 10 mL of buffer (tris–HCl buffer, 0.1 mol L ¹, pH 8). The medium was sterilized at 121C for 20 min. After cooling, the flasks were inoculated with 5% (v/w) inoculum (1.036 OD at 600 nm) and incubated at 37C for various time periods (12, 24, 36, 48, 60, 72, 84, and 96 h).

2.5. Optimization of process parameters

Cow dung was used as a substrate for the media optimization study. The process parameters studied were fermentation per- iod (12–96 h), pH (6–10), moisture content (60–180%, v/w), inoculum (2–12%, v/w), particle size (0.5–4.4 mm), carbon sources (1%, w/w) (glucose, lactose, trehalose, maltose, xylose, and starch), and nitrogen sources (1%, w/w) (gelatin, ammo- nium nitrate, peptone, yeast extract, urea, and casein). Results reported in this study are averages of triplicate findings.

2.6. Extraction and purification of an enzyme

Protease was extracted from the fermented medium using 100 mL of tris–HCl buffer (pH 8, 0.05 mol L ¹) by squeezing through a muslin cloth and centrifuging at 10,000g for 30 min at 4C. The clear supernatant was used as the crude en- zyme. This crude enzyme was precipitated with ammonium sulfate (70% saturation) and the enzyme precipitate obtained was centrifuged at 10,000gfor 10 min at 4C. The resulting pellet was dissolved in a small amount of 0.05 mol L ¹tris–

HCl buffer (pH 8), and this enzyme preparation was loaded for gel-filtration chromatography using a Sephadex G-75 col- umn (0.6·50 cm) equilibrated with the same buffer. The flow rate was adjusted to 0.5 mL min ¹. The fractions containing protease activity were pooled, concentrated and used for fur- ther studies.

2.7. SDS–PAGE and zymography

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was carried out according to Laemmli (1970) using a 11%

crosslinked polyacrylamide gel. Silver staining was performed to visualize the protein bands. Casein zymography was carried out using a 11% polyacrylamide slab gel containing SDS and 1% casein in separating gel as described by Heussen and Dowdle (1980) at 4C. After electrophoresis, the gel was soaked three times for 20 min in 2.5% (v/v) Triton x-100 to remove the SDS. The gel was stained with 0.1% coomassie brilliant blue R-250 in methanol–glacial acetic acid–water (40:7:53) followed by destaining with methanol–glacial acetic acid–water (40:7:53). Enzyme activity was visualized by incu- bating the gel for 12 h in 0.05 mol L ¹at room temperature.

2.8. Enzyme characterization

The protease activity was evaluated using the standard assay method in the following buffer systems at 0.1 mol L ¹concen- trations in the reaction mixture: citrate buffer, pH 4; succinate buffer, pH 5; sodium phosphate buffer, pH 6 to 7; tris-buffer,

pH 8, and glycine–NaOH buffer, pH 9 and 10, respectively. To check the pH stability, 25ll of the enzyme solution was mixed with 175ll of the buffer solutions (pH 4–10) and aliquots of the mixture were taken to measure the protease activity under standard assay conditions after incubation for 1 h. The effect of temperature on enzyme activity was studied by conducting the reactions at various temperatures (30, 40, 50, 60, and 70C) using the standard assay method. To evaluate the heat stability of the protease, the purified protease was denatured at various temperatures ranging from 30 to 70C for 1 h. To evaluate the effect of ions (0.01 mol L ¹) on enzyme activity, the enzyme sample was pre-incubated at 37C for 1 h and the relative activity was evaluated. To examine the effect of surfactants and detergents on the enzyme activity, several sur- factants and detergents were added to the enzyme solution at the indicated concentration and allowed to stand for 1 h at room temperature after which the remaining activities were measured. The enzyme activity of a control sample (without any surfactant and detergent) was taken as 100%. De-hairing property of the enzyme was carried out at room temperature (pH 9) for 18 h with crude enzyme preparation from an indus- trial point of view.

3. Results and discussion

3.1. Identification and screening of the isolate, B. cereus strain AT for protease secretion

TheB. cereusstrain AT was isolated from the fermented rice as described under Materials and Methods, which could be cata- lase-, and oxidase-positive. Indole was not produced and the strain was found to be positive for the fermentation of simple sugars like glucose, lactose, arabinose, and mannitol. The iso- late could hydrolyse casein and was negative for cellulose fer- mentation. Based on phenotypical characteristics and on the 16S rRNA sequence data, the strain was identified asB. cereus strain AT. The 970 bp nucleotide sequence was submitted to the Genbank under the accession number JQ 425477.

3.2. Evaluation of agro-residues for de-hairing protease production

The results in the present study indicated that the production of alkaline protease was higher in cow dung (4813 ± 62 U g ¹ material) than other agro-industrial residues (Fig. 1). Wheat bran supported 63.5 ± 4.3% protease produc- tion when compared with the cow dung substrate. The result showed that protease production byB. cereusstrain AT varied with the type of substrate. The selection of an ideal agro- biotech waste for enzyme production in a solid-state fermenta- tion process depends upon several factors, mainly related with cost and availability of the substrate material and this may in- volve screening of several agro-industrial residues (Pandey et al., 2000). The present study revealed the capability of cow dung as an ideal substrate for the production of alkaline prote- ase than the well studied wheat bran. It was interesting to note that, none of the reports was evidenced on the production of alkaline protease byB. cereususing cow dung as a substrate.

At first, we used cow dung as a substrate for the production of protease from Halomonas sp. PV1 (Vijayaraghavan and Vincent, 2012) andBacillus subtilisstrain VV (Vijayaraghavan

et al., 2012). The present study indicated the suitability of this substrate because of the presence of sufficient nutrients and the ability to remain loose under moisture conditions that pro- vide a large surface area for micro-organisms.

3.3. Effect of fermentation period and pH on de-hairing protease secretion

The effect of fermentation period on protease production was conducted for a period of 96 h of incubation at 37C and it reached maximum activity after 72 h of incubation (4035 ± 89 U g ¹ material) (Fig. 2). Results of this study showed that protease production increased with incubation time up to 72 h, after which the enzyme activity decreased con- siderably. These results are in accordance with observations made byKumar and Parrack (2003)withBacillussp. andOka- for and Anosike (2012)withBacillussp. SW2. This isolate was capable of producing protease in the pH range of 6–10.

The production of protease was maximum at pH 9 (5278 ± 103 U g ¹material) and substantially decreased when it was above and below pH 9. Hence, in subsequent experiments, the pH of the fermentation medium was kept at 9. At a higher pH, the metabolic action of the bacterium could have been suppressed, thus decreasing the enzyme production.

Similar trends have been observed in SSF of protease by Bacillus sp. (Prakasham et al., 2006; Okafor and Anosike, 2012).

3.4. Effect of moisture, inoculum and particle size

In the present study, maximum enzyme production was ob- served with 120% moisture content. Enzyme production was 3825 ± 75, 4515 ± 83, 5137 ± 152, 5514 ± 103, 5386 ± 149, 5311 ± 107, and 5241 ± 56 U g ¹ material at moisture levels of 60%, 80%, 100%, 120%, 140%, 160%, and 180%, respectively. Among the several factors that are important for microbial growth and enzyme production under solid state fermentation using particular substrates, moisture content/water activity is one of the most critical factors (Pandey et al., 2000;Nigam and Singh, 1994). Cow dung did not show much variation for the production of alkaline prote- ase at 100–140% of moisture. Even at 180% moisture level, no free water was found in the Erlenmeyer flask. These results are in accordance with the observations made with an alkalophilic B. subtilisstrain VV (Vijayaraghavan et al., 2012). The opti- mum moisture content varied with Bacillus sp. based on the substrate used for fermentation process (Rajkumar et al., 2011; Prakasham et al., 2006). The size of the inoculum is an important biological factor, which also determines the biomass production in the fermentation process. The result showed that there was a significant increase in alkaline protease production with an increase in inoculum size up to an optimum level of 6%, after which the enzyme yield reduced. The enzyme production was 3528 ± 102, 4903 ± 120, 5239 ± 134, 5144 ± 101, 5011 ± 168, and 4828 ± 140 U g ¹ material at 2%, 4%, 6%, 8%, 10%, and 12%, respectively. These results were in accordance with the observation made with other Bacillusstrains (Rajkumar et al., 2011). The particle size also greatly influenced the production of alkaline protease. The maximum enzyme production (5715 U g ¹material) was ob- served with approximately 1 mm particle size and decreased considerably in other particle sizes. The protease production was 4500 ± 143, 3892 ± 112, and 3740 ± 89 U g ¹ material for 0.5–0.7, 1.8–2.2 and 3.8–4.4 mm particle sizes respectively.

In the solid-state fermentation process, the availability of sur- face area plays a vital role for microbial attachment, mass transfer of various nutrients and the substrates and sub- sequent growth of microbial strain and product production (Prakasham et al., 2006). These results are in accordance with the literature data on particle size-mediated influence on microbial enzyme production in B. subtilis (Krishna and Chandrasekaran, 1996).

3.5. Effect of carbon and nitrogen

The choice of the carbon and nitrogen sources has a major influence on the yield of protease. The results showed that there was a significant increase in alkaline protease production with various tested carbon and nitrogen sources. Among the carbon sources tested, maltose supported maximum production of protease (10379 ± 168 U g ¹). The enzyme pro- duction was found to be 8370 ± 203, 8821 ± 229 and 9597 ± 152 U g ¹ for lactose, trehalose, and starch, respec- tively. In order to determine the optimum concentration of the carbon sources, enzyme production was investigated by supplementation of maltose (0.5–2%). The maximum produc- tion (10721 ± 217 U g ¹) was observed at 1% maltose. These results were in accordance with reported alkaline protease pro- Figure 2 Influence of fermentation period on alkaline protease

production under solid-state fermentation with cow dung.

Figure 1 Evaluation of agro-industrial residues and cow dung for the production of alkaline protease.

duction in the presence of different sugars (Ellaiah et al., 2002;

Vijayaraghavan et al., 2012). Among the nitrogen sources tested, yeast extract showed the maximum production of protease at 9920 ± 164 U g ¹, followed by peptone (9885 ± 203 U g ¹).

The enzyme production was found to be 6382 ± 152, 6328 ± 187, 8770 ± 192 and 9873 ± 178 U g ¹ for gelatin, ammonium nitrate, urea and casein, respectively. Similar observations were noticed in the case of protease production by different microbial species (Pandey et al., 2000; Prakasham et al., 2006). When different concentrations of yeast extract were tested, the yeast extract at 2% supported the maximum protease production with 11275 ± 237 U g ¹material. In or- der to understand the combined effect of carbon and nitrogen sources, the enzyme was produced in an optimized concentra- tion. To evaluate the combined effect of environmental and nutritional factors on protease production, the experiment was run under optimized conditions and the results are described in Fig. 3. Enzyme production was 11612 ± 163 U g ¹ after 72 h incubation at 37C in optimized conditions.

3.6. Enzyme purification and molecular weight

The crude protease sample was precipitated with 70% satura- tion of ammonium sulfate, dissolved in 0.05 mol l ¹tris–HCl buffer and loaded onto Sephadex G-75 column. In the crude extract, the enzyme activity was 11,400 U g ¹with 680 U mg ¹ protein with a yield of 100%. The ammonium sulfate purifica- tion step showed 1065 U mg ¹ protein with the yield being 67.2%. The specific activity increased to 2670 U mg ¹protein on Sephadex G-75 column chromatography with a 29% yield.

Gel filtration chromatography was still the major and conve- nient technique for protease purification of B. cereus strain AT. A similar kind of gel filtration chromatography has been used for the isolation of protease with Bacillus circulans (Subba Rao et al., 2009). In SDS–PAGE, the purified enzyme migrated as a single band with an apparent molecular weight of 46 kDa as well as zymography analysis (Fig. 4a and b).

These results are in accordance with literature reports where most of the molecular mass of protease from Bacillus genus is less than 50 kDa (Sousa et al., 2007). InB. cereus MCM B-326, the molecular weight of the protease was 45 kDa (Zambare et al., 2007) and was 39.5 kDa with B. circulans (Subba Rao et al., 2009).

3.7. Optimum pH and temperature of the enzyme

The enzyme was active in the pH range of 6–9, with the opti- mum activity at pH 9, suggesting that it is an alkaline protease.

At pH 8 and 9, the relative enzyme activity was 98.5 ± 0.8%

and 100%, respectively. Moreover, the enzyme activity fell to 47.5 ± 1.8% at pH 10. When the enzyme was incubated with different buffers for 1 h, the protease was very stable over a pH range of 6–10. However, there was more than 61 ± 3.9%

activity loss for pH 10 and more than a 95.4 ± 2.1% activity loss for pH below 6 (Fig. 5a). Similar results were reported withBacillus sp RRM1, B. cereusMCM B-326 and Bacillus sp. (Rajkumar et al., 2011; Nilegaonkar et al., 2007; Haile and Gessesse, 2012). The protease activity was highly stable at pH 9 after 1 h incubation. These results were in accordance with the observations made with Bacillus sp. (El-Hadj-Ali et al., 2007). Generally, the commercial microbial proteases have pH optima in the alkaline range; between 8 and 12 (Rao et al., 1998) and the Bacillussp. fell at this range. The maximum protease activity recorded was between 40 and 50C under standard reaction conditions, while the activity decreased rapidly above 60C. By analyzing the thermal sta- bility, the protease was found to be stable up to 50C for 1 h incubation, but lost approximately 69 ± 1.4% of its activ- ity at 60C (Fig. 5B). A similar result was reported withBacil- lus licheniformisRKK-04,B. subtilisRTSBA6.00 andBacillus sp. (Toyokawa et al., 2010; Shinde et al., 2012; Haile and Gessesse, 2012).

3.8. Effect of metal ions

In response to various chemical substances, results indicated that the stimulated activity by Ca²⁺ions, Na⁺and Mg²⁺ions showed 107 ± 0.7%, 103.5 ± 1.3% and 104.6 ± 0.9%, respectively. The enzyme activity was 87.5 ± 0.4% for Co²⁺

and was 73 ± 2.1% for Mn²⁺. Hg²⁺, Cu²⁺, Zn²⁺ and Fe²⁺ showed enzyme activities of 84 ± 0.4%, 78 ± 0.7%, 68.5 ± 1.1%, and 59.4 ± 1.4% respectively. Among the ions tested Ca²⁺ ions positively regulated enzyme activity. The Figure 4 (a) SDS–PAGE analysis of the purified protease. Lane 1. Crude enzyme. Lane 2. Ammonium sulfate precipitated sample.

Lane 3. Purified protease by Sephadex G-75. Lane 4. Molecular mass marker: 97.4-phosphorylase b: 66-bovine serum albumin: 43- ovalbumin: 29-carbonic anhydrase. (b) Zymography of purified protease.

Figure 3 Effect of maltose and yeast extract on alkaline protease production by Bacillus cereus strain AT under solid state fermentation.

Ca²⁺ ion dependent activity improvement indicated that the enzyme required calcium ions for its optimal activity and stability. This phenomenon might be attributed to calcium ion involvement in stabilization of the enzyme’s molecular structure as reported in some of the proteases derived from Bacillus sp. (Sana et al., 2006). These results were in accordance with the observations made withBacillus sp. and Bacillus megaterium RRM2 (Gupta et al., 2005; Rajkumar et al., 2011).

3.9. Effect of surfactants and detergents

The stability of alkaline proteases towards surfactants was determined by incubating the enzyme with surfactants (1%) and detergents (1% and 10%) for 1 h at room temperature.

The enzyme produced byB. cereusstrain AT was stable to- wards surfactants like SDS, Tween-20, Tween-80 and Triton X-100 and the relative enzyme activities were 118.2 ± 2.4%, 124 ± 1.1%, 107 ± 0.8%, and 128 ± 3.9%, respectively.

The protease from B. cereusstrain AT showed stability to- wards the surfactants like SDS. Similar kind of result was re- ported with B. circulans and B. cereus strain CA15 (Subba Rao et al., 2009; Uyar et al., 2011). Because of its stability in SDS, this enzyme may be of value for the formulation of deter- gents. Studies revealed that the purified enzyme was stable to- wards almost all tested detergents. The relative enzyme activities were 133 ± 5.2%, 129 ± 3.1%, 127 ± 2.9%, 124 ± 6.3%, 128 ± 0.8%, 72 ± 2%, and 137 ± 3.3% for

commercial detergents, namely, Ujala, Aircel-oxyblue, Tide, Henko, Mr. White, Surf excel, and Sun light, respectively. This result was in accordance with the result reported with other Bacillus sp. and B. licheniformis NCIM-2042 (El-Hadj-Ali et al., 2007; Bhunia et al., 2011). The enzyme activity decreased 5–18% at 10% detergent concentrations when compared with 1%.

3.10. De-hairing of skin

Protease from B. cereuswas found to be effective in leather processing (Zambare et al., 2010). To evaluate the effect of alkaline proteases on goat hide, it was incubated with a crude protease sample for 18 h (pH 9) at room temperature. The en- zyme produced byB. cereusstrain AT revealed its activity on goat hides and removed fine hairs. The alkaline protease from this organism effectively dehaired the goat hide within 18 h of incubation (Fig. 6). There is not much published literature available concerning enzymatic dehairing process gaining importance as an alternative chemical methodology and this process is significant in the reduction of toxicity, in addition to the improvement of leather quality (Sivasubramanian et al., 2008). This enzyme was non-keratinolytic and non- collagenolytic in nature. Thus these results indicate that the crude enzyme can be a better option for dehairing applications.

These results are highly significant than the observations made withB. licheniformisRP1 (Haddar et al., 2011) andB. circulans (Subba Rao et al., 2009).

Figure 5 (a) Optimum pH of the protease activity and stability. (b) Optimum temperature of the protease activity and its stability.

4. Conclusion

In conclusion, very few reports have been published on the use of cow dung as a substrate for enzyme bioprocess. In the pres- ent study, a de-hairing alkaline protease was produced using the cow dung substrate in solid-state fermentation.

Considering its cheap cost and availability, cow dung is an ideal substrate for enzyme bioprocess on an industrial point of view.

The alkaline protease fromB. cereusstrain AT was active in a range of temperatures and pH and was detergent stable and pos- sessed dehairing properties. The enzymatic properties of the alkaline protease suggest its suitable application as an additive in detergent formulation and in the leather industry.

Acknowledgements

One of the authors, P. Vijayaraghavan is thankful to the Council of Scientific and Industrial Research, New Delhi, India for financial support in the form of a Senior Research Fellowship.

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