De-hairing protease production by an isolated Bacillus cereus ... - Core

3 downloads 0 Views 832KB Size Report
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 ...

Saudi Journal of Biological Sciences (2014) 21, 27–34

King Saud University

Saudi Journal of Biological Sciences


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 a International Centre for Nanobiotechnology, Centre for Marine Science and Technology, Manonmaniam Sundaranar University, Rajakkamangalam 629 502, Kanyakumari District, Tamil Nadu, India b Department 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 cereus strain AT; Alkaline protease; De-hairing

Abstract Agro-industrial residues and cow dung were used as the substrate for the production of alkaline protease by Bacillus cereus strain AT. The bacterial strain Bacillus cereus strain AT produced a high level of protease using cow dung substrate (4813 ± 62 U g 1). Physiological fermentation 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 40–50 C and pH 6–9, with maximum activity at 50 C 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 effectively de-haired goat hides within 18 h of incubation at 30 C. The enzymatic properties of this protease 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: [email protected] (P. Vijayaraghavan). Peer review under responsibility of King Saud University.

Production and hosting by Elsevier 1319-562X ª 2014 Production and hosting by Elsevier B.V. on behalf of King Saud University.


P. Vijayaraghavan et al. 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. ª 2014 Production and hosting by Elsevier B.V. on behalf of King Saud University.

1. Introduction Proteases constitute one of the commercially important groups of extracellular microbial enzymes widely used in several industrial 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 processes 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 commercially 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 temperatures (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 from Bacillus sp. 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 purposes 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 enzymes 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 (Thanikaivelann 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 accounted for the cost of the growth medium (Joo et al., 2003). Solid-state fermentation (SSF) processes are therefore, of special 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 agroindustrial 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); proteinaceous shrimp shell powder (Wang et al., 2008); proteinaceous tannery solid waste (Ravindran et al., 2011); sardinelle (Sardinella 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 present 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 protease 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 cereus strain AT was isolated from fermented rice and was cultivated aerobically at 37 C in the medium for protease production which contained (g L 1): peptic digest of animal tissue 5, beef extract 1.5, yeast extract 1.5, sodium chloride 5, skim milk 10, pH 8. Morphological, physiological, 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 by Kumar et al. (1999) with little modification. The assay mixture contained 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 incubation for 30 min at 37 C, 4 mL of trichloroacetic acid (10%) was added and mixed. This mixture was filtered through Whatman 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 1 lg of tyrosine min 1 at 37 C under assay conditions. The total protein content was estimated as per the method of Bradford (1976), using Bovine Serum Albumin as standard. 2.3. Inoculum Inoculum was prepared by growing B. cereus strain 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 121 C for 20 min and cooled. After that, a loopful culture of B. cereus strain AT was inoculated into this nutrient medium with rotary shaking at 150 rpm at 37 C for 24 h and was stored at 2–8 C 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

De-hairing protease production by an isolated Bacillus cereus strain AT under solid-state protease. All substrates were dried (sun drying) for several days and further dried at 60 C 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 121 C for 20 min. After cooling, the flasks were inoculated with 5% (v/w) inoculum (1.036 OD at 600 nm) and incubated at 37 C 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 period (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, ammonium nitrate, peptone, yeast extract, urea, and casein). Results reported in this study are averages of triplicate findings.


pH 8, and glycine–NaOH buffer, pH 9 and 10, respectively. To check the pH stability, 25 ll of the enzyme solution was mixed with 175 ll 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 70 C) 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 70 C for 1 h. To evaluate the effect of ions (0.01 mol L 1) on enzyme activity, the enzyme sample was pre-incubated at 37 C for 1 h and the relative activity was evaluated. To examine the effect of surfactants and detergents on the enzyme activity, several surfactants 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 industrial point of view.

2.6. Extraction and purification of an enzyme 3. Results and discussion 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 4 C. The clear supernatant was used as the crude enzyme. This crude enzyme was precipitated with ammonium sulfate (70% saturation) and the enzyme precipitate obtained was centrifuged at 10,000g for 10 min at 4 C. The resulting pellet was dissolved in a small amount of 0.05 mol L 1 tris– HCl buffer (pH 8), and this enzyme preparation was loaded for gel-filtration chromatography using a Sephadex G-75 column (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 further 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 4 C. 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 incubating the gel for 12 h in 0.05 mol L 1 at 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 1 concentrations in the reaction mixture: citrate buffer, pH 4; succinate buffer, pH 5; sodium phosphate buffer, pH 6 to 7; tris-buffer,

3.1. Identification and screening of the isolate, B. cereus strain AT for protease secretion The B. cereus strain AT was isolated from the fermented rice as described under Materials and Methods, which could be catalase-, 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 isolate could hydrolyse casein and was negative for cellulose fermentation. Based on phenotypical characteristics and on the 16S rRNA sequence data, the strain was identified as B. 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 production when compared with the cow dung substrate. The result showed that protease production by B. cereus strain AT varied with the type of substrate. The selection of an ideal agrobiotech waste for enzyme production in a solid-state fermentation process depends upon several factors, mainly related with cost and availability of the substrate material and this may involve 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 protease than the well studied wheat bran. It was interesting to note that, none of the reports was evidenced on the production of alkaline protease by B. cereus using 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) and Bacillus subtilis strain VV (Vijayaraghavan


P. Vijayaraghavan et al. 3.4. Effect of moisture, inoculum and particle size

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

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 provide 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 37 C 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 considerably. These results are in accordance with observations made by Kumar and Parrack (2003) with Bacillus sp. and Okafor and Anosike (2012) with Bacillus sp. 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 1 material) 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).

In the present study, maximum enzyme production was observed 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 protease 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. subtilis strain VV (Vijayaraghavan et al., 2012). The optimum 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 Bacillus strains (Rajkumar et al., 2011). The particle size also greatly influenced the production of alkaline protease. The maximum enzyme production (5715 U g 1 material) was observed 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 surface area plays a vital role for microbial attachment, mass transfer of various nutrients and the substrates and subsequent 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

Figure 2 Influence of fermentation period on alkaline protease production under solid-state fermentation with cow dung.

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 production was found to be 8370 ± 203, 8821 ± 229 and 9597 ± 152 U g 1 for lactose, trehalose, and starch, respectively. In order to determine the optimum concentration of the carbon sources, enzyme production was investigated by supplementation of maltose (0.5–2%). The maximum production (10721 ± 217 U g 1) was observed at 1% maltose. These results were in accordance with reported alkaline protease pro-

De-hairing protease production by an isolated Bacillus cereus strain AT under solid-state


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

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 1 material. In order to understand the combined effect of carbon and nitrogen sources, the enzyme was produced in an optimized concentration. 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 37 C in optimized conditions. 3.6. Enzyme purification and molecular weight The crude protease sample was precipitated with 70% saturation of ammonium sulfate, dissolved in 0.05 mol l 1 tris–HCl buffer and loaded onto Sephadex G-75 column. In the crude extract, the enzyme activity was 11,400 U g 1 with 680 U mg 1 protein with a yield of 100%. The ammonium sulfate purification step showed 1065 U mg 1 protein with the yield being 67.2%. The specific activity increased to 2670 U mg 1 protein on Sephadex G-75 column chromatography with a 29% yield. Gel filtration chromatography was still the major and convenient 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). In B. 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).

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: 43ovalbumin: 29-carbonic anhydrase. (b) Zymography of purified protease.

3.7. Optimum pH and temperature of the enzyme The enzyme was active in the pH range of 6–9, with the optimum 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 with Bacillus sp RRM1, B. cereus MCM 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 Bacillus sp. fell at this range. The maximum protease activity recorded was between 40 and 50 C under standard reaction conditions, while the activity decreased rapidly above 60 C. By analyzing the thermal stability, the protease was found to be stable up to 50 C for 1 h incubation, but lost approximately 69 ± 1.4% of its activity at 60 C (Fig. 5B). A similar result was reported with Bacillus licheniformis RKK-04, B. subtilis RTSBA6.00 and Bacillus 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 5

P. Vijayaraghavan et al.

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

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 with Bacillus sp. and Bacillus megaterium RRM2 (Gupta et al., 2005; Rajkumar et al., 2011).

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%.

3.9. Effect of surfactants and detergents

Protease from B. cereus was 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 enzyme produced by B. cereus strain 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 noncollagenolytic 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 with B. licheniformis RP1 (Haddar et al., 2011) and B. circulans (Subba Rao et al., 2009).

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 by B. cereus strain AT was stable towards 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. cereus strain AT showed stability towards the surfactants like SDS. Similar kind of result was reported 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 detergents. Studies revealed that the purified enzyme was stable towards 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

3.10. De-hairing of skin

De-hairing protease production by an isolated Bacillus cereus strain AT under solid-state

Figure 6


Enzymatic dehairing of goat hide (a) Control; (b) After 18 h of incubation at room temperature.

4. Conclusion In conclusion, very few reports have been published on the use of cow dung as a substrate for enzyme bioprocess. In the present 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 from B. cereus strain AT was active in a range of temperatures and pH and was detergent stable and possessed 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. References Bradford, M.M., 1976. A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Bhunia, B., Dutta, D., Chaudhuri, S., 2011. Extracellular alkaline protease from Bacillus licheniformis NCIM-2042: improving enzyme activity assay and characterization. Eng. Life Sci. 11 (2), 207–215. De Azeredo, L.A., De Lima, M.B., Coelho, R.R., Freire, D.M., 2006. A low-cost fermentation medium for thermophilic protease production by Streptomyces sp. 594 using feather meal and corn steep liquor. Curr. Microbiol. 53 (4), 335–339. El-Hadj-Ali, N., Agrebi, R., Ghorbel-Frikha, B., Kanoun, S., Nasri, M., 2007. Biochemical and molecular characterization of a detergent stable alkaline serine-protease from a newly isolated Bacillus licheniformis NH1. Enzyme Microb. Technol. 40, 515–523. Ellaiah, P., Srinivasulu, B., Adinarayana, K., 2002. A review on microbial alkaline proteases. J. Sci. Ind. Res. 61, 690–704. Gessesse, A., 1997. The use of nug meal as a low-cost substrate for the production of alkaline protease by the alkaliphilic Bacillus sp. AR009 and some properties of the enzyme. Bioresour. Technol. 62, 59– 61. Ghorbel-Bellaaj, O., Manni, L., Jellouli, K., Hmidet, N., Nasri, M., 2012. Optimization of protease and chitinase production by Bacillus cereus SV1 on shrimp shell waste using statistical experimental design. Biochemical and molecular characterization of the chitinase. Ann. Microbiol. 62 (3), 1255–1268. Gupta, A., Roy, I., Patel, R.K., Singh, S.P., Khare, S.K., Gupta, M.N., 2005. One-step purification and characterization of an

alkaline protease from haloalkaliphilic Bacillus sp. J. Chromatogr. A 1075 (1–2), 103–108. Haddar, A., Hmidet, N., Bellaaj, O.G., Fakhfakh-Zouari, N.F., SellaiKamoun, V., Nasri, M., 2011. Alkaline Proteases Produced by Bacillus licheniformis rp1 grown on shrimp wastes: application in chitin extraction, chicken feather degradation and as a dehairing agent. Biotechnol. Bioprocess Eng. 16 (4), 669–678. Haile, G., Gessesse, A., 2012. Properties of alkaline protease C45 produced by alkaliphilic Bacillus Sp. isolated from Chitu, Ethiopian Soda Lake. J. Biotechnol. Biomater. 2, 136, doi:10.4172/2155952X.1000136. Han, X.Q., Damodaran, K., 1997. Stability of protease Q against autolysis and in sodium dodecyl sulfate and urea solutions. Biochem. Biophys. Res. Commun. 240, 839–843. Holt, J.G., Krieg, N.R., Sneath, P.H., Stanley, J.J., Williams, S.T., 1994. Bergey’s Manual of Determinative Bacteriology. Williams and Wilkins, Baltimore, USA. Heussen, C., Dowdle, E.B., 1980. Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal. Biochem. 102, 196– 202. Jacobs, M.F., 1995. Expression of the subtilis in Carlsberg-encoding gene in Bacillus licheniformis and Bacillus subtilis. Gene 152, 67–74. Johnvesly, B., Naik, G.R., 2001. Studies on production of thermostable alkaline protease from thermophilic and alkaliphilic Bacillus sp. JB-99 in a chemically defined medium. Process Biochem. 37, 139– 144. Joo, H.S., Kumar, C.G., Park, G.C., Paik, S.R., Chang, C.S., 2003. Oxidant and SDS-stable alkaline protease from Bacillus Clausii I52: production and some properties. J. Appl. Microbiol. 95, 267– 272. Kembhavi, A.A., Kulkarni, A., Pant, A., 1993. Salt-tolerant and thermostable alkaline protease from Bacillus subtilis NCIM No. 64. Appl. Biochem. Biotechnol. 38, 83–92. Krishna, C., Chandrasekaran, M., 1996. Banana waste as substrate for amylase production by Bacillus subtilis (CBTKL06) under solid state fermentation. Appl. Microbiol. Biotechnol. 46, 106–111. Kumar, C.G., Parrack, P., 2003. Arrowroot (Marantha arundinacea) starch as a new low-cost substrate for alkaline protease production. World J. Microbiol. Biotechnol. 19, 757–762. Kumar, C.G., Tiwari, M.P., Jany, K.D., 1999. Novel alkaline serine protease from alkalophilic Bacillus sp. purification and characterization. Process Biochem. 34, 441–449. Laemmli, U.K., 1970. Cleavage of structural proteins during assembly of head of bacteriophage T4. Nature (London) 227, 680–685. Nigam, P., Singh, D., 1994. Solid state (substrate) fermentation system and their applications in biotechnology. J. Basic Microbiol. 34, 405–423. Nilegaonkar, S.S., Zambare, W.P., Kanekar, P.P., Dhakephalkar, P.K., Sarnaik, S.S., 2007. Production and partial characterization of dehairing protease from Bacillus cereus MCM B-326. Bioresour. Technol. 98, 1238–1245. Okafor, U.O.G., Anosike, E.E.M., 2012. Screening and optimal protease production by Bacillus sp. Sw-2 using low cost substrate medium. Res. J. Microbiol. 7, 327–336.

34 Pandey, A., Soccol, C.R., Nigam, P., Brand, D., Mohan, R., Roussos, S., 2000. Biotechnological potential of coffee pulp and coffee husk for bioprocesses. Biochem. Eng. J. 6, 153–162. Prakasham, R.S., Subba Rao, C.h., Sarma, P.N., 2006. Green gram husk: an inexpensive substrate for alkaline protease production by Bacillus sp. in solid-state fermentation. Bioresour. Technol. 97, 1449–1454. Rajkumar, Rengananthan, Ranishee, J.K., Ramasamy, R., 2011a. Production and characterizaion of a novel proteases from Bacillus sp. RRM1 under solid state fermentation. J. Microbiol. Biotechnol. 21 (6), 627–636. Rajkumar, R., Jayappriyan, K.R., Rengasamy, R., 2011b. Purification and characterization of a protease produced by Bacillus megaterium RRM2: application in detergent and dehairing industries. J. Basic Microbiol. 51 (6), 614–624. Rao, M.B., Tanksale, A.M., Ghatge, M.S., Deshpande, V.V., 1998. Molecular and biotechnological aspects of microbial proteases. Microbiol. Mol. Biol. Rev. 62, 597–635. Ravindran, B., Ganesh Kumar, A., Aruna Bhavani, P.S., Ganesan Sekaran, 2011. Solid-state fermentation for the production of alkaline protease by Bacillus cereus 1173900 using proteinaceous tannery solid waste. Curr. Sci. 100 (5), 726–730. Riffel, A., Ortolan, S., Brandelli, A., 2003. De-hairing activity of extracellular proteases produced by keratinolytic bacteria. J. Chem. Technol. Biotechnol. 78, 855–859. Sana, B., Ghosh, D., Saha, M., Mukherjee, J., 2006. Purification and characterization of a salt, solvent, detergent and bleach tolerant protease from a new gamma-proteobacterium isolated from the marine environment of the Sundarbans. Process Biochem. 41, 208– 215. Sellami-Kamoun, A., Ghorbel-Frikha, B., Haddar, A., Nasri, M., 2011. Enhanced Bacillus cereus BG1 protease production by the use of sardinelle (Sardinella aurita) powder. Ann. Microbiol. 61 (2), 273–280. Shinde, A.A., Shaikh, F.K., Padul, M.V., Kachole, M.S., 2012. Bacillus subtilis RTSBA6.00, a new strain isolated from gut of Helicoverpa armigera (Lepidoptera: Noctuidae) produces chymotrypsin-like proteases.. Saudi J. Biol. Sci. 19 (3), 317–323. Shrinivas, D., Naik, G.R., 2011. Characterization of alkaline thermostable keratinolytic protease from thermoalkaliphilic Bacillus halodurans JB 99 exhibiting dehairing activity. Int. Biodeter. Biodegrad. 65, 29–35. Sivasubramanian, S., Murali Manohar, V., Rajaram, R., Puvanakrishna, 2008. A ecofriendly lime and sulfide free enzymatic dehairing of skins and hides using a bacterial alkaline protease. Chemosphere 70, 1015–1024.

P. Vijayaraghavan et al. Sousa, F., Ju, S., Erbel, A., Kokol, V., Cavaco-Paulo, A., Gubitz, G.M., 2007. A novel metalloprotease from Bacillus cereus for protein fibre processing. Enzyme Microb. Technol. 40, 1772–1781. Subba Rao, C.h., Sathish, T., Ravichandra, P., Prakasham, R.S., 2009. Characterization of thermo- and detergent stable serine protease from isolated Bacillus circulans and evaluation of eco-friendly applications. Process Biochem. 44, 262–268. Thanikaivelann, P., Rao, J.R., Nair, B.U., Ramasami, T., 2004. Progress and recent trends in biotechnological methods for leather processing. Trend Biotechnol. 22, 181–188. Tunga, R., Banerjee, R., Bhattacharyya, B.C., 2001. Optimization of some additives to improve protease production under SSF. Ind. J. Exp. Biol. 39, 1144–1148. Toyokawa, Y., Takahara, H., Reungsang, A., Fukuta, M., Hachimine, Y., Tachibana, S., Yasuda, M., 2010. Purification and characterization of a halotolerant serine proteinase from thermotolerant Bacillus licheniformis RKK-04 isolated from Thai fish sauce. Appl. Microbiol. Biotechnol. 86 (6), 1867–1875. Uyar, F., Porsuk, I., Kizil, G., Yilmaz, E.I., 2011. Optimal conditions for production of extracellular protease from newly isolated Bacillus cereus strain CA15. Eurasia J. Biosci. 5, 1–9. Vijayaraghavan, P., Vincent, S.G.P., 2012. Cow dung as a novel, inexpensive substrate for the production of a halo-tolerant alkaline protease by Halomonas sp. PV1 for eco-friendly applications. Biochem. Eng. J. 69, 57–60. Vijayaraghavan, P., Vijayan, A., Arun, A., Jenisha, J., Vincent, S.G.P., 2012. Cow dung: a potential biomass substrate for the production of detergent-stable dehairing protease by alkaliphilic Bacillus subtilis strain VV. SpringerPlus 1, 76. 2193-1801-1-76. Wang, S.L., Hsu, W.T., Liang, T.W., Yen, Y.H., Wang, C.L., 2008. Purification and characterization of three novel keratinolytic metalloproteases produced by Chryseobacterium indologenes TKU14 in a shrimp shell powder medium. Bioresour. Technol. 99 (13), 5679–5686. Yang, J.K., Shih, I.L., Tzeng, Y.M., Wang, S.L., 2000. Production and purification of protease from a Bacillus subtilis that can deproteinize crustacean wastes. Enzyme Microb. Technol. 26, 406–413. Zambare, V.P., Nilegaonkar, S.S., Kanekar, P.P., 2007. Production of an alkaline protease by Bacillus cereus MCM B-326 and its application as a dehairing agent. World J. Microbiol. Biotechnol. 23, 1569–1574. Zambare, V., Nilegaonkar, S., Kanekar, P., 2010. Application of protease from Bacillus cereus MCM B-326 as a bating agent in leather processing. IIOAB J. 1 (4), 18–21.

Suggest Documents