Cow dung: a potential biomass substrate for the production of detergent-stable dehairing protease by alkaliphilic Bacillus subtilis strain VV.pdf
RESEARCH Open Access Cow dung: a potential biomass substrate for the production of detergent-stable dehairing protease by alkaliphilic Bacillus subtilis strain VV Ponnuswamy Vijayaraghavan1*, Aija Vijayan2, Arumugaperumal Arun3, John Kennady Jenisha3 and Samuel Gnana Prakash Vincent1 Abstract Cow dung, a cheap and easily available source of energy, was used as the substrate for the production of alkaline protease by solid-state fermentation using the Bacillus subtilis strain VV. In order to achieve the maximum yield of this enzyme, the following optimum process parameters are needed: fermentation period (72 h), pH (10.0), moisture content (140%), inoculum (25%), temperature (30–40°C), carbon source (2% (w/w) maltose) and nitrogen source (1% (w/w) urea). The protease was stable over a broad temperature range (30–50°C) and pH (8.0-10.0), with maximum activity at 50°C and pH 10.0. Among the divalent ions tested, Ca2+ (0.01 M) increased enzyme activity. The purified protease, after being subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis, was found to have a molecular mass of 38.5 kDa. The enzyme was solvent-and surfactant-stable and showed activity even after 24 h incubation along with various commercially available detergents. This enzyme possessed dehairing properties for animal hide after 16 h of incubation at room temperature. From these results it is evident that cow dung is a potential substrate for the production of a detergent-stable, dehairing protease by B. subtilis. This enzyme has a lot of potential applications in the detergent and leather-processing industries. Keywords: Cow dung, Solid-state fermentation, Bacillus subtilis strain VV, Alkaline protease, Detergent-stable Background Proteases constitute one of the commercially important groups of extra-cellular microbial enzymes and are widely used in the detergent, food, pharmaceutical, chemical and leather industries (Scheuer 1990). These enzymes account for 40% of the total enzyme sales worldwide and this trend is expected to increase in the near future. This has led to increasing attention towards the exploitation of potent microbial strains for the production of alkaline proteases from an industrial point of view (Ellaiah et al. 2002). Although a wide range of microorganisms are known to produce proteases, a large proportion of the commercially available form of these enzymes is derived from Bacillus strains because of their ability to secrete large amounts of alkaline proteases having significant proteolytic activity and stability at considerably higher pH and temperatures (Jacobs 1995; Yang et al. 2000). The leather processing industry contributes significantly to the country’s economic development. Waste from the leather industry leads to environmental pollution. Alkaline proteases have dehairing properties and can be used in the leather processing industry. Conventional methods in leather processing involve the use of hydrogen sulphide and other chemicals which are pollutants. Thus, for envir- onmental reasons, the enzymatic dehairing process has more advantages over the chemical dehairing process (Andersen 1998). Proteases are used during the soaking, dehairing and bating states of preparing skins and hides. Pancreatic proteases are used in the bating process and the use of microbial alkaline proteases are popular (Varela et al. 1997). Alkaline proteases swell hair roots and attack hair follicle proteins, resulting in the easy removal of hair. These enzymes have been widely studied and their production from Bacillus sp. has gained momentum; * Correspondence: venzymes@gmail.com 1International Centre for Nanobiotechnology, Centre for Marine Science and Technology, Manonmaniam Sundaranar University, Rajakkamangalam–629 502, Kanyakumari DistrictTamil Nadu, India Full list of author information is available at the end of the article a SpringerOpen Journal © 2012 Vijayaraghavan et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Vijayaraghavan et al. SpringerPlus 2012, 1:76 http://www.springerplus.com/content/1/1/76 moreover, the high activity and stability of these enzymes at various temperature and pH ranges have also attracted the attention of researchers. Dehairing proteases have been characterized from various Bacillus sp., e.g. B. subtilis 11QDB32 (Varela et al. 1997), B. amyloliquefaciens (George et al. 1995), B. subtilis K2 (Hameed et al. 1996; Hameed et al. 1999) and B. circulans (Subba Rao et al. 2009). Proteases are generally produced using submerged fer- mentation not only due to its apparent advantages in con- sistent enzyme production but also for its cost- for medium components. From an industrial point of view, it is estimated that around 30-40% of the production cost of industrial enzymes can be attributed to the cost of the growth medium (Joo et al. 2003). Solid-state fermentation (SSF) has gained importance in the production of micro- bial enzymes owing to several economic advantages over submerged fermentation. The advantages of SSF include lower manufacturing costs with increased production, less pre-processing energy and effluent generation, along with easy process management and better product recovery (Prakasham et al. 2006; Oliveira et al. 2006). There are several reports describing the use of agro-industrial resi- dues for the production of alkaline protease (e.g. pigeon pea and Bacillus sp. JB-99 (Johnvesly et al. 2002); green gram husk and Bacillus sp. (Prakasham et al. 2006); Imperata cylindrical grass and potato peel and Bacillus subtilis (Mukherjee et al. 2008). Apart from these agro- industrial residues, increased attention has been paid in recent times to utilize other waste substances, e.g. feather meal, corn steep liquor (De Azeredo et al. 2006) and pro- teinaceous tannery solid waste, for the production of alka- line proteases (Ravindran et al. 2011). Even though cow dung is considered a waste, it contains essential nutrients (Misra et al. 2003); these include carbon, nitrogen, phos- phorus, potassium, calcium, magnesium, sulphur, manga- nese, copper, zinc, chloride, boron, iron and molybdenum. Most of the commercial proteases producing organisms are Bacillus sp. (Abo-Aba et al. 2006). The potential of cow dung as a biomass substrate for the production of alkaline protease by using Bacillus sp. has not yet been completely exploited. The main objective of the present study was the production of alkaline proteases by Bacillus subtilis utilizing cow dung as an energy source, and deter- mination of the optimum conditions necessary for the production of these enzymes. Results and discussion Isolation and identification of a best alkaline protease- producing organism Of the tested isolates, five were found to have the ability to produce alkaline protease. Among the positive isolates, the organism which produced a larger halo zone in re- sponse to the colony diameter was selected. The selected isolate was identified as Bacillus on the basis of various microscopic and biochemical investigations. The organism was a Gram-positive rod, spore-producing, VP-, catalase- and gelatin-positive. It fermented glucose, lactose and sucrose. It reacted negatively in the indole, methyl red, citrate, oxidase, starch and nitrate reduction test. All these results suggest that it belongs to the genus Bacillus. More- over, the organism was confirmed by its 16S rRNA gene sequence and identified as Bacillus subtilis strain VV. The 1071-bp sequence was submitted to GenBank (accession number: JQ 425476). Evaluation of cow dung as a cheap substrate for alkaline protease production This study has indicated that cow dung can be used as a potential substrate for alkaline protease production. En- zyme production by the B. subtilis strain VV was to the tune of 4030 ± 128 U/g solid substrate (cow dung) after 72 h of incubation at 37°C. The selection of a cheap sub- strate in SSF for the production of any metabolites is an im- portant factor from an industrial point of view. Apart from the cost, the availability of the substrate is a critical factor. An ideal substrate is one which is available in large quan- tities and throughout the year too. Although many cheap agro-industrial residues were evaluated (Prakasham et al. 2006; Johnvesly et al. 2002; Gessesse 1997) for the produc- tion of alkaline proteases, the availability of these substrates is seasonal. Apart from agro-industrial wastes, more atten- tion has been paid to the evaluation of solid wastes for the production of alkaline proteases (Ravindran et al. 2011; Ganesh Kumar et al. 2008). Waste water from the manufac- ture of shochu was also tried (Morimura et al. 1994) for production of proteases. In spite of evaluating these sub- strates, the search for a novel substrate continues. Recently, we used cow dung as a substrate for the production of a halo-tolerant alkaline protease using a alkalophilic isolate, Halomonas sp. PV1 (Vijayaraghavan and Vincent 2012). Of all the alkalophilic microorganisms that have been screened for use in various industrial applications, members of the genus Bacillus were found to be predominant and a prolific source of alkaline proteases (Kumar and Takagi 1999). Reports on SSF of cow dung for the production of alkaline protease using Bacillus sp. are limited or perhaps not avail- able. Hence, the present investigation aimed to exploit cow dung that is cheap and globally available for alkaline prote- ase production by Bacillus subtilis. The protein content of the cow dung medium was evaluated before and after fer- mentation. The cow dung possessed 80 ± 12 mg protein/g solid substrate, and the organism utilized 40 ± 4.5% of the protein content for the growth and synthesis of protease. Effect of fermentation period and pH on alkaline protease production To evaluate the effect of fermentation period on prote- ase production, the fermentation experiment was carried Vijayaraghavan et al. SpringerPlus 2012, 1:76 Page 2 of 9 http://www.springerplus.com/content/1/1/76 was added to the fermented substrate. This was placed in an orbital shaker at 150 rpm for 30 min for enzyme ex- traction. After this, the mixture was rapidly filtered using cotton and the cells were further harvested by centrifuga- tion at 10,000 g for 20 min. The supernatant was used as the enzyme source for protease assay. Determination of protease activity Alkaline protease activity was determined by standard assay. The reaction mixture contained 5 mL of casein (prepared in 0.05 M of glycine-NaOH buffer, pH 10.0) and an aliquot of 0.1 mL of the enzyme solution, and this mixture was incubated for 30 min at 37°C. The reac- tion was stopped by adding 5 mL of trichloroacetic acid solution (TCA) (0.11 M) and the mixture was filtered after 30 min. To 2 mL of the filtrate, 5.0 mL of 0.5 M so- dium carbonate and 1.0 mL of Folin-Ciocalteu’s phenol reagent were added, and this mixture was kept undis- turbed for 30 min at 37°C. The optical density of the solution was read against sample blank at 630 nm. One unit of enzyme activity was defined as the amount of en- zyme required to liberate 1 μg of tyrosine per minute under assay conditions (Chopra and Mathur 1985). The total protein content was estimated by Bradford’s method (Bradford 1976). Optimization of process parameters for protease production In the present study, solid-state protease production by the Bacillus subtilis strain VV was optimized by varying the physical parameters and nutrient sources. The prote- ase activity was determined in the fermented medium for every 12 h of fermentation up to 96 h in order to de- termine the fermentation period. To evaluate the effect of temperature on protease production, the substrate inoculated with bacterial culture was incubated at vari- ous temperatures (10–50°C). Addition of buffer (0.1 M) was performed so that the pH of the solid medium was varied from pH 6.0 to 11.0. To study the effect of the initial moisture content on protease production, the initial moisture content of the cow dung was adjusted to 60-180% using glycine-NaOH buffer (pH 10.0). To determine the effect of inoculum size on protease production, the inoculum concentration was increased accordingly (5-30%). In addition to the physical parameters, nutrient para- meters were also optimized. This included the effect of carbon sources (1%, w/w) (glucose, lactose, trehalose, mal- tose, xylose and starch) and nitrogen sources (1%, w/w) (gelatin, ammonium nitrate, peptone, yeast extract, urea, skimmed milk, and casein). The maximum production of protease at various concentrations (0.5-2.5%) of maltose as a carbon source and urea as a nitrogen source was investigated. The effect of the optimum concentration of maltose, urea and their combination on alkaline protease production for 24–96 h was also evaluated. The results reported in this study are averages of triplicate findings. Purification of protease The organism was grown aerobically in an optimized medium for 72 h at 37°C and extracted as described in materials and method section. Samples were centrifuged at 10,000 g for 10 min, and the supernatant was used as a crude enzyme preparation. It was precipitated with ammonium sulphate (40-80% saturation) and the enzyme precipitate obtained was centrifuged at 10,000 g for 10 min at 4°C. The precipitate obtained from the previous step was re-suspended in 5.0 mL of 0.025 M Tris–HCl buffer and dialysed against the same buffer. The dialysed sample was applied to a Sephadex G-75 gel filtration column (0.6 × 45 cm), and eluted with 0.025 M Tris–HCl buffer at pH 8.0, at a flow rate of 0.5 mL/min. Fractions of 2.0 mL were collected and the optical dens- ity of the sample was measured at 280 nm and analysed for proteolytic activity. SDS-PAGE and zymography Sodium dodecyl sulphate-polyacrylamide gel electrophor- esis was carried out according to (Laemmli 1970) using 11% crosslinked polyacrylamide gel. Silver staining was performed to visualize protein bands. Zymographic ana- lysis was performed by the enzyme pattern of proteins, obtained by zymogram with 1% casein substrate and detected using coomassie brilliant blue R-250 (Westergaar et al. 1980). Characterization of protease activity The effect of pH on the activity of the enzyme was studied by assaying the enzyme activity at different pH values ran- ging from 5.0 to11.0. To check the stability of the enzyme at various pH, 100 μL of the enzyme solution was mixed with 900 μL of buffer solutions (pH 5.0-11.0) and the mix- ture was taken to measure the protease activity under standard assay conditions after incubation for 1 h. The ef- fect of temperature on enzyme activity was studied by holding the reactions at various temperatures (30–70°C) using the standard assay method. To evaluate the heat stability of the protease, the sample was denatured at an optimized temperature (50°C) for 0–120 min. To study the effect of ions (0.01 M) on enzyme activity, the sample was pre-incubated with various divalent ions at 37°C for 1 h and the activity evaluated. To examine the effect of solvents, surfactants and detergents on enzyme activity, many agents were added to the enzyme solution at the indicated concentration, allowed to stand for 1 h and 24 h at room temperature and the activity measured. To evalu- ate the dehairing property of an enzyme, fresh goat-hide Vijayaraghavan et al. SpringerPlus 2012, 1:76 Page 7 of 9 http://www.springerplus.com/content/1/1/76 was incubated with 4.0 mg enzyme solution (pH 10.0) for up to 16 h at room temperature. Statistical analysis All experiments were performed in triplicate. Data were analyzed by correlation coefficient (r) and Student’s ‘t’ test. A significance level of 0.05 or less was considered statistically significant. Conclusions In conclusion, cow dung was utilized as a substrate for the production of alkaline protease in SSF. Cow dung is a cheaply available bioresource and this substrate is available in almost every country. So, this biomass could be effect- ively utilized for the production of alkaline protease an in- dustrial scale. Apart from the significance of the substrate, the enzyme secreted by B. subtilis strain VV is useful in the detergent and leather-processing industries. Competing interests The authors declare that they have no competing interests. Authors’ contribution PV designed and executed this project work. AV, AA and JJ gave technical assistance. SGPV guided this project work. All the authors have approved the submission of the manuscript. 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. Author details 1International Centre for Nanobiotechnology, Centre for Marine Science and Technology, Manonmaniam Sundaranar University, Rajakkamangalam–629 502, Kanyakumari DistrictTamil Nadu, India. 2Centre for Marine Science and Technology, Manonmaniam Sundaranar University, Rajakkamangalam–629 502, Kanyakumari District, Tamil Nadu, India. 3Department of Biotechnology, Kalasalingam University, Virudhunagar, Srivilliputtur, 626 126, Tamilnadu, India. Received: 5 October 2012 Accepted: 19 December 2012 Published: 22 December 2012 References Abo-Aba SEM, Soliman EAM, Nivien AA (2006) Enhanced production of extra cellular alkaline protease in Bacillus ciculance through plasmid transfer. Res J Agric Biol Sci 16:526–530 Andersen LP (1998) Method for dehairing of hides or skins by means of enzymes. US Patent 5:834,299 Aravindan R, Saravanabhavan S, Thanikaivelan P, Rao JR, Nair BUA (2007) Chemo enzymatic pathway leads towards zero discharge tanning. 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Title: Cow dung: a potential biomass substrate for the production of detergent-stable dehairing protease by alkaliphilic Bacillus subtilis strain VV ​
Authors: Ponnuswamy Vijayaraghavan, Aija Vijayan, Arumugaperumal Arun, John Kennady Jenisha, Samuel Gnana Prakash Vincent
Publisher: SpringerPlus
Date: 22 December 2012 ​

Abstract: Cow dung, a cheap and easily available source of energy, was used as the substrate for the production of alkaline protease by solid-state fermentation using the Bacillus subtilis strain VV. ​ In order to achieve the maximum yield of this enzyme, the following optimum process parameters are needed: fermentation period (72 h), pH (10.0), moisture content (140%), inoculum (25%), temperature (30–40°C), carbon source (2% (w/w) maltose) and nitrogen source (1% (w/w) urea). ​ The protease was stable over a broad temperature range (30–50°C) and pH (8.0-10.0), with maximum activity at 50°C and pH 10.0. ​ Among the divalent ions tested, Ca2+ (0.01 M) increased enzyme activity. ​ The purified protease, after being subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis, was found to have a molecular mass of 38.5 kDa. ​ The enzyme was solvent-and surfactant-stable and showed activity even after 24 h incubation along with various commercially available detergents. ​ This enzyme possessed dehairing properties for animal hide after 16 h of incubation at room temperature. ​ From these results it is evident that cow dung is a potential substrate for the production of a detergent-stable, dehairing protease by B. subtilis. ​ This enzyme has a lot of potential applications in the detergent and leather-processing industries. ​