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 response 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. Moreover, 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. Enzyme 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 substrate in SSF for the production of any metabolites is an important 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 quantities 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 production of alkaline proteases, the availability of these substrates is seasonal. Apart from agro-industrial wastes, more attention 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 manufacture of shochu was also tried (Morimura et al.1994) for production of proteases. In spite of evaluating these substrates, 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 available. Hence, the present investigation aimed to exploit cow dung that is cheap and globally available for alkaline protease production by Bacillus subtilis. The protein content of the cow dung medium was evaluated before and after fermentation. 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 protease production, the fermentation experiment was carried out for a period of 96 h. Results of this study showed that protease production increased with incubation time and was positively correlated (r = 0.842). Maximum alkaline protease production was achieved after 72 h of fermentation (4142 ± 172 U/g substrate) at 37°C (Figure 1a). The incubation time is governed by the characteristics of the culture and is also based on the growth rate and enzyme production. Similar findings have been reported with other Bacillus sp. (Ravindran et al.2011). The reduction in enzyme yield after the optimum period was probably due to the depletion of nutrients available to the microorganisms. Here, enzyme production gradually decreased after 72 h. The effect of pH on enzyme production was studied by culturing the organism at various pH levels (6.0-11.0). Enzyme production was 1502 ± 120, 2261 ± 142, 2945 ± 110 and 2291 ± 153 U/g substrate at pH 6.0, 7.0, 8.0 and 9.0, respectively. Enzyme synthesis increased with increase in medium pH (r = 0.839), and maximum enzyme production was achieved at pH 10.0 (4322 ± 148 U/g substrate). This trend clearly implies that this protease producer is alkaliphilic in nature. Enzyme production decreased at pH 11 (3229 ± 129 U/g substrate). At higher pH level (11), protease production decreased as the metabolic action of the bacterium may be suppressed. This result was in accordance with the observations made with other alkaliphilic protease-secreting Bacillus sp. (Uyar and Baysal 2004). Alkaline protease production by microbial strains strongly depends on extracellular pH because culture pH strongly influences many enzymatic processes and transport of various components across the cell membranes, which in turn support cell growth and product production (Ellaiah et al.2002).

Figure 1 (a) Effect of fermentation period on enzyme production. The organism was grown in the cow dung substrate and incubated at 37°C for 72 h. Error bar standard deviation. (b) Influence of initial moisture content on protease production by Bacillus subtilis strain VV on cow-dung substrate. Error bar standard deviation. Full size image

Influence of moisture content and inoculum on alkaline protease production

The maximum enzyme production was observed with 140% moisture content (5424 ± 116 U/g substrate). The moisture content positively regulated enzyme production (r = 0.763). The enzyme production decreased thereafter and it was 3722 ± 102 U/g substrate at 180% moisture (Figure 1b). This could be attributed to low microbial growth and anchoring on the surface of the solid medium at higher moisture content. Among the several factors that are important for microbial growth and enzyme production under SSF, moisture content is a critical factor (Pandey et al.2000;Nigam and Singh 1994). Cow dung has a high moisture-holding capacity. This could be the reason why the fermentation medium remained loose with higher moisture content and no free water. Even at a higher moisture level (180%), the yield had not decreased much, revealing that this substrate supports the growth of the organism and production of proteases. There was a significant increase in alkaline protease production with an increase in inoculum size and the correlation coefficient (r) was 0.931. Enzyme production was 609 ± 42, 2636 ± 189, 3879 ± 201, 4227 ± 146, 5626 ± 197 and 5094 ± 132 U/g substrate at in inoculum sizes of 5%, 10%, 15%, 20%, 25% and 30%, respectively. Increase of the inoculum level after 25% adversely affected enzyme production. This result was in accordance the results observed with other Bacillus sp. (Rajkumar et al.2011).

Effect of temperature

The effect of temperature on enzyme production was studied by culturing the organism at various temperatures (10–50°C) and the enzyme production was not significantly increased. Enzyme production was 103 ± 27, 2527 ± 91, 3281 ± 127, 4617 ± 101, 5630 ± 162 U/g substrate at temperatures of 10, 15, 20, 25 and 30°C, respectively. The optimum temperature for maximum protease production of 5842 ± 108 U/g substrate in SSF was recorded as 35°C. Incubation of temperatures below 35°C and above 40°C greatly reduced enzyme production. Enzyme production was recorded as 4015 ± 87, 2308 ± 133 and 1134 ± 104 U/g solid substrate at 40, 45 and 50°C, respectively. Incubation temperature is one of the most critical parameters that have to be controlled in the bioprocess as culture temperature influences protease production by microorganism (Ghorbel et al.2003).

Evaluation of supplementation of carbon and nitrogen sources

The solid medium was supplemented with several carbon sources such as glucose, lactose, trehalose, maltose, xylose and starch at 1% (w/w) level. Among these, the addition of maltose and starch supported maximum production of protease with 5482 ± 118 U/g, and 4360 ± 127 U/g solid substrate respectively. Enzyme production was 2768 ± 82, 4076 ± 103, 3642 ± 121 and 4074 ± 167 U/g substrate when glucose, lactose, xylose and trehalose were added, respectively. When different concentrations of maltose were added, maltose at 2% supported the maximum production with 5629 ± 120 U/g substrate which was statistically significant (r = 0.737). In SSF, addition of carbon sources increases enzyme production. However, addition of the carbon source in the cow dung substrate increased the total enzyme yield by a mere 18%. This clearly implies that cow dung contains more or less enough energy sources for the growth of microorganisms and protease production. The addition of maltose and starch enhanced protease production, with an increase of 30% and 5%, respectively. These results are in accordance with those of another study in which different sugars were supplemented (Ellaiah et al.2002).

Among nitrogen sources, addition of urea supported maximum protease production (5412 ± 142 U/g substrate). Addition of other sources such as gelatin (3827 ± 161 U/g), peptone (3485 ± 128 U/g), yeast extract (3587 ± 153 U/g) and casein (4830 ± 134 U/g) also supported protease production. Ammonium chloride repressed protease production (2787 ± 64 U/g). When different concentrations of urea were added, urea at 1% supported maximum protease production with 5489 ± 142 U/g. Enzyme production was not significantly increased in other concentrations and was not statistically significant. Results presented in Figure 2 show the influence of adding maltose, urea and their combinations on alkaline protease production under SSF at varying incubation times.

Figure 2 Effect of maltose, urea, and their combination influence on alkaline protease production by Bacillus subtilis strain VV under solid-state fermentation with cow dung. Error bar standard deviation. Full size image

Purification of the protease and SDS-PAGE

The alkaline protease of the crude extract was purified for homogeneity by a two-step procedure: ammonium sulphate precipitation and Sephadex G-75 gel filtration. In crude extract the specific activity was 16.11 U/mg protein, with an yield of 100%. The alkaline protease was purified 2.45 fold with ammonium sulphate precipitation and further chromatographic separation in Sephadex G-75 column to obtain an 18.37% yield. The specific activity of the purified enzyme was 152.61 U/mg protein. A typical purification experiment is summarized in Table 1. In the SDS-PAGE, the purified enzyme migrated as a single band with an apparent molecular weight of 38.5 kDa (Figure 3a); this was also the case with the zymography analysis (Figure 3b). These results are in accordance with literature reports where molecular masses of most proteases derived from Bacillus sp. are less than 50 kDa (Sousa et al.2007).

Table 1 Purification summary of extracellular alkaline protease from Bacillus subtilis strain VV Full size table

Figure 3 (a) SDS-PAGE analysis of the purified protease. Lane 1. crude enzyme. Lane 2. ammonium sulphate precipitated sample (upto 40% saturation). Lane 3. ammonium sulphate precipitated sample (80% saturation). Lane 4. purified protease by sephadex G-75. Lane 5. molecular mass marker: 205-myosin muscle rabbit 97.4-phosphorylase b: 66-bovine serum albumin: 43-ovalbumin: 29-carbonic anhydrase. (b) zymography of purified protease. Full size image

Effect of temperature and pH on the activity of alkaline protease

Enzyme activity was increased with increase in temperature and was statistically significant (p < 0.05). At around 50°C, the maximum protease activity (5922 ± 182 U/g) was determined and it declined at higher temperatures. The enzyme activity was 3062 ± 123, 4432 ± 138, 3802 ± 107, and 994 ± 42 U/g at 30, 40, 60 and 70°C, respectively. Enzyme activity was not detected at 80°C. This might be due to the total denaturation of the enzyme. This protease could be classified as thermostable, because of its optimal activity at 50°C. This result was in accordance with the results obtained with other Bacillus sp. (Rajkumar et al.2011). When analyzed for thermal stability, the protease was found to be stable for 40 min at 50°C (42 ± 3.8% activity), which decreased to 2 ± 0.13% after 120 min of denaturation at this temperature. The effect of pH on enzyme activity was evaluated in the pH range, 6.0 to 11.0 and optimal activity occurred in the alkaline range (pH 9.0-11.0). The enzyme produced by the Bacillus subtilis strain VV revealed robustness towards alkaline pH. Enzyme activity was found to be the high at higher pH (10.0) (7464 ± 169 U/g material) and was statistically significant (p < 0.05). The enzyme activity was 1156 ± 72, 3548 ± 112, 3631 ± 147, 4140 ± 118 and 4744 ± 152 for the pH 6.0, 7.0, 8.0, 9.0 and 11.0, respectively. This result was in accordance observations made with other Bacillus sp. (Ghorbel et al.2003; Arulmani et al.2007). The protease was stable over a pH range of 8.0 to 11.0, and was highly stable at pH 9.0 (100% relative activity). It lost approximately 15.7 ± 1.8% and 33.7 ± 2.6% activity at pH 10.0 and 11.0, respectively. However, more than 61 ± 4.1% of the enzyme activity was lost when the pH fell below 6.0. Similar pattern of pH stability of protease produced by Bacillus circulans was described earlier (Towatana et al.1999).

Effect of ions on enzyme activity

The effect of various divalent ions (0.01 M) on the activity of the enzyme was evaluated. Of the various divalent ions added, Ca2+ ions were found to increase enzyme activity (108 ± 4.9%). This result was in accordance with results observed with other Bacillus sp. in a similar study (Towatana et al.1999). Ions like Cu2+ (64 ± 2.8%), Fe2+ (73 ± 5.3%), Hg2+ (13.2 ± 1.6%) and Zn2+ (61.9 ± 3.4%) strongly inhibited enzyme activity. Enzyme activity was slightly affected by Mg2+ and Mn2+ ions. The characteristic features of the enzyme produced by B. subtilis in this study were similar to that produced by Bacillus circulans (Subba Rao et al.2009).

Effect of organic solvent, surfactants and detergents on the stability of protease

The enzyme was stable towards all tested organic solvent (1%) for 1 h at room temperature. Among the organic solvents tested, methanol and acetonitrile showed better stability (Table 2) but these were not significantly increased. The alkaline protease was stable towards the non-ionic surfactants like SDS, tween-20, tween-80 and triton X-100 and the enzyme activity was 104 ± 2.1%, 168.2 ± 7.8%, 111.5 ± 9.8% and 141.3 ± 21%, respectively. These results were statistically significant (p < 0.05). This finding gains significance because modern bleach-based detergent formulations are mainly composed of SDS. This result is in accordance with the observation made with Bacillus clausii (Joo et al.2003). This enzyme showed significant stability (p < 0.05) in the presence of commercially available detergent such as Sunlight and Ujala after 1 h of incubation. This alkaline protease was evaluated for its possible applications in detergent formulation as it showed stability after 1 h and 24 h incubation with these detergents and the results are presented in Table 2. A similar result was reported with Bacillus circulans (Subba Rao et al.2009). The enzymatic properties of the protease suggest its suitability as an addition to detergent formulations.

Table 2 Effect of solvent and detergents on protease activity from Bacillus subtilis strain VV Full size table

Dehairing of skin

In the present study, 4.0 mg enzyme solution effectively removed hair from the goat skin after 16 h of incubation at room temperature (30°C) (Figure 4). This enzyme has non-keratinolytic and non-collagenolytic in nature. Several microbial proteases were evaluated for their dehairing property (Sivasubramanian et al.2008; Aravindan et al.2007) and it was noticed that only those enzymes with stability under alkaline conditions especially between 9.0 and 11.0 are important. Bacillus subtilis proteases had many advantages when compared with proteases from other Bacillus sp. because of its stability at this range. There is not much published literature concerning enzymatic dehairing process. It is gaining importance as an alternative chemical process and is significant in the reduction of toxicity in addition to the improvement of the texture of leather (Sivsubramanian et al.2008). Alkaline proteases derived from B. circulans, B. cereus and B. subtilis dehaired the goat skin in 18, 21 and 12 h, respectively (Subba Rao et al.2009; Sivasubramanian et al.2008; Nilegaonkar et al.2007). Based on this fact, alkaline protease derived from B. subtilis strain VV can find great use in the leather processing industry.