Imperial Journal of Interdisciplinary Research (IJIR) Vol-3, Issue-11, 2017 ISSN: 2454-1362, http://www.onlinejournal.in Production and Optimization of Alkaline Protease by Bacillus spp. Isolated from Tannery Industry and Potential Biotechnological Application of the Enzyme Tahsin Khan* & Mihir Lal Saha Laboratory of Microbiology, Department of Botany, University of Dhaka, Dhaka- 1000, Bangladesh Abstract: Four Bacillus spp. strains isolated from the enzyme market most of which are alkaline proteases effluents of tannery industries (Dhaka, Bangladesh) [5]. Most commercial alkaline proteases are showing proteolytic activity were studied to produced by bacteria, especially Bacillus spp. [1] for determine their alkaline protease enzyme production several reasons, including their high growth rates capability. They were also optimized with respect to leading to short fermentation cycle time, initial medium pH and temperature. Their range of accompanied with extracellular secretion of enzymes enzyme activity were between 47.84 and 179.13 [6, 7]. U/ml, initially. The optimum initial media pH was Detergent industries are the primary consumers of 10.0 for B. subtilis B20, 9.0 for both B. subtilis PB18 enzymes, in terms of both volume and value [8]. The and Bacillus sp. BVC38 and 7.0 for B. use of enzymes in detergent formulations enhances amyloliqefaciens Egy25. Optimum temperatures the detergents’ ability to remove tough stains making were determined as 60° C for both B. subtilis B20 the detergent environmentally safe also. Nowadays, and PB18 and 40 °C for the other two organisms. many laundry-detergents contain cocktails of After optimization, alkaline protease production enzymes including proteases, amylases, cellulases capability of B. subtilis B20 and PB18 was increased and lipases. Microbial proteases, have major to 301.80 and 255.34 U/ml, respectively. The crude application in detergent formulations [9, 10]. enzyme of B. subtilis B20 with better wash Proteases also help to prevent the redeposition of performance analysis (visually) and an optimum pH proteins on fabrics, particularly hydrophobic ones 10.0 and 60° C temperature can be considered as a such as blood, thereby also providing a whiteness potential candidate for biotechnological application benefit [11]. Alkaline proteases with superior in detergent industry. The crude alkaline proteases performance for commercial exploitations, especially produced by the four organisms were also tested for for detergents, are being sought. Although such their dehairing potentiality of raw hides and enzymes enzymes have already been found in some strains of from B. subtilis B20 and PB18 showed better results Bacillus proteolyticus, B. thermantarcticus, and B. of dehairing like lime-sulphide chemical treatment, mojavensis [12-14], further exploration of protease which can be considered as environment friendly producers is warranted. alternative to the hazardous chemical treatment in Alkaline proteases are envisaged to have tannery industries. extensive applications in leather industries. The enzymes catalyze the breakdown of the protein Keywords: Alkaline protease; Bacillus spp.; keratin in the hair and allow it to be easily removed. detergent additive; dehairing agent. In a tannery, rawhides are subjected to series of chemical treatments prior to tanning and are 1. Introduction converted to finished leather. Proteases may play a vital role in these treatments by replacing these Proteases or proteinases are proteolytic enzymes hazardous chemicals especially involved in soaking, which catalyze the hydrolysis of proteins. Proteases dehairing and bating [15]. The conventional method have wide applications in leather processing, for depilation has now been clearly recognized to be detergent, food, pharmaceutical, and textile environmentally objectionable accounting for the industries [1, 2]. Proteases can be obtained from discharge of 100 % of sulfide and 80 % of the plants, animal organs and microorganisms, but the suspended solids in the tannery effluent [16]. But the application of bacterial proteases is more significant advantage of using proteases for dehairing of skins when compared to the proteases from other sources are the reduction of the sulfide contents in the [3]. Selection of the right organism plays a key role effluent, recovery of the hair/wool which is of good in obtaining high yield of desirable enzymes. Besides quality, an increased yield of leather area, easy the cultural parameters like temperature and pH also handling of the pelts by workmen, simplification of plays a major role in enzyme production [4]. the pretreatment, the elimination of the bate in the Proteases constitute 60–65% of the global industrial deliming stage and finally the production of a good Imperial Journal of Interdisciplinary Research (IJIR) Page 258 Imperial Journal of Interdisciplinary Research (IJIR) Vol-3, Issue-11, 2017 ISSN: 2454-1362, http://www.onlinejournal.in quality pelts/leather [17, 18]. Usage of enzymes for M Tris-HCl buffer was incubated with 400 μl of dehairing and bating not only prevents pollution crude enzyme for 60 min at 40 °C. The reaction was problems, but also is effective in saving energy. In stopped by the addition of 135 μl of 35% general, proteases play a vital role in leather trichloroacetic acid (TCA) keeping the mixture for processing starting from soaking of hides to finished 10 min at 4° C. After centrifugation at 13,000 rpm products [19, 20]. for 10 min 750 μl of supernatant was mixed with 750 Therefore, taking the demands into account and μl of 1 N NaOH and then absorbance was taken at knowing the geographic richness and biodiversity of 440 nm. The control was prepared by adding TCA our local environment, it was assumed that there is before the addition of enzyme in substrate. One unit potential for alkalophilic Bacillus spp. strains living of protease activity was determined as the amount of here. Discovering such strains and producing enzyme that produces an increase in absorbance of alkaline proteases with novel characteristics will be 0.01 under the above assay condition. of great value to the enzyme industry for different applications. With this as main objective, the 2.4. Characterization of the Crude Protease effluents of Hazaribagh tannery industries were Enzyme screened for isolating bacterial strains of interest and four potential proteolytic Bacillus sp. were found The crude proteases obtained from the four [21]. In this study, the goal was to estimate alkaline organisms were further subjected to preliminary protease enzyme production capability of the characterization study. Therefore, the effects of organisms regarding temperature and pH initial media pH and temperature on their activity optimization. Also, to exploit the potentiality of the were studied. The procedures are outlined below. crude enzymes as detergent additive and dehairing agent of rawhide. 2.4.1. Effect of Initial Media pH on Activity of Protease 2. Materials and Methods The effect of initial media pH on the proteolytic 2.1. Microorganism activity of crude alkaline proteases from the four Bacillus spp. were determined by assaying the Four proteolytic Bacillus sp. were isolated from enzyme activity at different pH values ranging from the effluents of leather processing industries and 7.0 to 12.0 using the following buffer systems: identified as B. subtilis B20, B. subtilis PB18, phosphate (pH 7.0), Tris-HCl (pH 8.0-9.0), glycine- Bacillus sp. BVC38 and B. amyloliqefaciens Egy25 NaOH (pH 10.0–11.0) and KCl-NaOH (pH 12.0) at [21]. 40° C and under standard assay conditions. The concentration of each buffer was 0.1 M. The relative activities were based on the ratio of the activity obtained at certain pH to the maximum activity 2.2. Media and Culture Conditions obtained at that range and expressed as percentage considering 0.5 % error. Seed culture for the experiment was prepared in a test tube containing 5 ml of nutrient broth medium 2.4.2. Effect of Temperature on Activity of (pH 8.0) by inoculating with one bacterial colony Protease from fresh culture and incubating at 37 °C for 24 h. 5 ml of seed culture was transferred aseptically to a The effect of temperature was determined by 250-ml conical flask containing 50 ml of modified incubating the reaction mixture (keeping the different alkaline protease producing broth (APPB) medium optimum pH for each organism) for 60 min at [22] consisting of 1% glucose, 0.5% peptone, 0.5% different temperatures ranging from 20 to 80° C (at yeast extract, 0.5% K2HPO4, 0.01% MgSO4.H2O and 20° C intervals) and under standard assay conditions. pH 8.5. The flask was then incubated in a rotary The relative activities (as % considering 0.5% error) shaker incubator at 40° C for 48 h at 150 rpm. After were expressed as the ratio of the proteolytic activity 48 h, the culture fluid was withdrawn and obtained at certain temperature, to the maximum centrifuged at 8000 rpm for 10 min at 4° C. The cell activity at the given temperature range. free supernatant was used for crude enzyme assay. 2.5. Evaluation of Washing Performance 2.3. Assay for Proteolytic Activity Clean cotton cloth was soiled with fresh bovine Protease activity was determined with azo-casein blood and dried overnight. Small pieces (5cm x 5cm) (Sigma Co. St. Louis Mo), according to the modified were cut from the cloth. The stained cloth pieces procedure described by Kreger and Lockwood [23]. were subjected to wash treatments with commercial In this method, 400 μl of azo-casein solution in 0.05 solid detergent (Jet, local detergent powder available Imperial Journal of Interdisciplinary Research (IJIR) Page 259 Imperial Journal of Interdisciplinary Research (IJIR) Vol-3, Issue-11, 2017 ISSN: 2454-1362, http://www.onlinejournal.in in Bangladesh market) diluted in tap water at 7 activity of PB18 was raised to 255.3 U/ml, which mg/ml, supplemented with and without crude was second highest among the four. enzyme. The stained cloth pieces were taken in separate flasks, with 100 ml as final volume, as mentioned above: flask with tap water only; flask with tap water and only commercial detergent (Jet) at final concentration of 7 mg/ml and flask with tap water commercial detergent and crude enzyme (10 ml).