Imperial Journal of Interdisciplinary Research (IJIR) Vol-3, Issue-11, 2017 ISSN: 2454-1362, http://www.onlinejournal.in

Production and Optimization of Alkaline by Bacillus spp. Isolated from Tannery Industry and Potential Biotechnological Application of the

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 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 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, , 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

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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

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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). Each flask was treated at 40° C for 20 min with agitation. After treatment, cloth pieces were taken out, rinsed with water and dried. Visual examination of the cloth pieces showed the capability of crude enzymes in removing stains. Figure 1. Enzyme activity of four organisms (values presented are the means of triplicate analyses). 2.6. Application of Protease for Dehairing

For dehairing test, goat skin was bought from Table 1. Enzyme activities of the organisms at different local market. Hides were cut in small pieces (3cm x media pH and incubation temperatures, after 24 h. 3cm) and were taken in conical flasks containing 70 Parameters Protease ml of water and 30 ml of crude enzyme. Control was Organisms activity Fixed Variable Levels maintained by treating the hide only with water. For (U/ml) conventional chemical treatment, one piece of the 7.0 123.90 skin was treated with 2% lime and 2% sulphide. The 8.0 169.70 flasks were treated for 24 h in shaking incubator at Temp. 9.0 193.74 150 rpm at 40° C. The treated skin pieces were pH washed with water and deharing was observed by 40° C 10.0 256.33 scratching the skin surface with a blunt knife. B. subtilis 11.0 143.87 B20 12.0 87.28 2.7. Statistical Analysis 20 71.60 pH Temp. 40 256.33 Statistical analysis was performed with the 10.0 (°C) 60 301.80 Statistical Package for the Social Sciences (SPSS) v. 80 26.20 16.0 for windows (SPSS, SAS Institute Inc. Cary, 7.0 63.78 USA). Effect of each parameter was studied in 8.0 99.85 triplicate and the data were presented as the mean ± Temp. 9.0 187.30 standard deviation (SD) of triplicates (n= 3). pH 40° C 10.0 155.90 3. Results B. subtilis 11.0 116.94 PB18 12.0 74.61 3.1. Protease Activity of the Selected Isolates 20 64.20 Temp. 40 187.30 pH 9.0 The comparative study of the four organisms for (°C) 60 255.34 the enzyme production was carried out separately in 80 20.90 modified APPB medium at pH 8.0 and their range of 7.0 50.49 enzyme activity were between 47.84 and 179.13 8.0 59.18 U/ml. B. subtilis B20 showed highest proteolytic Temp. 9.0 84.90 activity (169.70±9.43 U/ml) among the four pH 40° C 10.0 43.50 organisms. B. subtilis PB18 also showed better enzyme productivity of 99.85±0.89 U/ml. Alkaline Bacillus sp. 11.0 30.80 protease production capacity of Bacillus sp. BVC38 BVC38 12.0 20.57 and B. amyloliqefaciens Egy25 were relatively lower 20 54.70 Temp. 40 84.90 than the former two; 59.18 ± 6.19 and 48.38 ± 0.54 pH 9.0 U/ml, respectively (Figure 1). (°C) 60 60.50 After optimization of both pH and temperature for 80 30.80 alkaline protease enzyme production of all the four 7.0 70.90 B. organisms, B. subtilis B20 remained to be the highest 8.0 48.38 amyloliqef- Temp. crude enzyme producer; 301.80 U/ml at pH 10.0 and pH 9.0 35.27 aciens 40° C 60° C temperature (Table 1). After optimization of 10.0 13.80 Egy25 pH at 9.0 and temperature at 40° C proteolytic 11.0 10.52

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Imperial Journal of Interdisciplinary Research (IJIR) Vol-3, Issue-11, 2017 ISSN: 2454-1362, http://www.onlinejournal.in

12.0 6.89 BVC38 and B. amyloliqefaciens Egy25 have at 40° C 20 28.90 at their optimum pH levels determined previously. Temp. 40 70.90 One should also consider that the enzymes pH 7.0 (°C) 60 37.90 synthesized at higher temperatures might face the 80 16.30 risk of denaturation of the protein, as well as degradation due to the proteolytic activity of the 3.2. Effect of Initial Media pH on Protease protease produced. This could contribute to the lower Activity relative activity results obtained at higher temperature in this study as well. Lesser enzyme The effect of pH on proteolytic activities of the activity was seen by all the organisms at both higher crude enzyme produced by the organisms were (80° C) and lower (20° C) temperature points (Figure determined over a pH range from 7.0 to 12.0. The 3). pH-activity profile of B. subtilis B20 (Table 1) shows that the enzyme has a broader pH range and is highly active between pH 7.0 and 11.0 with an optimum around 10.0. The relative activities (Figure 2) at pH 9.0 were about 76% of that at pH 10.0. However, the protease activity decreased to about 56% at pH 11.0. The data also suggests that both B. subtilis PB18 and Bacillus sp. BVC38 might range to be a neutral to alkaline in terms of proteolytic activity and their optimum pH was 9.0. However, in case of B. amyloliqefaciens Egy25, there was an inverse relationship between the enzyme synthesis and pH increase (based on the specific protease activities) Figure 3. Effect of temperature on protease activity of and it had optimum pH of 7.0 (additional the organisms. experiments performed revealed that the optimum pH was not below 7.0; data not shown) for alkaline protease production. With this regard, B. subtilis B20 3.4. Wash Performance Analysis seemed to be a better candidate for further studies as pH of laundry detergents is generally in the range of As shown in Figure 4, limited washing 9.0–12.0. performance was observed with detergent only (D), however the treatment of stains washed with detergent supplemented with crude enzymes from the four organisms (E-H) gave a better stain removal. The degree of blood stain removal from the fabric was found in the order of: crude protease enzyme and sterile distilled water with detergent > sterile distilled water with detergent > sterile distilled water. Among the four, crude enzyme produced by B. subtilis B20 performed best in stain removal as supplement with detergent (Figure 4E).

Figure 2. Effect of pH on the relative protease activity of the organisms.

3.3. Effect of Temperature on Protease Activity

The effect of temperature on proteolytic activities of the crude enzyme produced by the organisms were determined over a range from 20-80° C at 20° C intervals. Based on the maximum protease activities (Table 1), both B. subtilis B20 and PB18 have temperature optima at 60° C whereas Bacillus sp.

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Imperial Journal of Interdisciplinary Research (IJIR) Vol-3, Issue-11, 2017 ISSN: 2454-1362, http://www.onlinejournal.in

Figure 4. Photographs showing wash performances of commercial detergent and the crude enzymes of four organisms. (A) Clean cotton cloth; (B) Dried blood stained cloth; (C) Washed with tap water; (D) Washed with detergent only; (E-H) Washed with detergent supplemented with the crude enzymes from the four organisms.

3.5. Dehairing Test Figure 5. Photographs showing dehairing of goat skin Figure 5 shows the dehairing test of goat skin. A by the enzyme produced by organisms. (A) Control; (B) comparative test was carried out by the conventional Chemically treated skin; (C-F) Enzyme treated skin by chemical and crude enzyme from the four organisms. the organisms. Crude enzymes were found to be effective for dehairing equally to that of lime-sulphide (B). 4. Discussion Among the four organisms, the enzyme produced by B. subtilis B20 and PB18 showed more than 98% In our previous work, we reported isolation and dehairing (C-D) and B. amyloliqefaciens Egy25 identification of four high proteolytic activity showed more than 90% dehairing (E). Crude alkaline showing Bacillus spp. [21]. Alkaline protease protease of Bacillus sp. BVC38 was not very activity of the four organisms was determined with effective for dehairing (F). The skins treated with the azo-casein and two of them viz. B. subtilis B20 and enzyme felt much softer and had better elasticity than PB18 showed better capacity of enzyme production. the chemically treated one (data not shown). On the Ferrero et al. [24] reported the production of two other hand, the skin treated by lime-sulphide was alkaline proteases by B. licheniformis MIR 29. B. rubbery and hard. licheniformis K-508 also secreted at least two types of proteases, a -like and a chymotrypsin-like protease [25]. The optimum pH for the proteolytic activities of two better alkaline protease producing isolates were 10.0 and 9.0, respectively (Table 1). These findings are in accordance with several earlier reports showing pH optima of 9.0–10.5 for proteases of Bacillus cereus [26], B. mojavensis [11] and B. licheniformis YP1A [27]. The higher relative activity of crude protease produced by the B. subtilis B20 over a broad pH range indicate its potential application for industrial purposes, which require stability over wide pH ranges. The growth and enzyme activity of microorganisms is greatly influenced by different incubation temperatures. The growth of microorganisms can be inhibited at one temperature but it can be activated at another temperature.

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Imperial Journal of Interdisciplinary Research (IJIR) Vol-3, Issue-11, 2017 ISSN: 2454-1362, http://www.onlinejournal.in

Temperature significantly regulates the synthesis and effects of hazardous chemical treatments in the secretion of bacterial extracellular protease by environment. changing the physical properties of the cell membrane [28]. Therefore, temperature is a critical 5. Conclusion parameter that should be controlled to obtain an optimum enzyme production. Li et al. [27] showed In this paper four Bacillus spp. viz. B. subtilis the production of an alkaline protease by B. B20, PB18, Bacillus sp. BVC38 and B. licheniformis YP1A with optimum temperature of amyloliqefaciens Egy25, with proteolytic activity 60° C. Optimum temperature of both B. subtilis B20 were studied to determine their alkaline protease and PB18 are in accordance with this data. enzyme production capability and their couple of The detergent proteases work best by hydrolyzing fermentation conditions (initial media pH and large insoluble proteins in the bulk wash liquor. temperature) were optimized. Based on the results, B. Proteins are initially removed from the fabric surface subtilis B20 had highest protease production capacity either by components of the detergent matrix or by of 301.80 U/ml at pH 10.0 and 60 °C temperature, water alone. Depending upon the size of the resulting 10% inoculum with an incubation time of 24 h. fragments, they are either solubilized into bulk Whereas second highest protease activity (255.34 solution or they deposit themselves back to the U/ml) was obtained from B. subtilis PB18 at pH 9.0 fabric. Hence, the best detergent enzymes provide and all the other parameters were same as the improved substrate hydrolysis, resulting in better previous one. So, B. subtilis B20 and PB18 showed a stain removal and anti-redeposition benefits. The closer performance with the main difference in their obtained results of this experiment demonstrate that pH profiles. In all experiments, B. amyloliqefaciens crude protease enzymes from the organisms can Egy25 and Bacillus sp. BVC38 were less effectively remove blood stains (Figure 4). This performing. Taking all the results into account, B. agrees with an earlier report on laundry performance subtilis B20 can be the strain of choice for further of Subtilisin proteases, where the protease acted optimization study and a potential candidate for synergistically with the detergent to efficiently future industrial applications. Overall, the highest remove stain by hydrolyzing large insoluble protein protease activity (301.80 U/ml) achieved in this fragments which strongly adhered to the fabric [29]. study was lower compared to the regular proteases Reports have mentioned the usefulness of alkaline used industrially, which are mostly in purified forms. proteases from B. circulans [30] in the facilitation of One should also consider that the alkaline protease blood stains removal from cotton cloth both in the activity in this study is from crude enzymes, obtained presence and absence of detergents. Abidi et al. [31] purely from natural strains, which did not go through showed that the supplementation of proteolytic any strain improvement study. Furthermore, a preparation of Botrytis cinerea in detergent (Henkel- characterization after purification and possible Alki) significantly improved the cleaning of blood, application study of this enzyme would be a task to egg yolk and chocolate stains. Therefore, crude do in future. Considering the biodiversity of our enzymes produced by the organisms of this research environment it is highly important to discover new could be considered as a potential candidate for use enzymes from isolates living under extreme as cleaning additive in detergents to facilitate the conditions, which could have novel properties that removal of proteinaceous stains. could contribute to the current enzyme potential. Leather processing is one of the most important industrial activities in Bangladesh. Dehairing of 6. Acknowledgements hides is an important and unavoidable step involved Gratitude to the Ministry of Science and in the processing of leather in the tanneries. The Technology, People s Republic of Bangladesh for conventional lime sulphide method employed for this ’ providing financial support for this research work purpose creates pollution hazards [32]. Hence an through NST fellowship program. Also, gratitude to attempt to minimize the use of lime sulphide in the the Biotechnology Research Centre, University of tanneries and to develop an environment friendly Dhaka for providing partial financial support for this method of dehairing was made in the present study. research project. Authors are grateful to Dr. Md. In a previous work by Mukhtar and Haq [20] alkaline Mozammel Hoq, professor, Department of protease from Bacillus subtilis IH-72 was used for Microbiology, University of Dhaka and Md. Arafat the complete depilation of the skin, which was Al Mamun, Senior Scientific Officer, Centre for achieved after 4 hrs of treatment and the resulting Advanced Research in Sciences, University of Dhaka pelt was of white color with no scud. Similar results for their support in estimation of the enzyme have been achieved by utilizing the crude protease activities. enzyme from B. subtilis B20 and PB18. So, the enzymes from these organisms can be also considered as potential candidates for dehairing of raw hides in tannery industries and thus reduce the

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Imperial Journal of Interdisciplinary Research (IJIR) Vol-3, Issue-11, 2017 ISSN: 2454-1362, http://www.onlinejournal.in

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