Microbial Production of Alkaline Proteases and Evaluation of Its Performances for Pretreatment of Leather Industry

Journal of Multidisciplinary Engineering Science and Technology (JMEST) ISSN: 3159-0040 Vol. 2 Issue 12, December - 2015 Microbial Production Of Alkaline Proteases And Evaluation Of Its Performances For Pretreatment Of Leather Industry 1 2 4 5 Uddin M. E.1, Ahmad T. , Sarkar M. K. I.2, Azim D. A. , Rahman S. S.3, Islam M. S. , Karim M. R. , Rahman M. M.1, Rahman M.6, Islam M. R.*1 1 Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia, Bangladesh 2Department of Biochemistry, Primeasia University, Dhaka, Bangladesh 3Department of Math and Natural Science (MNS), BRAC University, Dhaka, Bangladesh 4Department of Applied Nutrition and Food Technology, Islamic University, Kushtia, Bangladesh 5Department of Microbiology, Chittagong University, Chittagong, Bangladesh 6Department of Biochemistry and Molecular Biology, Dhaka University, Dhaka, Bangladesh * Corresponding author: Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia, Bangladesh, E. mail: [email protected] (Md. Rezuanul Islam) Abstract—A high alkaline protease producing Introduction bacterial strain was isolated and identified a local soil sample. The organism was gram positive and Over the last few decades leather industry is based on forms spore during adverse condition in the large scale chemicals treatment which created growth medium. After various tests it was worldwide environmental hazards. Leather suggested and the features agreed with the manufacturing is one of the industrial activities description of Bacillus subtilis. It was also globally wide spread, which involves the use of wide identified as B. subtilis with 99.9% identity by API range of chemicals many of which are hazardous, 50 CHB. The enzyme hydrolyses a number of highly toxic and obnoxiously odorous [6, 25]. Enzymatic proteins including azocasein which suggests that dehairing in tanneries has been envisaged as an it is an extracellular alkaline protease. The alternative to sulfides [26]. Use of enzymes for experimentally determined isoelectric point was industrial processing has received considerable 5.1 and the optimal enzyme activity was at 60°C attention in recent years owing mainly to and at pH 8.5. The esterase preferentially environmental concerns [11]. Proteases help in hydrolyzed short-chain fatty acids. Native enzyme breakdown of proteins into simpler form that exist preparations typically showed a Michaelis between two amino acids of a polypeptide chain by constant (Km) and Vmax of 0.40mM and 12,200 U the process of hydrolysis [21]. mg)-1, respectively. This microbial enzyme was partially purified by ammonium sulfate Leather industries are one of the most promising fields fractionation, dialysis, DEAE cellulose for export to earn foreign currency in Bangladesh. chromatography and electrophoretic analysis. Most of the tannery industries in Bangladesh use Enzyme purity was tested by SDS-PAGE. chemicals for dehairing that led great environmental Quantitative estimation has shown that 40mL of and health problem. Recently government of People’s culture supernatant could dehair 2×1 cm of leather Republic of Bangladesh has taken initiative to develop completely in 9 hours. In future the tanneries will the industry from outside the city and modernize it [18]. use a combination of chemical and enzymatic Enzymatic dehairing is suggested as an environment processes. In practical applications, protease is a friendly alternative to the conventional chemical useful enzyme for promoting the hydrolysis of process [18]. Enzymes have been pursued as one of proteins and showing significant industrial the promising alternates to lime and sodium sulfide [7]. applications. Enzymes display a high capability of degrading insoluble keratin substrates of their several potential Keywords—Bacillus subtilis, SDS-PAGE, uses associated to the hydrolysis of keratinous Alkaline proteases, Azocasein test, Leather substrates and other applications [3]. In recent years industry, Electrophoretic analysis proteases find application in leather making among the different industrial proteases the most widely used [5] enzymes in leather manufacturing . In the back drop of this scenario enzymes started replacing poisonous www.jmest.org JMESTN42350702 3370 Journal of Multidisciplinary Engineering Science and Technology (JMEST) ISSN: 3159-0040 Vol. 2 Issue 12, December - 2015 chemicals from tannery industries. A number of CHB (BioMerieux, France), was used to identify industries such as NOVO chemicals started producing species of bacteria. NOVOzymes for the tannery industries. Biochemical & Microbiological tests for the Higher cost of enzyme is one of the major factors for characterization of the organism: To identify the the system not being practiced through found biochemical properties of the organism different tests environmentally friendly. It is essential to develop a were performed. For correct interpretation of the cost effective and eco-friendly technology by screening for efficient enzymes from microbial results in every test Escherichia coli was taken as sources and producing them in large quantities by control. The carbohydrate tests that were performed applying recombinant DNA technology. Enzymes are the Glucose, Lactose, Ribose, Sucrose, Mannitol, found in nature are quite often not readily available in Adonitol, Arabinose, Sorbitol, and Maltose. Others quantities sufficient for industrial use, so use of gene Biochemical tests that were performed are the expression methods to express recombinant proteins Hydrogen sulfide test, Motility Test, Indole Production in suitable heterologous expression systems is [2] Test, Citrate Utilization Test, Nitrate Reduction Test, required . Genetic engineering could be used to increase the gene copy number as an effective Oxidase test (young culture), Catalase Test, Urease method for improving enzyme productivity [13]. test, Indole (SIM) test, Methyl Red (MR) Voges- Secretion of recombinant proteins is a common Proskauer (VP) Test, Starch Hydrolysis Test and strategy for heterologous protein expression. The Gelatin Liquefaction Test. Some Microbiological tests major goal of the research showing that proteases that were performed are the Gram staining for the enzyme can be utilized in enzymatic dehairing of cow Bacteria, Spore staining, colony morphology and skin in tannery industry to control the environment growth curve determination. from pollution, which is a prerequisite for biotechnological applications. Production of Protease and Proteolytic Activity Materials and Methods Evaluation by Azocasein Test: The microorganism was cultivated in sterile nutrient broth medium. The Microorganism, Culture Medium and Growth culture was grown overnight on a rotary shaker at 150 Conditions: Soil samples were collected from the rpm and incubated at 37°C for 15-20 hours. The poultry wastes in Savar, after serial dilution, culture culture was then centrifuged at 10000 rpm for 10 was given in LB broth media from the sample for 16 h minutes at 4°C. The supernatant was collected and at 37°C. At the next day single colony was found. used as crude enzyme sample. Proteolytic activities were assayed by Azocasein test, described by Kreger Among them few colonies were identified on the basis and Lockwood (1981) was done. Here azocasein is of different colony morphology. Each colony was used as a substrate. Optical density was measured at inoculated into screw capped test tubes containing 440 nm. autoclaved feather with liquid broth media and incubated overnight at 37°C with shaking at 160 rpm. Evaluation of Growth profile and protease activity One media was used as negative control. Chemicals of the organism at 37ºC: The organism was grown in used in the experiment were from Oxoid Ltd. nutrient broth at 37ºC. Samples were taken at (Basingstoke, UK), Merck AG (Darmstadt, Germany), different time interval and absorbance was taken at and Sigma (USA). Azokeratin was synthesized based 600nm to measure the growth profile. The growth on the method described in a previous study [20]. profile of the organism showed that the organism showed optimum growth after about 24 hours and the Isolation and Identification of Bacteria from Local protease activity was the maximum after 26 hours of Soil Sample: The soil sample was collected from the incubation. In the initial stage of growth there was poultry wastes in Savar, after serial dilution, culture basal level of extracellular protease which increased were given in LB broth media from the sample for 16 h with the increase of time. The result showed that there at 37°C. At the next day single colony was found. Among them few colonies were identified on the basis was differential synthesis of enzyme with growth time. of different colony morphology. Each colony was inoculated into screw capped test tubes containing Effect of pH and temperature on enzymatic activity autoclaved feather with liquid broth media and and stability: For determining the effect of pH on incubated overnight at 37°C with shaking at 160 rpm. protease activity different buffer system with different One media was used as negative control. Gram’s pH were used. Azocasein was dissolved in different staining; morphological studies, physiological and buffer solution and the enzyme assay was carried out biochemical characteristics of the isolate were within pH range (4.0 to 10.5) by azocasein assay investigated according to Bergey’s Manuals [24]. A method. All of them were used at 0.05M rapid bacterial identification test kit for Bacillus, API 50 concentration.

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