Online - 2455-3891 Vol 10, Issue 9, 2017 Print - 0974-2441 Research Article ISOLATION, PURIFICATION, AND OPTIMIZATION OF THERMOPHILIC AND ALKALIPHILC PROTEASE ORIGINATING FROM HOT WATER SPRING BACTERIA ASHWINI N PUNTAMBEKAR, MANJUSHA S DAKE* Protein Biochemistry Laboratory, Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth, Pune - 411 033, Maharashtra, India. Email: [email protected] Received: 05 April 2017, Revised and Accepted: 31 May 2017 ABSTRACT Objective: The main objective of this study is to investigate the industrial applications of a thermophillic alkaline protease from a hot water spring bacterial isolate “A” and to study its production, optimization, and purification. Methods: The alkaline protease was produced using shake flask studies maintaining a pH of 9.0 and a temperature of 50°C. Optimization studies of the enzyme were carried out using variable pH, temperature, organic carbon, and nitrogen sources followed by purification of the enzyme using DEAE-cellulose ion exchange chromatography technique. Stability of the enzyme was analyzed in the presence of organic solvents and surfactants. The efficiency of the enzyme in the removal of proteinaceous stains in the presence of strong detergents under extreme conditions was assessed. The fibrinolytic activity of the enzyme in dissolving the blood clot was confirmed. Results: The isolated alkaline protease was purified to homogeneity with a 16-fold increase. Media optimization studies revealed that 1% glucose and 1 % casein-induced the production of alkaline protease. The purified enzyme retained stability in the presence of ethanol, methanol, and acetone and surfactants such as 0.5% (w/v) sodium dodecyl sulfate (SDS) and 0.5% (v/v) Triton-X-100. The isolated alkaline protease successfully removed the proteinaceous stains and showed significant results in the dissolution of blood clot. Conclusion: The above experimental results confirm that the isolated enzyme has both thermophilic and alkaliphilic protease properties. Thereby the enzyme finds promising industrial applications even in extreme conditions. Keywords: Protease, Thermophillic, Alkaliphillic, Purification, Applications. © 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ajpcr.2017.v10i9.19717 INTRODUCTION Microbial proteases have immense applications as compared to other hydrolytic enzymes in various spheres of science and industries and Proteases (EC 3.4.2.1), are the protein hydrolyzing enzymes and there is a high demand of alkaline protease in detergent industries constitute a large group of industrially important enzymes. They catalyze which make it as a potent enzyme [12]. Insoluble fibrous keratin the cleavage of peptide bonds in proteins. Based on their mechanism of protein is the primary substance found in chicken feathers, and cannot action proteases are divided as endopeptidases and exopeptidases. The be degraded by common proteases. Keratinases belong to the group of exopeptidases act only near the terminal amino or carboxyl position. extracellular serine proteases capable of degrading keratin [13]. Endopeptidases are further categorized as serine, cysteine, aspartate, and metallopeptidases based on their catalytic mechanism. Bacterial Thermophilic bacteria from hot water springs produce unique proteases are the most significant [1] and account for nearly 60% thermostable enzymes where proteases originating from such bacterial of the total worldwide enzyme sales [2,3]. , , Aeromonas Alcaligenes species are of particular interest due to their wide range of commercial , , , and are the Arthrobacter Bacillus Halomonas Pseudomonas, Serratia applications [14-16]. major bacterial genera which contribute to proteases. Bacterial alkaline proteases, are specialized due to their higher activity under alkaline Very few reports are available on the bacterial enzymes showing pH range (6-13) specialized and broad substrate specificity. Most of both thermophilic and alkaliphilic nature. Therefore, the present the alkaline proteases have active serine center, whereas some others work aims to study the biotechnological and industrial applications are of a metallo-type where metal ions contribute to enhanced activity of a thermophilic alkaline protease isolated from hot water spring in and stability at higher temperatures [4]. Enhanced thermostability the removal of proteinaceous stains and its medicinal importance as was observed for alkaline proteases from sp., sp., Bacillus Streptomyces a fibrinolytic agent. Production of enzyme to a significant extent was and Thermus sp. after the addition of CaCl [5]. Serine proteases find 2 achieved using wheat bran as a cheaper agro based residues. Entire industrial importance [6]. Thermal stability and activity at alkaline pH study aims to carry out production, optimization, and purification of ensures applications of alkaline protease in detergent formulation [7]. the isolated alkaline protease enzyme. Studies have revealed that bacteria capable of producing alkaline proteases have keratinolytic activity [8]. Alkaline proteases have METHODS wide-scale industrial applications including food processing, leather processing as a dehairing agent, textile industry, diagnostic reagents, Isolation of thermophilic strain household waste management, recovery of silver from X-ray film, Water and soil samples were collected from hot spring region near and bioremediation [9,10]. Proteases have wide applications in many Mumbai (Maharashtra, India) in sterile containers and transported to industries, namely, detergents, and food processing, especially for laboratory and maintained at 4°C microorganisms were isolated from cheese ripening, meat tenderization, animal nutrition, pharmaceutics, collected soil and water samples using nutrient agar plates (pH 7.0) by paper industry, and food industry [11]. serial dilution technique. Plates were incubated at 37°C and 50°C for Puntambekar and Dake Asian J Pharm Clin Res, Vol 10, Issue 9, 2017, 284-291 24-48 h. Pure cultures for seven bacterial isolates (A, B, C, D, E, F, and G) (w/v) concentration in the production medium. Growth of bacterial obtained as individual colonies were screened for the production of isolates and subsequent production of enzyme was monitored at pH 9.0 alkaline protease using skim milk agar (pH 9.0) which were incubated at and a temperature of 50°C after 24, 48, and 72 h. The caseinolytic assay 50°C for 24-48 h. Isolates showing maximum zone of hydrolysis, maximum was performed and the activity was recorded. pH, and temperature tolerance were further selected for production of alkaline protease by submerged fermentation process. The biochemical Effect of variable buffers on enzyme activity characterization was performed for selected bacterial isolates. The stability of protease was analyzed using variable buffers (tris NaOH, Carbonate-bicarbonate, Glycine-NaOH, and Borax) with pH = 9.0 Production of protease by submerged fermentation process at 50°C for 24 h. The enzyme was preincubated along with buffers for The production media (pH 7.0; pH 9.0) comprising glucose 0.1%, 1 h. The activity of protease without buffer was considered 100%. The peptone 1%, yeast extract 0.02% (w/v), MgSO4 0.01% (w/v), CaCl2 0.5% residual proteolytic activity was measured. (w/v), and FeSO4. 7H2O 0.01% (w/v), were inoculated using all seven bacterial isolates (A, B, C, D, E, F, and G) and incubated at 37°C and 50°C Effect of organic solvents and surfactants on the activity of alkaline in a shaking incubator (Remi, Thane, Maharashtra, India) maintained at protease 150 rpm. Media were harvested after 24, 48, and 72 h and centrifuged The stability of alkaline protease from isolate “A” was analyzed in the (± 4°C) at 8000 rpm for 15 min. Supernatant derived from the bacterial presence of 5% (v/v) of different organic solvents (hexane, methanol, culture served as a source of extracellular protease. isopropanol, ethanol, toluene, dimethyl sulfoxide (DMSO), and acetone), 0.5% and 1% (w/v) of surfactants such as (sodium dodecyl Selected bacterial isolates with higher activity were used further for sulfate [SDS], Triton X-100, and Tween-80) and hydrogen peroxide optimization of enzyme production under variable pH and temperature as an oxidizing agent (H2O2). The enzyme was preincubated along conditions. with solvents, surfactants, and oxidizing agents for 1 h. The activity of protease without solvent, surfactant, and the oxidizing agent was Protease assay considered 100%. The residual proteolytic activity was measured. Protease activity was determined by following the regular assay method using casein as a substrate [17]. The protease activity was Purification of alkaline protease assayed by incubating 1 ml of the enzyme with 2.5 ml of 1% (w/v) The cultural supernatant derived from isolate “A” containing alkaline casein (prepared in 100 mM sodium phosphate buffer; pH 7.0 and protease activity was subjected to partial purification using 80% 100 mM Tris-HCl buffer; pH 9.0) at 37°C for 30 minutes. Reaction was ammonium sulfate fractionation. The precipitated proteins were terminated by adding 2.5 ml tricarboxylic acid (TCA). TCA soluble separated by centrifugation of extract at 10,000 rpm for 20 minutes. fraction containing soluble peptides was measured using the Lowry Resultant precipitate was dissolved in Tris buffer (0.02 M, pH 9.0) and dialyzed extensively