ISOLATION and CHARACTERIZATION of HYDROLYTIC ENZYME PRODUCING HALOPHILIC BACTERIA Salinicoccus Roseus from OKHA

International Journal of Microbiology Research ISSN: 0975-5276 & E-ISSN: 0975-9174, Volume 7, Issue 2, 2015, pp.-616-622. Available online at http://www.bioinfopublication.org/jouarchive.php?opt=&jouid=BPJ0000234 ISOLATION AND CHARACTERIZATION OF HYDROLYTIC ENZYME PRODUCING HALOPHILIC BACTERIA Salinicoccus roseus FROM OKHA RAVI R.K.1* AND PATEL P.2 1Department of Biotechnology, Shree M. & N. Virani Science College, Rajkot - 360 005, Gujarat, India. 2Department of Biotechnology, Christ College, Rajkot - 360 005, Gujarat, India. *Corresponding Author: Email- [email protected] Received: January 15, 2015; Revised: May 14, 2015; Accepted: May 18, 2015 Abstract- Halophilic bacteria were isolated and purified from marine water of Okha region of Gujarat, India and further subjected to screen for extracellular hydrolytic enzyme production. Two strains (R1 & R2) showed extracellular hydrolytic enzyme activities, such as amylases, proteases, lipases and gelatinase, where as optimum activity observed with amylase. They were characterized on the basis of morphology, physiol- ogy and biochemical tests. The phylogenetic tree revealed that the strains R1 & R2 fit in to an evolutionary cluster comprising members of Salinicoccus roseus with 99% similarity. 16S rDNA sequence was submitted at NCBI and named as Salinicoccus roseus strain rvscokh1 & rsk1 respectively. The DNA G+C content rvscokh1 & rsk1 was 55.6 & 55.7 mol% respectively. The GenBank accession number of the 16S rDNA sequences of strain rvscokh1 & rsk1 is HQ190916 & HQ258884 respectively. Salinicoccus roseus is gram positive coccus bacteria. Strain rvscokh1 & rsk1 sustain to 10% and 6% NaCl concentration respectively while optimum pH was 7.4 for growth. Optimum amylase activity was found at temperature 37°C, pH 8 and 5% salt concentration for both the strains reveals that these strains can produce potentially industrially important enzymes. Keywords- Phylogenesis, Halophiles, Hydrolytic enzymes, Amylase, Salinicoccus roseus Citation: Ravi R.K. and Patel P. (2015) Isolation and Characterization of Hydrolytic Enzyme Producing Halophilic Bacteria Salinicoccus roseus from Okha. International Journal of Microbiology Research, ISSN: 0975-5276 & E-ISSN: 0975-9174, Volume 7, Issue 2, pp.-616-622. Copyright: Copyright©2015 Ravi R.K. and Patel P. This is an open-access article distributed under the terms of the Creative Commons Attribu- tion License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. Introduction efficient processes [5]. The purpose of our study was to isolate and In recent years, there has been more interest in studying the micro- characterize pure cultures of extreme or moderate halophiles from biology of halophiles since these naturally occurring hypersaline enrichments obtained from marine water samples of coastal region environments are the potential source of diverse microorganisms of Gujarat and potentials to check extracellular hydrolytic enzyme [1]. Halophilic bacteria are found in marine, great salt lakes, natural production. Extracellular hydrolytic enzymes such as amylases, or artificial salterns, salina etc. Those halophiles whose optimum proteases, lipases etc have diverse potential usages in industrial salt concentration for growth is above approximately 5-20% (w/v) and chemical sectors. Bacterial amylases are potentially beneficial NaCl are considered moderate halophiles and 20-30% (w/v) NaCl in terms of ease to obtain, higher yield, ease to purify and thermo- are considered extreme halophiles which are of valuable use for stablity. Screening of marine microorganisms with higher amylase research regarding hereditary, physiological and biochemical fea- activity could therefore facilitate the discovery of novel amylases tures and strain resources [2]. These extreme environments have suitable for new biotechnological and industrial applications [6]. In been found to contain halophilic organisms that developed strate- present investigation, extracellular hydrolytic enzyme producing gies or live optimally in such environments and lives occurring in halophilic bacteria Salinicoccus roseus strain rvscokh1 & rsk1 was such environments have found to possess an elite biotechnological isolated and purified from marine water of Okha region of Gujarat potential [3]. Many halophiles and halotolerant microorganisms can and optimization study for amylase activity has been performed. grow over a wide range of salt concentrations depending on envi- Based on determining parts of physiological and biochemical index, ronmental and nutritional factors for the growth and tolerance [4]. 16S rDNA sequence was obtained, after which the homology was Studies on halophilic bacterial extracellular enzymes are important compared and phylogenetic analysis was established. with respect to their industrial applications mainly in fermented food, Materials and Methods textile, pharmaceutical, leather industries, nutrient recycling and biodegradation process. Halophilic microorganisms have increased, Source of Strains and additional applications of industrially important enzymes are still Halophilic bacteria were isolated from a Marine water sample col- under development. These uses have placed greater stress on lected from Okha region (22°28'11"N 69°4'47"E) at Gujarat, India. increasing indigenous amylase production and search for more The samples were collected using a sterile container and were International Journal of Microbiology Research ISSN: 0975-5276 & E-ISSN: 0975-9174, Volume 7, Issue 2, 2015 || Bioinfo Publications || 616 Isolation and Characterization of Hydrolytic Enzyme Producing Halophilic Bacteria Salinicoccus roseus from Okha transferred to laboratory. All the samples were stored in cold condi- ed by addition of 3, 5 dinitrosalicylic acid and followed by boiling for tion. 10 minutes [14]. The final volume was made up to 12 ml with dis- tilled water and optical density was measured at 540 nm. One unit Culture and Isolates of amylase activity was defined as amount of µg of maltose equiva- The samples were serially diluted and each diluted samples were lent liberated per minute per ml of enzyme under assay condition. plated on Nutrient agar (NA) and Seghal and Gibbons (SG) agar The amount of maltose was determined from the maltose standard media, supplemented with 5%-15% NaCl for salinity and incubated curve. Enzyme productions were further optimized using various at 37˚C for 2-4 days. After incubation, representative isolated colo- parameters like temperature, pH and salinity in different ranges. nies were transferred to nutrient broth medium. Isolates were rou- tinely cultured aerobically at 37°C in Nutrient broth medium [7] con- Characterization and Growth Condition taining (g/L): Peptic digest of animal tissue 5.0 g, Sodium chloride The isolates were examined for their colony shape, spore formation, 5.0 g, Beef extract 1.5 g and Yeast extract 1.5 g, pH 7.4 supple- motility and pigmentation. Gram staining was performed by using mented with 5-15% NaCl for 72 h. Solid medium was prepared by acetic acid-fixed samples as described by Dussault [15]. Growth adding 15 g/L agar. The selected colonies were purified three or rates at different salt concentrations were determined by using four times in the solid medium before they were put into the liquid maintenance medium prepared with salt concentrations of 5-15% medium to culture for 3 days at 37°C at 120 rpm. Same method (w/v). Biochemical tests were performed according to Bergey’s followed for Seghal and Gibbons media containing Yeast extract Manual of Systemic Bacteriology (1994). (HiMedia) 1.0 g, Casamino acids (HiMedia) 0.75 g, Na citrate 0.3 g, The optimal conditions for growth of isolates and amylase produc- MgSO4· 7 H2O 2.0 g, KCl, 0.2 g, FeCl2 0.0023 g, NaCl 25 g, dis- tion were tested with varying parameters. The pH growth range was tilled water 100 ml [8]. pH 5-10, NaCl growth range 5-15 % and temperature range was 15°C-50°C taken for optimization. To determine antibiotic sensitivi- Screening of Extracellular Hydrolytic Enzymes ty, an active culture was inoculated onto Mueller-Hinton (Hi-media) The isolates were screened for the production of extracellular hy- agar plates, octadiscs impregnated with antibiotics were placed on drolytic enzymes such as amylase, protease, lipase and gelatinase their surface. The plates were incubated and the zones of inhibition using different plate assays method as detailed below: around the antibiotic discs were recorded [16]. All the growing tests were carried out according to the following Amylase Production methods: 5 ml liquid medium was interfused with 0.5 ml bacterium The presences of amylolytic activity on plates were determined liquid in each tube; then, the strain was cultured for 3 days at 37°C, using method described by Coronado [9] using HM medium supple- optical density was detected at λ=600 nm, test was repeated three mented with 0.5% (w/v) soluble starch. After incubation at 37°C for times. 3 days, the plates were flooded with 0.3% - 0.6% KI solution, a clear zone around the growth indicated hydrolysis of starch. DNA Extraction DNA extraction was performed according to following methods: Protease Production Cells were cultured to approximately the late exponential phase and Proteolytic activity of the cultures was screened qualitatively in a then harvested by centrifugation in 15 ml portions at 10,000 rpm for saline medium containing 2% (w/v) sterile skim milk [10]. Formation 10 min.

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