ISOLATION, SCREENING, AND CHARACTERIZATION OF CHITINASE PRODUCING BACTERIA FROM MARINE WASTES Original Article S. KRITHIKA 1 , C. CHELLARAM 2,3 * 1 Sathyabama University, Chennai 600119, Tamil Nadu, India, 2 Vel Tech Multitech Engineering College, Chennai 600062, Tamil Nadu, India, 3 Applied Biotechnology Department, Sur College of Applied Sciences, Sur-411, Oman Email: chellaramc.sur@cas.edu.om Received: 28 Nov 2015 Revised and Accepted: 15 Mar 2016 ABSTRACT Objective: Aim of this study deals with screening and characterization of chitinase-producing bacteria from marine waste and its deposited soil along the coastal regions in Chennai. Methods: The soil samples were collected aseptically and subjected to serial dilution to isolate the bacterial strains. Totally, 35 morphologically different microorganisms were isolated and were screened for their chitinolytic activity in colloidal chitin incorporated media through zone assay using Congo red stain. The biochemical tests were performed for the isolated to prove their validity and further with sequencing to determine the species. Results: The isolates were screened based on the size of the zone formed. Best chitinase producers were subjected to biochemical tests and 16s ribosomal RNA sequencing. A novel strain, Acinetobacter ASK18, a gram-negative, motile organism was identified. Thus, the isolate may be a potent producer of chitinase, and the marine wastes can be utilized efficiently to generate a high value-added product. Conclusion: A novel strain, Acinetobacter ASK18, would further be subjected to purification of the enzyme produced, and hence the active principle could be evaluated as an effective pharmacological drug in anticancer and antibacterial properties. Keywords: Marine wastes, Chitinase, Congo red, Biochemical tests, 16s rRNA sequencing © 2016 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/) INTRODUCTION Chitin is considered as the second natural polymer next to cellulose with the structural unit of N-acetylglucosamine linked by β-1,4 bonds. It is present in the cell wall of higher fungi, exoskeletons of insect, and shells of crustaceans [1, 2]. Most of the seafood processing industries contain chitin in the processed marine wastes. These wastes may cause major environmental problems due to its easy deterioration. Such chitinous wastes are degraded through chemical processes like demineralization and deproteinization, which causes corrosive problems, low yield, and high costs [3]. Being more eco-friendly and cost effective method as compared to the chemical method for chitinous degradation, the enzymatic method can be adopted as an alternative. Chitinases (EC 3.2.1.14) are a set of enzymes that are produced by several bacteria, actinomycetes, fungi, and also by higher plants [4-8]. Chitinases play a major role in degrading the chitinous waste from the seafood industry and thus retains the carbon-nitrogen balance in the environment through the utilization of crustacean waste [9]. Shrimp waste is considered as a major source of chitin. The presence of chitinolytic microbes indicates the availability of chitin in the soil. Chitinases also play a major role in many areas such as the production of single cell protein, growth factors [10, 11], mosquito control, a biocontrol agent of fungal pathogens, and isolation of fungal protoplasts [12, 13]. Thus, the need of microbial chitinase production has increased, and it serves two purposes: (i) reduce environmental hazards and (ii) increases production of industrially important value-added products. Thus, the present study has been narrowed on isolation, screening, and characterization of chitinase producers from marine waste samples collected from Chennai. MATERIALS AND METHODS Chemicals The materials, media, reagents used for this study were procured from Titan, SRL and Hi-Media, India. Methods Collection of soil samples Prawn shell and depositing premises in the fish market of Tambaram and Vanagaram areas in Chennai, Tamil Nadu, India were selected for the soil collection. Soil at the depth of approximately 9– 12 cm was collected in a sterile zip-lock cover with the help of a sterile spatula and placed in an ice pack for transportation to the laboratory and it was processed [14]. Preparation of colloidal chitin To 10 g of chitin powder (Titan, India), added 120 ml of conc. HCl, incubated at 37 °C, 180 rpm for 1 h. The mixture was transferred through glass wool to 50% ethanol and thoroughly mixed to obtain a homogenous suspension. This was further transferred through filter paper and washed with distilled water until the colloidal chitin reaches pH 7. Colloidal chitin was collected and stored at 4 °C until use [15]. Isolation of chitinase producers Chitin utilizing bacteria from the collected soil sample was isolated by serial dilution and spread plate technique. 1 ml of each dilution was plated in triplicates on nutrient agar medium supplemented with 1% colloidal chitin and incubated at room temperature (27 °C) for 3 d, and isolation of bacteria was carried out from the third day onward. The chitinase producers were selected based on the morphology, color, and growth in the colloidal chitin-incorporated medium [16]. Screening of chitinase-producing bacteria Quadrant streak of all the isolates was carried out in nutrient agar plate supplemented with colloidal chitin to isolate the potential organism based on the chitinase produced. Single streak inoculation measuring 2 cm length was performed for all the bacterial isolates on nutrient agar medium supplemented International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 8, Issue 5, 2016