*Corresponding Author: S.Kavi Karunya, Email: kavi.vit@gmail.com, Phone No: +91- 9629144780 ISSN 0976 – 3333 ORIGINAL RESEARCH ARTICLE Available Online at www.ijpba.info International Journal of Pharmaceutical & Biological Archives 2011; 2(6):1680-1685 Optimization and Purification of Chitinase Produced by Bacillus subtilis and Its Antifungal Activity against Plant Pathogens S.Kavi Karunya*, D.Reetha, P.Saranraj and D. John Milton Department of Microbiology,Annamalai University,Chidambaram – 608 002, India Received 24 Aug 2011; Revised 26 Oct 2011; Accepted 07 Nov 2011 ABSTRACT Microorganisms, which secret a complex of mycolytic enzymes are considered to be possible biological control agents of plant diseases. Since Chitinases are digestive enzymes that break down glycosidic bonds in chitin. In this present study, an industrial enzyme chitinase produced by Bacillus subtilis was optimized, purified and its antifungal activity was investigated against plant pathogens. The bacteria Bacillus subtilis was isolated from soil sample and it was identified by staining techniques, plating on selective media and biochemical characteristics. The Bacillus subtilis showed highest chitinolytic activity in Colloidal chitin agars that degrade chitin in 0.6 mm zone of clearance. The production of chitinase by Bacillus subtilis was optimized under different media, substrates, substrate concentrations, pH, temperature and incubation period. The maximum chitinase production was observed in Luria Bertaini Broth amended with 0.3% colloidal chitin at pH 7.0 and temperature 35˚C after four days of incubation. The enzyme was partially purified by Dialysis method. Protein concentration of 200μg/ml was estimated according to Lowry’s method. The Chitinase has antifungal activity against plant pathogens viz, Aspergillus niger, Aspergillus flavus and Penicillium chrysogenum. Key words: Chitinase, Chitin, Bacillus subtilis, Fungal plant pathogens and Antifungal activity. 1. INTRODUCTION Enzyme technology is an interdisciplinary field, and enzymes are routinely used in many environmental-friendly industrial sectors. With the advancement in biotechnology especially in the area of genetics, protein engineering, developments in bioinformatics, and the availability of sequence data have opened a new era of enzyme applications in many industrial processes [1] . Chitinase, a group of enzymes capable of degrading chitin directly to low molecular weight product [2] and found in a broad range of organisms, including bacteria (Bacillus, Aeromonas, Vibrio, Pseudomonas, Serratia, Enterobacter, Actinomycete), fungi (Trichoderma and Aspergillus), and higher plants, insects, crustaceans, invertebrates and some vertebrates. The roles of these chitinases may be divided into several categories; for example, a major role of fungi, crustaceans and insects is modification of the organisms structural constituent chitin [3, 4, 5] Chitin (C . 8 H 13 O 5 N)n, the (1-4)-β-linked homopolymer of N-acetyl-D-glucosamine, is produced in enormous amounts in the biosphere. It is the most abundant biopolymer next to cellulose with an annual production of 10 10 to 10 11 tons per annum. Shrimp and crab shell, are waste products from canning industries. They are the best sources of chitin. Cell walls of many microorganisms and plants are made of chitin cross-linked with other biopolymers. Chitin binds easily with other materials and can be cast into sheets or films. Chitin and most of its derivatives have a strong positive charge on them. This property has been exploited for many uses. Chitin and its derivatives are biodegradable and biocompatible to humans and most of the animals. For these reasons, there are lot of emphasis on studies on possible uses of Chitin and its derivatives in recent years [6] The production of chitinase by plants has been suggested to be a part of their defense mechanism against fungal pathogens . [7] . Chitinases have received increased attention due to their wide range of biotechnological applications, especially in the biocontrol of fungal phytopathogens [8] . Chitinase have been implicated in plant resistance against fungal pathogens because of their inducible nature and antifungal activities in vitro. Chitinase in fungi is thought to have autolytic,