MOLECULAR CLONING AND SEQUENCE ANALYSIS OF CHITINASE GENE chiA FROM LOCAL ISOLATE OF Bacillus licheniformis MS1

Abstract

Detection of chitin-degrading bacteria from natural sources such as rhizosphere soil is useful for the isolation of bacteria that produce antifungal or other novel compounds. A high correlation between chitinolysis and production of bioactive compounds has been reported (Chen et al., 1991; Pisano et al., 1992; Hoster et al., 2005). Microorganisms, which secret a complex of mycolytic enzymes, are considered to be used as bio- logical control agents of plant diseases (Helisto et al., 2001; Chang et al., 2003; Hoster et al., 2005). Chitin hydrolysis is performed by a major pathway composed of three separate enzymes to break down polymeric chitin to chitin oligosaccharides, diacetylchitobiose and N- actylglucosamine, separately or synergistically and consecutively in the degradation of chitin to free N-actylglucosamine. Endochitinase (E.C. 3.2.1.14) is a poly (1,4-(N-acetyl-β-D-glucosaminide))-glycanohydrolases,  which produce multimers of β-N-acetylglucosamine by random hydrolysis of β-1-4-linkages in chitin and chitodextrins  while Exochitinase (E.C. 3.2.1.52) β-1,4-N-acetylhexosaminidase, is catalyzing the sequential release of soluble dimers starting at the non-reducing end of the polymer. Chitobiases (E.C. 3.2.1.30) or β-N-acetylglucosaminidase, helps the hydrolysis of chitobiose into monomers of N-actylglucosamine (Souza et al., 2003). It is though that chitin hydrolysis occurs either by a sequential or synergetic action of endochitinase, exochitinase, and chitobiases (Tanaka et al., 2003). While exoand endo-chitinases are able to depolymerize chitin alone. The presence of both activities significantly increases the efficiency of chitinolytic system (Howard et al., 2003). Recently, the cloning and expression of the chitinase gene and its introduction into the biologically susceptible species or the construction of recombinant strains with new capacities have been recommended to be one of the interesting areas of chitinase studies and applications. Chitinase genes have been cloned and characterized from many microorganisms (Ueda et al., 2003; Hobel et al., 2005; Yano et al., 2005; Yong et al., 2006). Some of which were either transformed into plants and/or bacterial strains to increase their ability to control phytopathogens (Koby et al., 1994; Punja, 2001) or were high level of expression in Escherichia coli cells to enhance the activity of Bacillus thuringiensis to control pests (Regev et al., 1996; Sampson and Gooday, 1998). Chitinase has received increased attention because of their potential application in the biological control of plants-pathogenic fungi and pests, as well as in the bioconversion of shellfish chitin wastes (Chang et al., 2003; Hoster et al., 2005). In our previous study, local isolate of Bacillus licheniformis MS1 isolated from agricultural fields, Giza, Egypt, was found to be one of the most producing a large amount of chitinase enzyme (Kamil et al., 2007). For this reason this isolate was selected in the present study for cloning, sequencing and molecular analysis of its chitinase gene (chiA).