Characterization of Chitinase and Partial Gene Cloning from Microbacterium sp. TU05

Abstract

A bacterium TU05 was isolated from soil in Bangkok, Thailand. It can produce chitinase (EC. 3.2.1.14) that catalyzes the degradation of chitin. Gram-stain, biochemical characteristics, and sequence of 16S rRNA gene, identified this bacterium as Microbacterium sp.. Microbacterium sp. TU05 produced highest chitinase activity on the second day in CCMM and the tenth day in FCMM. Hydrolysis products analyzed by HPLC, crude enzyme showed two enzymatic activity, chitinase and chitobiase. The optimum pH and temperature were pH 5.0 and pH 7.0 and 40°C, respectively. Substrate specificity of crude enzyme illustrated it was exochitinase, because it had the highest hydrolytic activity on colloidal chitin. After SDS-PAGE and activity staining, at least two bands of chitinase activity were found with molecular weight 65 and 30 kDa. When partially purified by DEAE-cellulose column chromatography, two peaks of chitinase activity were found. After SDS-PAGE and activity staining of the partially purified chitinase peak1, one band has chitinase activity with molecular weight of 65 kDa. Partially purified chitinase peak2 has 2 bands of chitinase activity at 55 and 30 kDa. Partially purified chitinase peak1 was selected for characterization. The optimum pH and optimum temperature of partially purified chitinase peak1 were 5.0 and 40°C. Shotgun cloning technique was employed for chitinase gene cloning. After screening 30,000 transformants, no positive clones were found. Partial chitinase gene amplification of Microbacterium sp. TU05 by PCR amplification using primers designed from conserved amino acid sequence of Bacillus sp. Family 18 chitinase gene was performed. The PCR products was 636 bp with 71% similarity to chitinase from Arthrobacter sp.