Mutagenetic Study on Chitinase Producing Chi-a Gene From Chromobactenrium Sp. Strain C-61 Expressed in Escherichia coli XL-1 Blue

摘要

A gene, Chi 54 encoding a chitinase was derived from the chromosomal DNA of soil borne bacterium, Chromobacterium sp. strain C-61. The gene Chi 54 was composed of 1,611 nucleotides and encoded a protein that included a type 3 chitin binding domain ChBD, and a CD (catalytic domain). From the chitinase gene Chi-54, some already formed recombinant strains, formed by either addition or deletion of domains were grown in LB medium and studied for enzyme assays. The Chi-a, an original clone of chitinase expressed in the strain of Escherichia coli (XL-1 Blue) grown on LB/Kanamycin plates, having ChBD and CD domains. The cell extract of 13-1- a point mutant, Chi-a, the original clone, and the new transformant Chi a-2 showed good chitinase activity. The strain Chi-a, had been selected for mutagenetic studies. The error-prone PCR was succeeded when MnCl_2 concentrations were 0.025, 0.050, and 0.075 mM/1. The standard and error-prone PCR transformants were tooth picked on 1/2 strength LB medium, supplemented with colloidal chitin medium plates showed clearing zones around the growing bacteria. The enzyme activities of both the PCR transformants were compared and the maximum chitinase activity was obtained in the mutated Chi-a clone. The mutated Chi-a clone was sequenced and the mutated sequences were recorded.