Molecular Cloning and Gene Expression of the Chitinase from Bacillus cereus BCRC 11026
Date Issued
2009
Date
2009
Author(s)
Lin, Zih-Shiou
Abstract
N-acetyl-D-glucosamine (GlcNAc) and N-acetyl chitooligosaccharides, the products of chitin, are important to several industries and biotechnology sectors. They could be hydrolyzed from chitin by chitinase. The aim of this study was to purify and clone the gene encoding chitinase from Bacillus cereus BCRC 11026 and to perform recombinant expression in a prokaryotic E. coli system. Chitinase was purified from B. cereus BCRC 11026 through ammonium sulfate fractionation and Mono S ion-exchange chromatography. Its molecular mass was about 61 kDa, as determined by SDS-PAGE. The N-terminal amino acid sequence of the purified chitinase was further determined. Cloning was carried out using PCR technique, using the homogeneous gene sequences of the purified chitinase as the primers. The chitinase fragment (GD1A4) amplified from the genomic DNA of B. cereus BCRC 11026 was ligated with pET-32a (+) vector to generate an expression vector, pET32a-GD1A4. In order to achieve successful expression of the recombinant chitinase, the plasmid pET32a-GD1A4 was then transferred into E. coli BL-21 to produce the recombinant protein with a molecular weight of 38.5 kDa. Furthermore, the recombinant chitinase were purified and concentrated by 48.78 fold with a recovery of 1.67 % and a specific activity of 12.536 U/mg. The optimal pH and temperature for the recombinant chitinase wase 6.0 and 60 ℃, respectively, and the enzyme activity was relatively stable below 60 ℃. This chitinase could be utilized for the production of N-acetyl-D-glucosamine and N-acetyl chitooligosaccharides.
Subjects
chitin
chititnase
Type
thesis
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