| Summary: | 碩士 === 國立交通大學 === 理學院碩士在職專班應用科技學程 === 96 === CBP21 is a chitin-binding protein from Serratia marcescens, a Gram-negative soil bacterium. When S. marcescens is grown in the presence of chitin as a carbon source, CBP21 (197 amino acids, including a 27-residue leader peptide) is one of the major proteins produced and secreted along with other chitinases. In recent years, researchers have found that the non-catalytic CBP21 can bind to the insoluble crystalline substrate and lead to structural change of substrate. Consequently, CBP21 can increase the substrate accessibility and strongly promote the hydrolysis of crystalline β-chitin by other chitinases. Previous study also showed that CBP21 can bind on chitin at a pH value around 8.0 and can be eluted in acid condition (pH < 7). This feature is potentially useful for establishing a system for protein purification and for serving as a tool in enzyme immobilization. Through the application of PCR amplification, a DNA fragment containing the CBP21 gene, a downstream peptide-linker and a genease cut site was obtained. The fragment was successfully inserted into the pRSET_A expression vector. Several genes of glycoside hydrolases, including chitosanase, chitinase, and laminaripentaose-producing β-1,3-glucanase (LPHase), were inserted into the multiple cloning site of the constructed plasmid and further transformed into the E. coli BL21 (DE3) for fusion-protein expression. The capability of this system was then evaluated using β-form chitin as matrix for affinity-column purification. Results showed that proteins with high purity (> 90% homogeneity for all cases) were easily obtained. The recovery yield was more than 40 percent.
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