Molecular cloning of a cellobiohydrolase from Piromyces rhizinflatus and heterologous expression

• Main Authors: Chih-Yuan Chu, 朱致遠
• Other Authors: 劉嚞睿
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/28405025856078805951

碩士 === 國立臺灣大學 === 動物科學技術學研究所 === 99 === Abstract Cellulose is the most abundant renewable polysaccharide with a high potential for degradation to useful end products. In nature, most cellulose is produced as crystalline cellulose. Therefore, cellulases with high hydrolytic activity against crystalline cellulose are of great interest. In this study, a crystalline cellulose degradation enzyme was investigated. The cDNA encoding a β-glucanase, CbhYW23-2, was cloned from the ruminal fungus Piromyces rhizinflatus. The cDNA sequence of cbhYW23-2 contained an open reading frame of 1,557 bp encoding a protein of 519 amino acids with a predicted molecular weight of 57 kDa. A putative conserved domain of glycosyl hydrolase (GH) family 6 was observed at the C-terminus and a putative conserved domain of cellulose binding domain (CBD) family 1 was observed at the N-terminus of CbhYW23-2. These two domains were separated by an Asn-rich linker. To examine the enzyme activities, CbhYW23-2 was expressed in E. coli as recombinant fusion protein and purified by immobilized metal ion-affinity chromatography. Response surface modeling (RSM) combined with central composite design (CCD) and regression analysis were then employed for the planned statistical optimization of the β-glucanase activities of CbhYW23-2. The optimal conditions for the highest β-glucanase activity of CbhYW23-2 were observed at 46.4°C and pH 6.0. At this condition, the specific activity of CbhYW23-2 was 2411.1±197.9 U/mg. CbhYW23-2 also showed hydrolytic activities toward Avicel, carboxymethyl cellulose (CMC), lichenan, and pachyman. The results also proved that the highly activity of CbhYW23-2 on crystalline cellulose makes it a promising candidate enzyme for biotechnological and industrial applications. Then cbhYW23-2 was constructed into the expression vector of S. cerevisiae. CbhYW23-2 was displayed on the cell surface of S. cerevisiae via a-agglutinin. The localization of a-agglutinin-CbhYW23-2 fusion protein on the cell surface was confirmed by analysis of β-glucanase activity assay, enzyme diffusion method, western blot, and immunofluorescence microscopy. In summary, the results suggested that RSM combined with CCD and regression analysis were effective in determining optimized temperature and pH conditions for the enzyme activity of CbhYW23-2. The a-agglutinin-CbhYW23-2 fusion protein was expressed on the cell surface of S. cerevisiae successfully and still remained the β-glucanase activity.