瘤胃真菌Piromyces rhizinflatus之外切聚葡萄糖酶基因選殖及其外源性表現

Abstract

中文摘要 纖維素為地球上最豐富的多醣且具有高度潛力成為再生能源，然而在自然界中的纖維素大多為結晶型。因此，具有對結晶型纖維素較高水解能力的酵素成為許多研究者追尋的目標。 由台灣水牛瘤胃真菌Piromyces rhizinflatus篩選出的β-聚葡萄糖酶基因cbhYW23-2，其序列長度為1557個含氮鹼基，表現出的酵素由519個胺基酸組成，分子量約為57 kDa。其胺基酸序列的C端具有屬於醣基水解酶第6族的酵素區，N端為屬於碳水化合物結合單元第1族的結合區，兩區由富含天門冬醯胺酸的胜肽連接。為測定酵素活性，將帶有His6的CbhYW23-2重組蛋白由Escherichia coli表現並以親和性管柱純化，再以反應曲面法結合中央合成設計及迴歸分析酵素之最適溫度及pH值，結果分別為46.4°C及pH 6.0，於此反應條件下，酵素比活性為2411.1±197.9 U/mg。CbhYW23-2也展現了對於Avicel、羧甲基纖維素 (carboxylmethyl cellulose, CMC)、lichenan、以及pachyman的活性。故證明CbhYW23-2對於結晶型纖維素具有極高的活性，因此具有應用於工業上的潛力。接著將cbhYW23-2基因構築於Saccharomyces cerevisiae表現質體上，並使CbhYW23-2接合於a凝集素，使CbhYW23-2能夠表現於S. cerevisiae的細胞壁上。經酵素活性測定、酵素擴散法、西方轉漬及間接免疫螢光染色分析，證實CbhYW23-2可成功表現並錨定在S. cerevisiae細胞表面。 綜上所述，本研究成功利用反應曲面法得到此β-聚葡萄糖酶之最適反應條件，並利用S. cerevisiae細胞表面蛋白a凝集素，將CbhYW23-2固定於S. cerevisiae表面並仍具有對β-聚葡萄糖之活性。

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.