酿酒酵母表面展示β-淀粉酶及其酶学性质研究

Study on cell surface display of β-amylase on Saccharomces cerevisiae and its practical properties

通过PCR技术扩增来源于大麦的β-淀粉酶基因,将其与酿酒酵母细胞壁蛋白α凝集素基因在读框内融合,构建得到表面展示载体pBA-AG,进一步将该重组质粒通过遗传转化,整合到酿酒酵母W303-1A的染色体中,获得了β-淀粉酶经过α凝集素锚定信号结合到细胞壁上的重组酵母。重组酵母表面展示的β-淀粉酶活力为131U/g干细胞。对展示的β-淀粉酶酶学性质研究表明,其最适反应温度为50℃,最适作用pH为5.0,与游离酶相比,其温度稳定性和pH稳定性均得到提高。本研究利用α凝集素系统首次将β-淀粉酶成功展示在酿酒酵母表面,为以酿酒酵母为基础的全细胞催化剂研究与应用打下了一定基础。

The gene encoding mature β-amylase from barley was cloned via PCR and then fused with the α-agglutinin of Saccharomyces cerevisiae in frame and a recombinant plasmid named pBA-AG was constructed. Recombinant plasmid pBA-AG was successfully transformed into S. cerevisiae W303-1A and was integrated into the chromosome. The expressed β-amylase was successfully anchored on the cell wall and displayed on the surface of recombinant S. cerevisiae. The measured activity of displayed β-amylase was 131U/g dry cells. The optimal temperature and pH for displayed β-amylase was 50℃ and 5.0,respectively. The recombinant β-amylase displayed on cell surface exhibited enhanced thermostability compared to free enzyme. In this study,the firstly constructed recombinant S. cerevisiae strain displaying β-amylase on the cell surface with a-agglutinin as carrier protein showed a great potential for industrial application as a whole-cell biocatalyst.