壳聚糖酶基因在解脂耶氏酵母中的表达及重组酶性质研究

Expression of Chitosanase Gene in Yarrowia lipolytica and Characterization of the Recombinant Enzyme

为构建高效表达壳聚糖酶工程菌株,以Microbacteriumsp.基因组为模板扩增壳聚糖酶基因,目的基因与表达载体pINA1317连接构建重组质粒,重组质粒NotⅠ酶切线性化后转化解脂耶氏酵母Y.liPolytica Po1h。所得结果为PCR扩增得到约1000bp的特异性片段,序列分析表明含有壳聚糖酶全长基因,开放阅读框801bp,编码266个氨基酸残基。转化子酶活力为10.4U/mL,是原菌株酶活力的3.15倍。重组蛋白经DEAE-Sepharose FF分离纯化,达到电泳纯,SDS-PAGE显示单一条带,分子量约41kDa。重组酶反应最适温度为45℃,最适pH为5.6,Km和Vmax分别是0.926mg/mL和6.15U/mL。质谱分析酶解产物主要为三糖和五糖。实现了壳聚糖酶在解脂耶氏酵母中的分泌表达,为壳聚糖酶的工业化生产奠定了理论基础。

This study was carried out to overexpress of a chitosanase in Yarrowia lipolytica Po1h.The chitosanase gene was obtained using the genomic DNA of Microbacterium sp.as a template.The gene was ligated into the vector pINA1317 to get the recombinant plasmid.The expression plasmid was transformed to Y.lipolytica Po1h after linearized with Not I.A 1000-length fragment was obtained,which consisted of 801bp encoding a polypeptide of 266 amino acids.The activity of transformnant reached 10.4 U/mL.The recombinant chitosanase was purified by DEAE-Sepharose FF and the molecular weight was estimated to be 41 kDa by SDS-PAGE.The optimum pH and temperature were pH 5.6 and 45 ℃,respectively.The Km and Vmax values with soluble chitosan as substrate were 0.926 mg/mL and 6.15 U/mL,respectively.Chitotriose and chitopentaose were the major enzymic products.In conclusion,the chitosanase was successfully expressed and secreted in Y.lipolytica,making an important step towards the production of chitosanase on industrial scale.