| 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 |
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| 引用本文： | 刘万顺,王娇,杨艳,韩宝芹,董文,刘光磊,池振明.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[J].中国海洋大学学报(自然科学版),2011,41(12):58-62. |
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| 作者姓名： | 刘万顺 王娇 杨艳 韩宝芹 董文 刘光磊 池振明 |
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| 作者单位： | 中国海洋大学海洋生命学院,山东青岛,266003 |
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| 摘 要： | 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。
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| 关 键 词： | 壳聚糖酶 解脂耶氏酵母 表达 酶学性质 |
Expression of Chitosanase Gene in Yarrowia lipolytica and Characterization of the Recombinant Enzyme |
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| LIU Wan-Shun,WANG Jiao,YANG Yan,HAN Bao-Qin,DONG Wen,LIU Guang-Lei,CHI Zhen-Ming.Expression of Chitosanase Gene in Yarrowia lipolytica and Characterization of the Recombinant Enzyme[J].Periodical of Ocean University of China,2011,41(12):58-62. |
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| Authors: | LIU Wan-Shun WANG Jiao YANG Yan HAN Bao-Qin DONG Wen LIU Guang-Lei CHI Zhen-Ming |
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| Institution: | LIU Wan-Shun,WANG Jiao,YANG Yan,HAN Bao-Qin,DONG Wen,LIU Guang-Lei,CHI Zhen-Ming(College of Marine Life Science,Ocean University of China,Qingdao 266003,China) |
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| Abstract: | 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 ch... |
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| Keywords: | chitosanase Yarrowia lipolytica Po1h expression characterization |
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