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乙醛脱氢酶的克隆表达及其酶学性质 被引量:3

Expression and enzymatic properties of acetaldehyde dehydrogenase
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摘要 将Gene Bank中人源乙醛脱氢酶2的基因序列根据酿酒酵母(Saccharomyces cerevisiae)的密码子偏好性进行密码子优化后合成目的基因ALDH2,采用融合PCR技术,构建人源ALDH2基因的表达组件TRP1L-URA3-TPIp-ALDH2-TPIt-TRP1R。通过电转化的方法将基因表达组件通过同源重组整合到酿酒酵母W303-1A的基因组上,获得基因工程菌W303-ALDH2。工程菌发酵后的粗酶液经His TrapTMexcel亲和层析柱纯化获得重组ALDH2,其活性为20.70 U/mg;重组酶的相对分子质量是56 k Da;最适反应pH和温度分别为5.0和35℃;除Na+外,K+、Ca2+、Mg2+、Mn2+均能不同程度地提高ALDH2的酶活。以乙醛为底物测得酶的Km值为0.908 mmol/L,最大反应速度Vmax为114.94 U/mg;以辅酶NAD+为底物测得酶的Km值为0.282 mmol/L,最大反应速度Vmax为58.82 U/mg。 The target gene ALDH2 was synthesized according to the sequences of human acetaldehyde dehydrogenase 2 in Gene Bank after codon optimization of Saccharomyces cerevisiae. Then the ALDH2 gene expression cassette TRP1 L-URA3-TPIp-ALTH2-TPIt-TRP1 R was constructed using fusion PCR and electro-transformed into S. cerevisiae W303-1 A to produce the modified strain W303-ALDH2. The recombinant ALDH2 was obtained after purification by His TrapTMexcel affinity chromatography,and its molecular weight and activity was 56 k Da and 20. 70 U/mg,respectively. The results showed that the optimal reaction pH value and temperature for recombinant ALDH2 were 5. 0 and35 ℃,respectively. Except Na+,the K+,Ca2 +,Mg2 +,Mn2 +could increase the activity of ALDH2 in different degrees. When acetaldehyde was used as substrate,the Kmvalue and maximum reaction rate Vmaxof recombinant ALDH2 was 0. 908 mmol/L and 114. 94 U/mg,respectively. When NAD+was used as substrate,the Kmvalue and maximum reaction rate Vmaxof recombinant ALDH2 was 0. 282 mmol/L and 58. 82 U/mg,respectively.
作者 豆欣喜 张明 林材 吴殿辉 李晓敏 孙军勇 蔡国林 陆健 DOU Xin-xi;ZHANG Ming;LIN Cai;WU Dian-hui;LI Xiao-min;SUN Jun-yong;CAI Guo-lin;LU jian(The Key Laboratory of Industrial Biotechnology, Jiangnan University, Wuxi 214122, China;National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China;School of Bioteehnology, Jiangnan Univelsity, Wuxi 214122, China;Jiangsu Agribusiness Malt Co. Ltd, Sheyang 224300, China;China Resources Snow Breweries Co. Ltd, Qinhuangdao 066001 , China)
出处 《食品与发酵工业》 CAS CSCD 北大核心 2018年第4期22-28,共7页 Food and Fermentation Industries
基金 啤酒用新酶创制与低碳制造关键技术研究,国家高技术发展(863)计划(2013AA102109) 基于酿酒酵母代谢工程改造降低黄酒中氨基甲酸乙酯含量的研究(31701588) 高等学校学科创新引智计划(111计划)资助项目(111-2-06) 江苏高校优势学科建设工程资助项目 江苏省现代工业发酵协同创新中心资助
关键词 人源乙醛脱氢酶2 酿酒酵母 克隆表达 酶学性质 human acetaldehyde dehydrogenase 2 Saccharomyces cerevisiae cloning and expression enzymatic properties
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