黄酒酵母β-苯乙醇合成途径醇脱氢酶Ⅰ的异源表达及酶学特性  

Heterologous Expression and Enzymatic Analysis of Alcohol Dehydrogenase Ⅰ inβ-Phenylethanol Biosynthesis Pathway from Huangjiu Yeast

作　　者：杨琪琳 刘双平[1,2,3,4,5] 赵禹宗 孙海龙 毛健 Yang Qilin;Liu Shuangping;Zhao Yuzong;Sun Hailong;Mao Jian(School of Food Science and Technology,National Engineering Research Center of Cereal Fermentation and Food Biomanufacturing,Jiangnan University,Wuxi 214122,Jiangsu;Jiangnan University(Shaoxing)Industrial Technology Research Institute,Shaoxing 312000,Zhejiang;National Engineering Research Center of Huangjiu,Zhejiang Guyuelongshan Shaoxing Wine Co.,Ltd.,Shaoxing 312000,Zhejiang;Jiangsu Provincial Engineering Research Center for Bioactive Product Processing,Jiangnan University,Wuxi 214122,Jiangsu;Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province,Wuxi 214122,Jiangsu)

机构地区：[1]江南大学食品学院粮食发酵与食品生物制造国家工程研究中心,江苏无锡214122 [2]江南大学(绍兴)产业技术研究院,浙江绍兴312000 [3]国家黄酒工程技术研究中心,浙江古越龙山绍兴酒股份有限公司,浙江绍兴312000 [4]江南大学江苏省生物活性制品加工工程技术研究中心,江苏无锡214122 [5]江南大学江苏省食品安全与质量控制协同创新中心,江苏无锡214122

出　　处：《中国食品学报》2022年第6期83-94,共12页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家自然科学基金青年科学基金项目(32072205,31701593)。

摘　　要：醇脱氢酶Ⅰ(Adh1p)是黄酒酵母艾利希途径最后合成β-苯乙醇的一个关键酶。为探究黄酒酵母Adh1p的差异及酶学特性,以工业生产菌株黄酒酵母HJ和模式菌株酿酒酵母S288C的基因组为模板,设计引物PCR扩增醇脱氢酶编码区基因ADH1,构建pET-28a(+)表达质粒,转化至大肠杆菌BL21(DE3)中,IPTG诱导表达获得Adh1p。通过超声破碎菌体后获取粗酶液,亲和层析纯化后获取纯酶,进而探究酶学特性。以苯乙醛为底物,酶活性测定表明:来自黄酒酵母的Adh1p^(HJ)纯酶的酶活(231.51 U/g)比来自酿酒酵母的Adh1p^(S288C)(203.48 U/g)高13.79%,且Adh1p^(HJ)的K_(m)值(0.524μmol/L)小于Adh1p^(S288C)的K_(m)值(0.759μmol/L),表现为Adh1p^(HJ)与底物的亲和力更大。从k_(cat)/K_(m)值发现Adh1p^(HJ)(0.406 L/(μmol·min))的催化效率更高。Adh1p^(HJ)耐受β-苯乙醇和乙醇的能力较高于Adh1p^(S288C)。本研究结果为Adh1p的结构以及酶学性质分析提供方法和理论依据,同时也对β-苯乙醇工业生产开发提供借鉴。Alcohol dehydrogenase is a key enzyme in the final synthesis of β-phenylethanol in Huangjiu yeast,which comes from the Ehrlich pathway.To explore the differences and enzymatic analysis of Adh1p^(HJ),the ADH1 of Saccharomyces cerevisiae HJ and Saccharomyces cerevisiae S288C were used as templates respectively to amplify the target gene by PCR.The expression plasmid pET-28a(+)-ADH1 were constructed,and transformed into Escherichia coli BL21(DE3)cells to obtain high expression induced by IPTG.The crude enzymes were obtained after ultrasonic fragmentation,and then purified by affinity chromatography with AKTA pure.Finally the enzymatic analysis were explored.Using phenylacetaldehyde as substrate,it was found that the enzyme activity(231.51 U/g)of Adh1p^(HJ)was 13.79%higher than that of Adh1p^(S288C)(203.48 U/g),and the K_(m)value of Adh1p^(HJ)(0.524μmol/L)was less than that of Adh1p^(S288C)(0.759μmol/L),which indicated that the affinity between Adh1pHJ and substrate was higher.From the k_(cat)/K_(m)value,it is found that the catalytic efficiency of Adh1p^(HJ)[0.406 L/(μmol·min)]is higher.The tolerance of Adh1p^(HJ)toβ-phenylethanol and ethanol was higher than that of Adh1p^(S288C).This study provides a method and theoretical basis for the analysis of the structure and properties of Adh1p,and also provides a reference for the industrial production and development ofβ-phenylethanol.

关 键 词：醇脱氢酶Ⅰ 异源表达 酶学性质 黄酒 黄酒酵母 

分 类 号：TS262.4[轻工技术与工程—发酵工程]