摘要
为降低白酒中杂醇油含量,构建高活性杂醇油酯化酶,酯化杂醇油为较高沸点的酯从而降低酒体中杂醇油含量。该研究利用逆转录-聚合酶链式反应(RT-PCR)扩增酯化酶基因,酶切、以其为模板,进行易错聚合酶链式反应,连接、转化大肠杆菌(Escherichia coli)DH5α,筛选酶活显著提升的酯化酶。结果表明,酯化酶氨基酸中146位缬氨酸突变为天冬氨酸,198位苏氨酸突变为异亮氨酸,376位天冬氨酸突变为谷氨酸。初始酯化酶和构建酯化酶合成乙酸正丙酯、乙酸异丁酯、乙酸异戊酯酶活分别为10.83 U/L、24.72 U/L,2.58 U/L、6.24 U/L,3.83 U/L、8.78 U/L,构建酯化酶酶活分别是初始酯化酶酶活的2.28倍、2.42倍、2.29倍。构建杂醇油酯化酶在白酒生产中具有极大的应用潜力。
In order to decrease fusel oil of Baijiu, the esterifying enzyme was reconstructed to efficiently transform fusel oil into the esters with higher boiling points. The gene of the esterifying enzyme was amplified by reverse translation-polymerase chain reaction (RT-PCR). The mutated sequences were cut and linked with the plasmid cut by restricted enzymes and it was used as template and error-prone PCR was performed. The constructed vector was transformed into Escherichia coli DH5α, and the esterifying enzyme with significantly enhanced enzyme activity was selected. The results showed that the 146 site valine, 198 site theronine and 376 site aspartic acid amino acids of esterifying enzyme were mutated into aspartic acid, isoleucine and glutamic acid. The enzyme activity of n-propyl acetate, isobutyl acetate, isoamyl acetate of the original esterifying enzyme and the reconstructed esterifying enzyme was 10.83 U/L and 24.72 U/L, 2.58 U/L and 6.24 U/L, 3.83 U/L and 8.78 U/L, respectively. The esterifying enzyme activity of the reconstructed esterifying enzyme was 2.28, 2.42, 2.29 times that of the original esterifying enzyme. The esterase designed by molecular reconstruction was promising for application in the modern production of Baijiu brewing.
作者
张瑞瑞
ZHANG Ruirui(Intangible Cultural Heritage Research Centre of Hubei Province,School of Marxism,Hubei University of Technology,Wuhan 430068,China)
出处
《中国酿造》
CAS
北大核心
2019年第5期86-89,共4页
China Brewing
基金
湖北省非物质文化遗产研究中心2018年开放基金项目(HGFY20180101,HGFY20180302)
湖北省教育厅2018人文社科青年项目(18Q055)
关键词
白酒
杂醇油
酯化酶
分子构建
Baijiu
fusel oil
esterase
molecular reconstruction
