黑曲霉单宁酶TahA的克隆表达和酶学特性解析  

Cloning,expression and biochemical characterization of tannase TahA from Aspergillus niger

作　　者：李梦迪 张志萌[1] 董自星 田康明 金鹏 刘晓光 王正祥[1,2] LI Meng-di;ZHANG Zhi-meng;DONG Zi-xing;TIAN Kang-ming;JIN Peng;LIU Xiao-guang;WANG Zheng-xiang(College of Biotechnology,Tianjin University of Science&Technology,Tianjin 300457,China;Department of Biochemical Engineering,College of Chemical Engineering and Materials Science,TianjinUniversity of Science&Technology,Tianjin 300457,China)

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]天津科技大学化工与材料学院生物化工系,天津300457

出　　处：《食品与发酵工业》2018年第11期15-21,共7页Food and Fermentation Industries

基　　金：天津市高等学校创新团队培养计划"酶与生物催化关键技术"(TD12-5002)

摘　　要：单宁酶在茶饮料和医药等行业的应用受到了许多因素的限制,包括酶学性质研究的缺乏和较高的生产成本等。为此,该研究通过基因工程技术将黑曲霉CICIM F0510的单宁酶基因tah A在毕赤酵母中进行了克隆表达,构建获得了重组酶GS115 (pPIC-tahA)。摇瓶水平上,重组菌的单宁酶酶活为1. 04 U/mL。酶学性质的解析表明,重组酶TahA的最适作用温度和pH值分别为30℃和4. 5;分别在20～50℃和pH值3. 5～5. 0孵育1 h和2 h后,TahA的相对酶活仍能保持在60%以上; Cu^(2+)、Mn^(2+)和K^+对重组酶的活性有明显的促进作用,而Co^(2+)、Fe^(3+)、Ca^(2+)、Mg^(2+)、Zn^(2+)、Sn^(2+)、EDTA和SDS对其酶活有不同程度的抑制作用;以没食子酸甲酯为底物时,重组酶的Vmax和Km值分别为0. 436μmol/(mL·min)和5. 143 mmol/L。此外,该重组酶还可以水解茶叶提取物中的酯型儿茶素。这些良好的生化特征为重组单宁酶TahA在茶饮料行业中的应用奠定了坚实的基础。The practical use of tannase in tea beverage and pharmaceutical industries has been limited by a number of factors,including insufficient knowledge about its properties and high production costs.In the present study,gene tahA encoding tannase of Aspergillus niger CICIM F0510 was successfully cloned and expressed in Pichia pastoris,generating the recombinant strain GS115(pPIC-tahA).In shaking flask experiments,the activity of the recombinant enzyme was 1.04 U/mL.The temperature and pH optima of TahA was shown to be 30℃and 4.5,respectively.After incubation at 20-50℃or pH 3.5-5.0 for 1 h and 2 h,respectively,the recombinant enzyme still retained more than 60%of its initial activity.Cu^2+,Mn^2+and K+significantly enhanced the activity of TahA,whereas Co^2+,Fe 3+,Ca^2+,Mg^2+,Zn^2+,Sn^2+,EDTA and SDS exerted different inhibitory effects on its activity.The V max and K m of TahA toward methyl gallate were determined to be 0.436μmol/(mL·min)and 5.143 mmol/L.Besides,TahA hydrolyzed the ester catechins in tea extracts.All these distinct biochemical properties lay a solid foundation for the application of TahA in tea beverages.

关 键 词：黑曲霉 单宁酶 分子克隆 酶学特征 茶饮料 

分 类 号：TQ925[轻工技术与工程—发酵工程]