柿单宁酶PsnTanA异源表达及酶学特性表征  

Exogenous expression and characterization of a novel tannase PsnTanA from persimmon

作　　者：卢海强[1] 陈伟 田洪涛[1] 谷新晰[1] 谷子林[2] LU Haiqiang;CHEN Wei;TIAN Hongtao;GU Xinxi;GU Zilin(College of Food Science and Technology,Hebei Agricultural University,Baoding 071000,China;College of Animal Science and Technology,Hebei Agricultural University,Baoding 071000,China)

机构地区：[1]河北农业大学食品科技学院,河北保定071000 [2]河北农业大学动物科技学院,河北保定071000

出　　处：《河北农业大学学报》2022年第3期69-74,共6页Journal of Hebei Agricultural University

基　　金：国家自然科学基金项目(31901631);山东省泰山产业领军人才(LJNY202023);国家兔产业技术体系资助项目(CARS-44-B-3)。

摘　　要：单宁酶在食品、饲料和制药等领域具有巨大应用潜力。本研究通过对柿树单宁酶基因PsnTanA进行密码子优化,使得该基因的GC含量由60.2%调整到57.1%,CAI值由0.62调整到0.76,优化前后基因序列的一致性为78.3%。单宁酶PsnTanA经大肠杆菌BL21表达后,重组酶PsnTanA在40℃和pH 7.0下表现出最大活性,比酶活力为1.53 U/mg,表观分子量约为39 kD。多种金属离子抑制了重组酶PsnTanA的活力,并随着金属离子浓度的增大而增强,其中Cu;和Fe;表现出的抑制效应最为明显,部分有机试剂也表现出类似的现象。以没食子酸正丙酯为底物时,单宁酶PsnTanA的Km值和Vmax值分别为1.43 mmol/L和3.28 mmol/(L·min)。除此之外,研究发现单宁酶PsnTanA能够有效降解高粱、茶和绿豆单宁等底物。本研究不仅丰富了现有单宁酶资源,也为进一步为单宁酶在食品和饲料中的应用提供了必要的理论支撑。Tananse are extensively applied in food processing,feed and pharmaceutical industries and many other fields.In this study,the coding region of persimmon PsnTanA gene was optimized according to the codon usage in E.coli resulting in decrease of GC content from 60.2% to 57.1% and increase of the CAI from 0.62 to 0.76.The resulting sequence shared 78.3% sequence similarity with the origin sequence.Then the optimized sequence was expressed in E.coli BL21 and the novel recombinant tannase PsnTanA showed maximal catalytic activity with a high specific activity of 1.53 U/mg at pH 7.0 and under 40℃.The purified PsnTanA had a molecular mass of 39 kD.The activity of tannase was drastically inhibited by several metal ions among which Cu;and Fe;displayed pronounced inhibition.Likewise,a similar inhibition was identified with organic chemicals.The Km and Vmax values of the tannase were 1.43 mmol/Land 3.28 mmol/(L·min) respectively with the substrate n-propyl gallate.The tananse,it was able to degrade a broad range of substrates from sorghum,tea and mung bean.The work reported here has not only enriched the available resources of enzyme,but has also provided theoretical support for the application of tannase in the food and feed industry.

关 键 词：柿 单宁酶 异源表达 酶学特性 底物谱 

分 类 号：Q814.4[生物学—生物工程]