黑曲霉果糖基水解酶的重组表达及酶学特征分析

董自星; 肖华; 王静培; 张志萌; 王君; 路福平

doi:10.13386/j.issn1002-0306.2017.10.026

黑曲霉果糖基水解酶的重组表达及酶学特征分析

1. 天津科技大学化工与材料学院生物化工系
2. 天津科技大学生物工程学院与工业发酵微生物教育部重点实验室

详细信息

作者简介:
董自星 (1986-) , 男, 博士, 助理研究员, 研究方向:酶工程与技术, E-mail:dzx@tust.edu.cn。;

路福平 (1967-) , 男, 博士, 教授, 研究方向:应用微生物与酶工程, E-mail:lfp@tust.edu.cn。;

中图分类号: TS201.25
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出版历程
- 收稿日期: 2016-10-10

Recombinant expression and biochemical characterization of fructosyl hydrolases from Aspergillus niger

Department of Biochemical Engineering, College of Chemical Engineering and Materials Science, Tianjin University of Science & Technology
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science & Technology

摘要

摘要: 目的:果糖基转移酶在新型果糖基衍生品的制备过程中具有重要作用,获得酶活高、性能优良的果糖基水解酶是关键。方法:利用MEGA 4.0以及Clustal X2等软件对黑曲霉的基因组进行分析,遴选了5个果糖基水解酶基因,接着将它们在毕赤酵母中进行了克隆表达,并研究了重组酶的酶学性质。结果:在摇瓶水平上,重组酶Fru1和Fru5的酶活分别为1360和1560 U/m L,远高于目前已报道的果糖基水解酶的酶活。重组酶Fru1的最适反应温度和p H分别为45℃和5.5,EDTA对它有微弱的促进作用,Fe²⁺、Na+、Co²⁺、Cu²⁺和Ca²⁺会轻微地抑制酶活,该酶对蔗糖既有水解作用,又有转苷活性,还可以水解菊粉中的二糖到五糖;重组酶Fru5的最适反应温度和p H分别为50℃和5.0,Li⁺、Na⁺和EDTA对其酶活有促进作用,但Fe²⁺则强烈抑制酶的活性,它还可以水解蔗糖以及菊粉中几乎所有的大分子糖。结论:本研究为果糖基水解酶的工业化生产及其在新型果糖基衍生品制备过程中的应用提供了可能途径。
- 果糖基水解酶 /
- 新型果糖基衍生品 /
- 黑曲霉 /
- 重组表达 /
- 酶学性质
Abstract: Objective: Fructosyl hydrolase plays a significant role in the preparation of novel fructose derivatives.It is important to obtain an efficient fructosyl hydrolase with high activity.Methods: Five fructosyl hydrolase-encoding genes in the Aspergillus niger genome were selected and analyzed by MEGA 4.0, Clustal X2 and other softwares. Subsequently, these genes were successfully cloned and expressed in Pichia pastoris followed by comprehensive investigation of their biochemical properties.Results: At the shake-flask fermentation level, the activities of recombinant Fru1 and Fru5 were determined to be 1360 and1560 U/m L, respectively, which were much higher than those of the fructosyl hydrolases ever reported.The optimum temperature and p H of recombinant enzyme Fru1 were 45 ℃ and 5.5, respectively.Its enzymatic activity was slightly enhanced by EDTA and inhibited by Na+, Co²⁺, Cu²⁺and Ca²⁺. Interestingly, this enzyme showed both hydrolytic and transglycosylation activities toward sucrose, and hydrolyzed short-chain inulin with degree of polymerization of 2 ~ 5. The temperature and p H optima of recombinant Fru5 were 50 ℃ and 5.0, respectively.It was remarkably enhanced by Li⁺, Na⁺and EDTA, and strongly inhibited by Fe²⁺.Fru5 could also hydrolyze sucrose and almost all polysaccharides in inulin.Conclusions: Our results have paved the way for the industrial manufacturing of fructosyl hydrolases and their applications in the production of novel fructose derivatives.
- fructosyl hydrolases /
- novel fructose derivatives /
- Aspergillus niger /
- recombinant expression /
- enzymatic properties

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