低温木糖苷/阿拉伯呋喃糖苷酶AX543的基因克隆及性质研究

doi:10.13560/j.cnki.biotech.bull.1985.2016.11.025

生物技术通报 ›› 2016, Vol. 32 ›› Issue (11): 215-223.doi: 10.13560/j.cnki.biotech.bull.1985.2016.11.025

低温木糖苷/阿拉伯呋喃糖苷酶AX543的基因克隆及性质研究

张明慧, 李中媛, 仇海燕, 王翠琼, 王惠, 张同存

天津科技大学生物工程学院工业微生物重点实验室,天津 300457

收稿日期:2016-03-08 出版日期:2016-11-25 发布日期:2016-11-11
作者简介:张明慧,女,硕士,研究方向：微生物与生化药学,E-mail：zmh415751532@126.com;李中媛同为本文第一作者
基金资助:
天津市高等学校科技发展基金计划项目（20140604）,天津科技大学青年教师创新基金项目（2014CXLG06）

Cloning and Characterization of a Novel Cold-active Bifunctional Xylosidase/Arabinfuranosidease AX543

ZHANG Ming-hui, LI Zhong-yuan, QIU Hai-yan, WANG Cui-qiong, WANG Hui, ZHANG Tong-cun

Key Laboratory of Industrial Microbiology,Ministry of Education and Tianjin City,College of Biotechnology,Tianjin University of Science and Technology,Tianjin 300457

Received:2016-03-08 Published:2016-11-25 Online:2016-11-11

摘要/Abstract

摘要： 旨在获得在低温条件下具有高催化能力的低温木糖苷酶,并对其进行异源表达研究和酶学性质分析。利用Touch down PCR和TAIL PCR方法,从枝顶孢菌中克隆得到一个序列新颖的GH43家族双功能木糖苷酶/阿拉伯呋喃糖苷酶基因ax543,该酶基因在毕赤酵母中成功表达。酶学性质分析发现重组酶AX543的最适温度为25℃,在15℃和4℃仍有54%和21%的相对酶活;具有木糖苷酶和阿拉伯呋喃糖苷酶活性,并可以降解木二糖、木三糖、桦木木聚糖、榉木木聚糖和小麦阿拉伯木聚糖;可以与木聚糖酶Xyn11-1协同作用,协同度达1.46;具有高木糖/阿拉伯糖耐受性,抑制常数K_i分别为84.78 mmol/L和54.01 mmol/L。

关键词: 木糖苷酶, 阿拉伯呋喃糖苷酶, 双功能, 低温, 协同作用, 产物耐受性

Abstract: This work aims to obtain the cold-active xylosidase with high catalytic activity at low temperature,and to study its heterologous expression,as well as analyze its enzymatic characteristics. A novel psychrophilic bifunctional xylosidase/arabinofuranosidase gene ax543 in GH43 family was cloned using Touch down PCR and TAIL PCR,and was successfully expressed in Pichia pastoris. Analysis of biochemical characterization suggested that recombinant enzyme AX543 exhibited optimal activity at 25℃,and remained 54% and 21% relative activities at 15℃ and 4℃. Moreover,AX543 with xylosidase and arabinofuranosidase activity hydrolyzed xylobiose,xylotriose,birchwood xylan,beechwood xylan,and wheat arabinoxylan,and showed a 1.46 fold synergic effect with xylanase Xyn11-1. AX543 also presented xylose and arabinose tolerance with K_i 84.78 mmol/L and 54.01 mmol/L.

Key words: xylosidase, arabinofuranosidase, bifunctional, cold-active, synergic effect, product tolerance

张明慧, 李中媛, 仇海燕, 王翠琼, 王惠, 张同存. 低温木糖苷/阿拉伯呋喃糖苷酶AX543的基因克隆及性质研究[J]. 生物技术通报, 2016, 32(11): 215-223.

ZHANG Ming-hui, LI Zhong-yuan, QIU Hai-yan, WANG Cui-qiong, WANG Hui, ZHANG Tong-cun. Cloning and Characterization of a Novel Cold-active Bifunctional Xylosidase/Arabinfuranosidease AX543[J]. Biotechnology Bulletin, 2016, 32(11): 215-223.

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低温木糖苷/阿拉伯呋喃糖苷酶AX543的基因克隆及性质研究

Cloning and Characterization of a Novel Cold-active Bifunctional Xylosidase/Arabinfuranosidease AX543

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