耐酸Bacillus velezensis P7木聚糖酶基因在枯草芽孢杆菌中的表达及优化  

Expression of the Acid-resistant Xylanase Gene from Bacillus velezensis P7 in Bacillus subtilis and Optimization of Its Production

作　　者：黄雪芹 左勇 张强 徐佳 杨建飞 易媛 HUANG Xueqin;ZUO Yong;ZHANG Qiang;XU Jia;YANG Jianfei;YI Yuan(Sichuan University of Science&Engineering,School of Biological Engineering,Yibin 644000,China;Sichuan Normal University College of Life Science,Chengdu 610101,China)

机构地区：[1]四川轻化工大学生物工程学院,四川宜宾644000 [2]四川师范大学生命科学学院,四川成都610101

出　　处：《现代食品科技》2021年第12期40-48,32,共10页Modern Food Science and Technology

基　　金：四川省科技厅重点研发项目(2018NZ0050);固态发酵资源利用四川省重点实验室应用研究项目(2018GTY004);宜宾市科技计划项目(2018NY009)。

摘　　要：为获得适应于酒糟酸性环境的木聚糖酶,将内源微生物耐酸性环境的野生Bacillus velezensis P7木聚糖酶基因克隆到枯草芽孢杆菌WB800中,纯化后通过SDS-PAGE电泳显示出约40 ku的蛋白多肽。为增加枯草芽孢WB800-P7工程菌株在高密度发酵中细胞外分泌木聚糖酶的产量,进行诱导和培养条件优化,用正交试验分析得出温度对产酶影响最大,确定菌株分泌重组蛋白的条件结果表明:最佳诱导条件为OD6000.8、IPTG 1.2 mmol/L,最佳发酵条件为pH 6.0、培养温度35℃、接种量2%、摇床转速160 r/min。在该条件下发酵16 h,经重复验证,酶活力达到4.21 IU,与优化前相比,酶活力提高了64.45%。并探究重组酶的耐受性,结果表明在强酸性到中性条件下(pH 5.0~7.0),其残余酶活力达到初始酶活力的80%以上。试验成功构建了木聚糖酶工程菌并较好表达蛋白,且重组木聚糖酶在酸性条件的耐受性良好。To obtain a xylanase protein that can adapt to acidic environments(e.g.,in distiller’s grains),the endogenous xylanase gene from acid-resistant Bacillus velezensis P7 was successfully cloned using the polymerase chain reaction.The xylanase gene fragment and the pHT43-HIS shuttle plasmid were first digested with the restriction enzymes Bam HI and Xba I.After purification of the xylanase gene fragments with a gel recovery kit,they were then ligated with the vector fragments using T4 DNA ligase,and the recombinant plasmid was subsequently transfected into Bacillus subtilis WB800 cells.SDS-PAGE analysis of the purified xylanase revealed that it was a 40 ku polypeptide.To increase the extracellular production of xylanase by Bacillus subtilis WB800-P7 in high-density fermentation,the effects of temperature,pH,inoculum size,rotation speed,and other individual factors on the production process were studied,and orthogonal experiments were then used to determine the interactions among the factors and to predict the optimal fermentation conditions for promoting the secretion of the recombinant protein.The results suggested the optimal fermentation conditions of strain WB800-P7 to be as follows:inoculum OD600 of 0.8,IPTG concentration of 1.2 mmol/L,fermentation pH of 6.0,culture temperature of 35℃,inoculum size of 2%,and rotation speed of 160 r/min.Among these factors,the culture temperature had the most significant effect on enzyme production.Under these optimal conditions,the enzyme activity reached 4.21 IU after 16 h of fermentation,which was 64.45%higher than that before optimization.The acid resistance of the recombinant xylanase was analyzed,whereupon its residual enzymatic activity was found to be more than 80%of that of the original enzyme under strongly acidic and neutral conditions(pH 5.0~7.0).In conclusion,a modified B.subtilis strain with better protein expression capability and which produces a highly acid-resistant xylanase was successfully engineered in this study.

关 键 词：克隆 枯草芽孢杆菌 产酶优化 木聚糖酶 

分 类 号：TQ925[轻工技术与工程—发酵工程]