纤维素酶CxGH7A在枯草芽孢杆菌中的分泌表达与酶学性质  

Secretory expression and enzymatic properties of cellulase CxGH7A in Bacillus subtilis

作　　者：李蓉 刘珊君 顾劲博 唐文竹[1] LI Rong;LIU Shanjun;GU Jinbo;TANG Wenzhu(School of Biological Engineering,Dalian Polytechnic University,Dalian 116034,China)

机构地区：[1]大连工业大学生物工程学院,辽宁大连116034

出　　处：《大连工业大学学报》2024年第6期391-396,共6页Journal of Dalian Polytechnic University

基　　金：国家自然科学基金项目(31600640);辽宁省教育厅基本科研业务费项目(LJKMZ20220886).

摘　　要：实验室早期从一株纤维素降解菌Arthrobotryssp.CX1中获得了纤维素酶CxGH7A基因并构建毕赤酵母重组菌株pPICZαA-CxGH7A/X-33。但酵母发酵表达外源蛋白周期较长,经济成本高。为实现CxGH7A的高效外源表达,本研究构建了重组质粒pHT43-CxGH7A,并转入枯草芽孢杆菌WB600中,获得枯草芽孢杆菌重组菌pHT43-CxGH7A/WB600,对其酶学性质进行表征并对发酵条件进行优化。结果表明,重组菌pHT43-CxGH7A/WB600表达后胞外分泌液在55ku处有差异蛋白条带,酶活力达到47.25U/mL。优化后,重组菌在TB培养基、pH7.0、诱导温度30℃、诱导剂浓度0.5mmol/L、诱导时间36h条件下,上清液中的酶活力达到123.73U/mL,且枯草芽孢杆菌表达系统生产CxGH7A的效益达到毕赤酵母表达系统的4.6倍。CxGH7A gene was obtained from a cellulose degrading bacterium Arthrobotrys sp.CX1,and a recombinant strain pPICZαA-CxGH 7 A/X-33 of Pichia pastoris has been constructed early before.However,yeast fermentation for expressing exogenous proteins has a longer cycle and higher economic costs.To achieve efficient exogenous expression of CxGH7A,a recombinant plasmid pHT43-CxGH7A was constructed and transferred into Bacillus subtilis WB600 to obtain the recombinant strain pHT43-CxGH7A/WB600.Its enzymatic properties were characterized and fermentation conditions were optimized.The results showed that there was a protein band of 55 ku in the extracellular secretion of the recombinant strain pHT43-CxGH7A/WB600 after expression,and the enzyme activity was 47.25 U/mL.The enzyme activity in the supernatant could reach to 123.73 U/mL in the conditions of TB medium,pH 7.0,induction temperature of 30℃,inducer concentration of 0.5 mmol/L and induction time of 36 h after optimization.The efficiency of CxGH7A produced by Bacillus subtilis expression system was 4.6 times higher than that of the Pichia pastoris expression system.

关 键 词：纤维素酶 枯草芽孢杆菌 毕赤酵母 酶学性质 

分 类 号：Q815[生物学—生物工程]