摘要
从中温α-淀粉酶生产菌株枯草芽孢杆菌(Bacillus subtilis)264中克隆得到中温α-淀粉酶基因(amy L)并构建了重组表达质粒p AX01-amy L。该质粒转化枯草芽孢杆菌264后利用同源重组机制将由木糖操纵子引导的amy L表达盒整合入枯草芽孢杆菌染色体的lac A位点,以增加中温α-淀粉酶基因的拷贝数。经淀粉平板产酶初筛及Southern blot鉴定,成功分离获得了可高效分泌表达中温α-淀粉酶的基因工程菌ZHWY。该菌株在摇瓶发酵75 h后α-淀粉酶酶活可达到730 U/m L,比酶活为156 U/mg总蛋白,与原始菌株264相比提高了70%,且继代过程中表达水平稳定。在5 L发酵罐中,枯草芽孢杆菌ZHWY发酵所产中温α-淀粉酶酶活最高达到1450 U/m L,具有良好的工业应用前景。
In this study,the gene of mesophilic α-amylase( amy L) from a mesophilic α-amylase-producing strain( Bacillus subtilis 264) has been cloned into an expression plasmid p AX01 at Bam HI and Sac II sites,forming an integrative recombinant plasmid p AX01- amy L. Following its transformation into B. subtilis 264,the recombinant plasmid p AX01- amy L mediates chromosomal integration of the amy L expression cassette by homologous recombination at lac A locus,and therefore,increases the copy number of the amy L genein B. subtilis 264. As a proof of concept,a high-level expression transformant,designated as B. subtilis ZHWY,was isolated and verified by Southern blot. It was shown that the secreted α-amylase activity of B. subtilis ZHWY in a 500 m L shake flask reached 730 U /m L( 156 U / mg protein) which was an improvement of 70% when compared with the wildtype strain B. subtilis264. Moreover,in a 5 L fermenter,B. subtilis ZHWY afforded a high α-amylase activity of 1450 U / m L,demonstrating its good potential for the industrial production of mesophilic α-amylase.
出处
《北京化工大学学报（自然科学版）》
CAS
CSCD
北大核心
2015年第4期78-82,共5页
Journal of Beijing University of Chemical Technology(Natural Science Edition)
基金
国家自然科学基金青年科学基金(21406005)
