[Objective] Acetolactate synthase (ALS) is the key enzyme in isobutanol biosyn-thetic pathway.Efficient expression of ALS is of great significance for the regulation of isobutanol metabolic pathway.[Methods] The acetolactate synthase gene (alsS) from Bacillus subtilis was amplified by PCR with primers designed according to the sequence of alsS in GeneBank, which is 1 713 bp.Then the alsS was cloned into the expression vector of pET-30a(+).The resulted recombinant plasmid was transformed into Escherichia coli BL21(DE3) for the overexpression of alsS.[Results] The heterologous expression condition was optimized to be inducted at an OD600 of 0.6-0.8, 30 ℃ with 1 mmol/L IPTG for 6 h.ALS was mostly expressed in the supernatant with the activity of 24.4 U/mL, which was improved for 7.13 times.Electrophoretically pure ALS was obtained after HisTrapTMFF affinity chro-matography with the specific activity of 95.2 U/mg.[Conclusion] These results contributed to the construction of isobutanol biosynthetic pathway in E.coli.%[目的]乙酰乳酸合成酶(ALS)是异丁醇生物合成中的关键酶,实现ALS的高效表达对调控异丁醇代谢途径有重要意义.[方法]根据GenBank中ALS的基因序列(alsS)设计引物,以枯草芽孢杆菌168基因组DNA为模板通过PCR扩增技术得到目标酶基因,目的片段全长为1713 bp.将alsS连接到pET-30a(+)上,得到重组质粒pET-30a(+)-alsS,并在Escherichia coli BL21(DE3)中实现表达.[结果]对表达条件进行了优化,获得最佳表达条件为:诱导温度30℃,诱导起始菌体OD600为 0.6-0.8,诱导剂 IPTG 浓度为1 mmol/L,诱导时间为6h.表达的乙酰乳酸合成酶大部分以可溶性形式存在于菌体内,优化后酶活可达到24.4 U/mL,比优化前提高了7.13倍.经HisTrapTMFF亲和层析后获得电泳纯的ALS,比活为95.2 U/mg.[结论]ALS的有效表达为在大肠杆菌体内构建异丁醇代谢途径打下了基础.
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