摘要
【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1 713 bp。将alsS连接到pET-30a(+)上,得到重组质粒pET-30a(+)-alsS,并在Escherichia coli BL2l(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°C,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6 h。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。
[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 iso- butanol 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 006oo 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 HisTrapVMFF 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.
出处
《微生物学通报》
CAS
CSCD
北大核心
2012年第11期1589-1596,共8页
Microbiology China
基金
国家973计划项目(No.2011CB710800)
