重组大肠杆菌内PDC和ADH表达条件优化  

Optimization of Expression Conditions for Pyruvate Decarboxylase and Alcohol Dehydrogenase

作　　者：叶广彬 葛菁萍[1] 秦锐[1] 宋刚[1] 平文祥[1] 

机构地区：[1]微生物黑龙江省高校重点实验室黑龙江大学生命科学学院,哈尔滨150080 [2]右江民族医学院科学实验中心,广西百色533000

出　　处：《中国食品学报》2015年第11期34-40,共7页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家自然科学基金面上项目(31270534);国家自然科学基金项目(31270143);国家自然科学基金-青年科学基金项目(31300355);黑龙江省高等学校科技创新团队(2012td009)

摘　　要：丙酮酸脱羧酶和乙醇脱氢酶II可将丙酮酸定向转化成乙醇。将已构建的含有丙酮酸脱羧酶和乙醇脱氢酶II基因的表达载体p ET-28a(+)-pdc-RBS-adh B转入大肠杆菌BL21中,实现在大肠杆菌体内高产乙醇的目的。对该工程菌株进行定性检测,优化诱导表达条件,定量检测,SDS-PAGE分析和做发酵试验,结果表明丙酮酸脱羧酶和乙醇脱氢酶II的最佳表达条件为IPTG浓度1.0 mmol/L,诱导时间7 h,诱导温度37℃。其最大酶活分别为1.34 U/mg和3.88 U/mg。重组子利用葡萄糖、木糖和混合糖发酵。在葡萄糖中发酵72 h,获得最大乙醇产量6.86 g/L,最高乙醇得率0.40 g/g。Pyruvate decarboxylase and alcohol dehydrogenase Ⅱ could convert pyruvate into ethanol. In this study,the constructed plasmid p ET-28a（＋）-pdc-RBS-adh B was transformed into Escherichia coli BL21 to improve the ethanol production. The recombinant strain was further studied for its qualitative characteristics, optimal enzyme expression conditions, quantitative characteristics, SDS-PAGE analysis and fermentation ability. The results showed that the optimal expression conditions for pyruvate decarboxylase and alcohol dehydrogenase Ⅱ were IPTG 1.0 mmol/L, enzyme inducing 7 h and incubating at 37 ℃. The highest enzyme activities of the pyruvate decarboxylase and alcohol dehydrogenase Ⅱ were1.34 U/mg and 3.88 U/mg, respectively. The recombinant strains can ferment glucose and the maxium ethanol production and ethanol yield were 6.86 g/L, 0.40 g/g, respectively.

关 键 词：大肠杆菌 丙酮酸脱羧酶 乙醇脱氢酶Ⅱ 

分 类 号：TQ920.1[轻工技术与工程—发酵工程] TQ223.122