重组大肠杆菌产双乙酰还原酶的发酵条件优化

Optimization of Fermentation Conditions for Production of Diacetyl Reductase Using Recombinant Escherichia coli

DOI：10.3969/j.issn.1673-1689.2017.09.009

中文关键词: 重组大肠杆菌 双乙酰还原酶 发酵培养基优化 诱导条件优化

英文关键词: recombinant Escherichia coli,diacetyl reductase,medium optimization,induction condition

基金项目:

作者

单位

边雅倩

江南大学 教育部工业生物技术重点实验室，江苏 无锡 214122

许国超

江南大学 教育部工业生物技术重点实验室，江苏 无锡 214122

倪晔

江南大学 教育部工业生物技术重点实验室，江苏 无锡 214122

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中文摘要:

光学纯2，3-丁二醇是重要的手性合成砌块。采用双乙酰还原酶催化还原双乙酰底物是一种合成光学纯2，3-丁二醇的绿色方法。作者在摇瓶中对产双乙酰还原酶重组大肠杆菌的发酵培养基及诱导条件进行了优化。优化后的发酵培养基组成为：甘油5 g/L，酵母浸膏10 g/L，鱼粉蛋白胨5 g/L，（NH4）2SO4 1.5 g/L，柠檬酸钠5 g/L，KH2PO4 1 g/L，MgSO4·7H2O 0.5 g/L，NaCl 2 g/L；优化后的诱导条件为37 ℃，IPTG终浓度为0.1 mmol/L。在优化培养基中，37 ℃和0.1 mmol/L IPTG条件下诱导发酵6 h，单位菌体（干重）产双乙酰还原酶量达13.84 kU/g，产酶水平达33.65 kU/L，分别为基础培养基的2.04和4.23倍，LB培养基的1.16和1.71倍。在此最优条件，在3 L发酵罐中进行了重组大肠杆菌产双乙酰还原酶的验证，发酵8 h时酶活最高，单位菌体产双乙酰还原酶量达14.09 kU/g，产酶水平达64.62 kU/L。本研究为高效制备可用于不对称还原合成光学纯2，3-丁二醇的双乙酰还原酶奠定了基础。

英文摘要:

Optically pure 2，3-butanediol is one of the important building blocks. Bioreduction of diacetyl using diacetyl reductase is an environmentally friendly method for chiral 2，3-butanediol production. The medium components and induction conditions of diacetyl reductase produced by recombinant Escherichia coli were optimized in flasks. The optimal medium composition for the production of diacetyl reductase was listed as follows：glycerol 5 g/L，yeast extract 10 g/L，peptone 5 g/L，（NH4）2SO4 1.5 g/L，sodium citrate 5 g/L，KH2PO4 1 g/L，MgSO4·7H2O 0.5 g/L and NaCl 2 g/L. The optimal induction conditions were listed as follows：37 ℃ and 0.1 mmol/L IPTG. After induced for 6 h under the optimized conditions，the enzyme activities reached to 13.84 kU/g dry cell weight and 33.65 kU/L，which were 2.04 and 4.23 folds of those in basic medium，1.16 and 1.71 folds of those in LB medium. In a 3 L fermenter，under the optimized conditions，the maximum enzyme activities achieved at 14.09 kU/g and 64.62 kU/L after cultured for 8 h，which were both higher than those in flasks. This study provides useful guidance on the highly efficient preparation of diacetyl reductase by recombinant Escherichia coli for the preparation of chiral 2，3-butanediol.

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