摘要
目的:在大肠杆菌宿主中过量表达丁二酮还原酶(DAR),同时构建辅酶NADH原位再生系统,利用全细胞高效催化丁二酮不对称还原合成(S)-乙偶姻。方法:PCR克隆多黏芽孢杆菌(Paenibacillus polymyxa) dar基因连到质粒pETDuet-1,转化至大肠杆菌(Escherichia coli) BL21(DE3),构建重组菌E. coli BL21(DE3)-DAR;通过Hi Trap TALON柱亲和层析纯化表达产物DAR酶蛋白,测定DAR的比酶活和分子动力学参数。在重组菌E. coli BL21(DE3)-DAR中构建辅酶NADH原位再生系统,协同表达枯草芽孢杆菌(Bacillus subtilis)的葡萄糖脱氢酶(GDH),构建重组菌E. coli BL21(DE3)-DAR/GDH,并以此重组菌为全细胞生物催化剂,优化催化条件,提高(S)-乙偶姻的产量和产率。结果:获得重组工程菌E. coli BL21(DE3)-DAR和E. coli BL21(DE3)-DAR/GDH。DAR以NADH为辅酶还原丁二酮的米氏常数Km、最大催化速率Vmax、催化常数Kcat分别为2. 59mmol/L、1. 64μmol/(L·min·mg)、12. 3/s,还原丁二酮生成(S)-乙偶姻光学的纯度为95. 86%,具有较好的催化效率和立体异构体选择性。构建辅酶NADH原位再生系统后,重组菌E. coli BL21(DE3)-DAR/GDH可高效催化丁二酮合成乙偶姻。在最优催化条件下分批补料,乙偶姻产量达51. 26g/L,转化率为81. 37%,生产速率为5. 13g/(L·h)。结论:使用非手性化合物原料丁二酮生产高附加值的手性化合物(S)-乙偶姻,以重组菌为全细胞生物催化剂合成(S)-乙偶姻,不需额外添加昂贵的辅酶,具有较高的生产应用价值。
Objective: The whole-cell biocatalyst,overexpressing diacetyl reductase (DAR) and introduced in situ-NADH regeneration systems was applied to improve (S)-acetoin production from prochiral diacetyl.Methods: The gene encoding DAR from Paenibacillus polymyxa was cloned and expressed in Escherichia coli.Recombine DAR was purified by Hi Trap TALON affinity chromatography,then enzyme activities and molecular kinetic parameters of purified DAR were measured. NADH in situ regeneration system based on glucose dehydrogenase (GDH) from Bacillus subtilis was introduced. The whole-cell biocatalyst,overexpressing DAR and GDH was applied to (S)-acetoin produce and the reaction conditions were optimized. Results: DAR showed a high catalytic efficiency and enantioselective (enantiomeric purity 95. 86%). The Km,Vmaxand Kcatvalues of DAR for diacetyl were 2. 59 mmol/L,1. 64μmol/(L · min · mg) and 12. 3/s,respectively. The whole-cell biocatalyst, introduced in situ-NADH regeneration systems resulted in higher (S)-acetoin concentration,productivity and yield form diacetyl. Under optimal conditions in fed-batch bioconversion,51. 26 g/L (S)-acetoin was produced from 63 g/L diacetyl with a productivity of 5. 13 g/(L·h). Conclusion: The compound of prochiral diacetyl was used as substrate for asymmetric synthesis of high value chiral (S)-acetoin. The results demonstrated that whole-cell biocatalyst,introduced in situ-NADH regeneration systems,can effectively improve the production of (S)-acetoin with good applicability and economic performance.
作者
李检秀
陈先锐
陈小玲
黄艳燕
莫棋文
谢能中
黄日波
LI Jian-xiu;CHEN Xiao-ling;HUANG Yan-yan;MO Qi-wen;XIE Neng-zhong;HUANG Ri-bo(National Engineering Research Center for Non-food Biorefinery,State Key Laboratory of Non-food Biomass Energy and Enzyme Technology,and Guangxi Key Laboratory of Biorefinery,Guangxi Academy of Sciences,Nanning 530007,China;State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources,College of Life Science and Technology,Guangxi University,Nanning 530004,China)
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2019年第4期60-68,共9页
China Biotechnology
基金
广西科技计划(桂科合14125008-2-22)
国家自然科学基金(21466007
31400079)资助项目
