基于分子伴侣策略提高NADPH依赖型醇脱氢酶的异源可溶性表达

Enhance Soluble Heteroexpression of a NADPH-Dependent Alcohol Dehydrogenase Based on the Chaperone Strategy

目的:生物催化的氧化还原反应广泛应用于手性化合物的制备,其中很多反应涉及辅酶NADPH的原位再生。以异丙醇为辅助底物,利用醇脱氢酶再生NADPH,具有比酶活高、副产物丙酮易于分离等优势,受到越来越多的关注。选择极具应用潜力的来源于Clostridium beijerinckii的醇脱氢酶CbADH作为研究对象,针对其在大肠杆菌中的可溶性表达差、酶活低的瓶颈问题开展研究。方法:首先通过引入诱导型质粒p Gro7表达分子伴侣GroES-GroEL,将pET-28a(+)质粒表达CbADH的可溶性提高了3.57倍,酶活达到出发菌株的4.83倍。其次,考察了另外三种不同的分子伴侣表达策略:pET-28a(+)单质粒共表达、基因组强化表达GroES-GroEL和组成型改造pGro7/GroES-GroEL和pET-28a(+)/CbADH双质粒共表达。结果:组成型改造pGro7和pET-28a(+)双质粒共表达策略的效果最优,其CbADH的可溶性表达提高了8.07倍,酶活达到了21.79U/mg DCW,是出发菌株的9.43倍。结论:为CbADH的工业应用奠定了良好的基础,也为外源蛋白的可溶性表达提供了参考。

Objectives:Many of biocatalytic redox reactions which are widely used in the production of chiral chemicals involve the regeneration of the coenzyme NADPH in situ.Alcohol dehydrogenases that regenerate NADPH with isopropanol as substrate have the advantages of high specific activity and easy separation of byproduct acetone,attracting more and more attention.Therefore,an alcohol dehydrogenase from Clostridium beijerinckii,namely CbADH,was chosen as the research object for its more considerable specific activity and the most applicable potentiality within present literatures.To solve the problem of poor soluble expression of CbADH in E.coli genetically engineered strains and the consequent enzyme activity as low as 2.31 U/mg DCW,the following studies were carried out.Methods:Firstly,different chaperone proteins were expressed by inducible plasmids to increase the soluble expression level of CbADH,and the results showed that molecular chaperone GroES-GroEL significantly improved the soluble expression of CbADH by 3.57 times more than the original strain,with enzyme activity of 11.18 U/mg DCW which is 4.83 times more than the original strain.Secondly,three other different GroES-GroEL expression strategies were examined:pET-28 a(+)single plasmid coexpression,genomic enhancing expression of chaperone,and constitutive-pGro7/pET-28 a(+)dual plasmid coexpression.Results:The results indicated that the constitutive-pGro7/pET-28 a(+)dual plasmid co-expression strategy had the best effect which improved the soluble expression of CbADH by 8.07 times more than the oringinal strain,with a CbADH activity of 21.79 U/mg DCW,which was 9.43 times higher than the oringinal strain.Conclusions:This study not only lays the foundation for the industrial application of CbADH but also provides a reference for heterologous soluble protein expression.