前手性酮全细胞转化体系中甲酸脱氢酶C-端无序结构与转化效率的关系研究

Study of the Relationship between the Disordered C Terminusin of Formate Dehydrogenase and the Conversion Rate in the Whole-cell Catalysis of Prochiral Acetone

目的:构建前手性酮类化合物的重组全细胞催化体系,并研究CbFDH C-端无序的11个氨基酸与酶活性的关系。方法:利用基因工程方法构建野生型LbADH,CbFDH及C-端11个氨基酸缺失的CbFDH截短突变体的表达菌株;SDS-PAGE分析其表达方式及表达量,利用分光光度计法测定全菌破碎上清中的酶活性;将含野生型LbADH及CbFDH的菌株与底物混合孵育,考察双菌全细胞体系的催化效果;并将含野生型CbFDH及CbFDHΔ354-364的菌液分别与含野生型LbADH的菌株混合催化苯乙酮的转化,比较两种菌的协助转化效率。结果:构建了重组表达载体pETDuet-1-adh,pETDuet-1-fdh及pETDuet-1-fdhΔ354-364,并实现了在大肠杆菌中的异源表达,含野生型LbADH及CbFDH的两种菌株能够实现苯乙酮的协同转化,转化率为24.4%,产物对映体纯度为96.79%;CbFDH及CbFDHΔ354-364分别占上清总蛋白的28.54%及37.72%,C-端的缺失未影响CbFDH的可溶性表达,但CbFDHΔ354-364全菌上清催化NAD+转化为还原型NADH的效率降低,为CbFDH粗酶液的29.82%;且重组菌株Rossetta(DE3)-pETDuet-1-fdhΔ354-364与Rossetta(DE3)-pETDuet-1-adh协同转化苯乙酮的转化率降低至8%。结论:重组菌株Rossetta(DE3)-pETDuet-1-adh与Rossetta(DE3)-pETDuet-1-fdh能够协同催化苯乙酮转化,转化效率为24.4%,产物对映体纯度为96.79%;重组菌株Rossetta(DE3)-pETDuet-1-fdhΔ354-364与Rossetta(DE3)-pETDuet-1-adh协同转化苯乙酮的效率为Rossetta(DE3)-pETDuet-1-fdh与Rossetta(DE3)-pETDuet-1-adh协同转化效率的32.79%,且Rossetta(DE3)-pETDuet-1-fdhΔ354-364全菌上清催化还原型辅酶再生的效率降低,提示C-端无序的11个氨基酸残基对CbFDH酶活性的发挥起着重要的作用。

Objective： Construct the whole cell biocatalysis system for the reduction of prochiral carbonyal compounds. And valuate the function of the disordered C terminus in the catalysis of formate dehydrogenase from Candida boidinii. Methods： The recombinant strains for the expression of LbADH , CbFDH and CbFDH△354-364were constructed by the genetic engineering methods. The expression of recombinant proteins were analyzed by SDS-PAGE, and the activity of the whole soluble proteins was determined photometrically at 340 nm. Rossetta （DE3）-pETDuet-1-adh and Rossetta （DE3）-pETDuet-1-fdh were mixed and incubated with acetophenone, the products were analyzed by high performance liquid chromatography （HPLC）. To compare the NADH regeneration rate of Rossetta （ DE3 ） -pETDuet-1 -fdh△354-364 and Rossetta （ DE3 ） -pETDuet-1-fdh, the two strains were mixed with Rossetta（ DE3 ）-pETDuet-1-adh separately, and incubated with acetophenone, the products were detected timely. Results： The recombinant plasmid pETDuet-1-adh, pETDuet-l-fdh and pETDuet-1-fdh△354-364 were constructed correctly. The recombinant proteins LbADH, CbFDH and CbFDH△354-364 could be expressed solublely. The two recombinant strains harboring LbADH and CbFDH separately can be used coupled for the asymmetric reduction of acetophenone, the conversion rate reached 24.4% and enantiomeric excess reached 96.79% ; the soluble expression of CbFDH△354-364 has no significant difference compared with CbFDH, but when using the whole soluble proteins of Rossetta （DE3）-pETDuet-l-fdh△354-364 catalyzed the regeneration of NADH, the conversion rate was sharply decreased to 8% when compared with Rossetta （DE3）-pETDuet-l-fdh. Conclusions： The recombinant strains Rossetta （ DE3 ） -pETDuet-1-fdhA354364 and Rossetta （ DE3 ） -pETDuet-1 -adh could be used coupled for the reduction of acetophenone, resulted in a chemical yield of 24. 4% and an enantiomeric excess of 96. 79%. Rossetta （ DE3 ）-pETDuet-1-adh and Rossetta （ DE3 ）-pETDuet-1-fdh were mixed and incubated with acetophenone, the conversion rate was decreased 32.79% compared with Rossetta （DE3）-pETDuet-1-fdh mixed with Rossetta （DE3）-pETI）uet-1-adh. The disordered residues in C terminus of CbFDH may play an important role in the catalysis process of CbFDH.