Immobilization Of Recombinant E. Coli For Enhanced NADPH Regeneration And Its Catalytic COBE Asymmetric Reduction

Insufficient supply of intracellular coenzyme NADPH,poor catalyst stability and unreusability were the main limiting factors for the industrial production of chiral alcohols.In this research,a catalytic system for the efficient synthesis ofβ-hydroxyesters was constructed by coupling the NADPH regeneration system and the immobilization of microbial cells.The research contents were as follows:Firstly,the regeneration coupling system of carbonyl reductase and NADPH were constructed with double gene tandem vector and double plasmid co-expression system,which metabolically regulated EMP and NAD kinase to promote the endogenous synthesis of NADPH.Using ethyl 4-chloroacetoacetate as the model substrate（COBE）,the engineered E.coli BL21（DE3）/p ETDue T-1-gap B-bc CR&p ET-28a-yfj B enhanced NADPH synthesis ability by 2.19 times,and catalytic efficiency was increased by 1.69times,in which the yeild ofβ-Hydroxyester was 98.18%and ee>99%.Secondly,different types of support materials were selected to immobilize recombinant E.coli to improve the stability of the catalyst.Using response surface analysis,the optimal immobilization conditions were as follows:the concentration of Ca Cl₂ is 1.19%（m/V）,immobilized time was 10 h,the concentration of polyethyleneimine is 3.81%（m/V）,and the cross-linking time is 34.42 min,and cell load was 17.5 g_cell/g _SA.On this basis,the performance of the immobilized cells was evaluated.The catalytic activity of the immobilized cells remained above 45.98%when stored for14 days at 4℃,and showed good thermal stability and acid/alkali resistance.Finally,the above system was used for asymmetric reduction of COBE.After optimizing the reaction temperature,p H,substrate concentration,amount of immobilized cells and reaction time,the catalytic efficiency of immobilized cells was increased to67.41%.Under the optimal reaction conditions,the immobilized cells still retained more than 20%catalytic activity after 6 batches of reuse.This research showed that the use of polyethyleneimine modified sodium alginate to immobilize recombinant E.coli based on enhanced NADPH regeneration can greatly improve the catalytic performance,and provide a feasible solution for the continuous industrial production ofβ-hydroxyesters.