Constructions Of Multi-enzymatic Cascade Reactions Of Transaminase By Heterodimerizing Leucine Zippers

The deepening of environmental pollution has enabled the sustainable and green development to become the mainstream of the current society.Compared with traditional chemical processes,it is now gradually being replaced by biological catalysis.Compared with the single enzyme system,the multi-enzyme system uses the substrate channel effect to obtain higher catalytic efficiency,increase production,reduce the production of toxic by-products,and save energy.At present,the synthesis of chiral amines is very important in the pharmaceutical industry.In view of the asymmetric synthesis of omega-aminotransferase(ω-TA)chiral amines,it is of interest to study them.In this paper,a pair of heterodimeric affinity peptides with strong binding ability was selected to use its affinity self-assembly property as a protein dimerization tool.By linking the affinity polypeptides to the corresponding three model enzymes(R-type transaminase,alanine dehydrogenase,and formate dehydrogenase)on the gene sequence,the fusion vectors are used to separately express the three modified enzymes.After purification,the three enzymes were divided into two groups for in vitro aqueous phase mixing(R-type transaminase and alanine dehydrogenase,alanine dehydrogenase and formate dehydrogenase).By performing non-denaturing electrophoresis on the sample,the self-assembly of the sample was confirmed based on the results of the banding,and there were various combinations.In the fluorescence quenching experiment,it was found that the stability of the mixed bienzyme modified by the polypeptide was better than that of the free bienzyme mixture.Two groups of enzymes after incubation and self-assembly were mixed for the multi-enzyme cascade reaction to investigate the catalytic ability of four amino receptors.Amines were generated from aminotransferases as the main reaction,and alanine dehydrogenase was introduced to remove by-products.Dehydrogenase is responsible for the regeneration of cofactors.When catalyzed with different prochiral ketone substrates,the conversion of acetophenone was 32%,the conversion of furfural was 41%,the conversion of tetralone was 30%,and there was no obvious activity of dihydroindanone.