Construction Of Double Enzyme System By Splicing Saccharomyces Cerevisiae Aldehyde Ketone Reductase And Glucose Decyanation

With the development of modern bioengineering technology,the production of complex compounds catalyzed by biological enzymes has played an increasingly important role in the engineering production field.Among many chiral molecules,diketones,dialdehydes,and aldehydes and ketones occupy a place,such as the important lipid-lowering drug statin.Saccharomyces cerevisiae(Gre2)is a co mMon aldehyde and ketone reductase,which has a good catalytic capacity for many aldehydes and ketones,especially dialdehydes,diketones,aldehydes and ketones.Through Gre2,we can efficiently produce chiral drug intermediates or drug effective molecules.Glucose dehydrogenase(GDH)is a protease that is very co mMon in organisms and widely used in enzyme-catalyzed processes.It is widely involved in a variety of coenzyme/reducing coenzyme(NADP/NAPDH)dependent enzyme catalytic systems.By supplying hydrogen to other enzymes,the reaction continues to proceed efficiently.In this article,firstly,through gene engineering technology,GRE2 and GDH genes were connected on the plasmid expression vector together and transformed into the receptive Escherichia coli.Colony PCR technology and enzyme digestion are used to verify the completion of the connection and induce Escherichia coli to express the double enzyme splicing body protein.The double enzymes splicing protein can complete the self-splicing process in the cell through the self splicing effect of the containing peptide.The results showed that the gre2-gdh splicing enzyme protein could express in E.coli and form a stable double enzyme system by self splicing.On the basis of the expression system of double enzyme splicer,the splicing of double enzyme protein was completed and the double enzyme splicer was obtained by making full use of the coordination effect of phosphate buffer and zinc ion.SEM,EDS and 2D-IR were used to study the characterization of double enzyme splices,and LC was used to study the enzyme activity and catalytic ability of the system.According to a series of characterization results and enzyme activity reaction,the enzyme activity and enzyme structure stability of double enzyme splicing system were significantly improved compared with free double enzyme.