Enzymatic Synthesis Of Deoxyguansine-5'-Triphosphate And Guansine-5'-Triphosphate

Deoxyguanosine triphosphate(dGTP)and guanosine triphosphate(GTP),the precursors for synthesizing DNA or RNA in vivo,are important raw materials for various modern biotechnologies based on PCR.In this study,we used permeabilized whole-cell co-expressing guanylate kinase and acetate kinase to biosynthesize dGTP and GTP.At the same time,we investigated the application of whole cell catalysts constructed by bacterial cell surface display in biosynthetic reactions.Gene gmk encoding the guanylate kinase(GMKase)of Lactobacillus bulgaricus and ack encoding the acetate kinase(ACKase)of E.coli were cloned into plasmid pET28-a to construct two recombinant strains.Then the recombinant strains co-expressing GMKase and ACKase,which named as pAG and pGA,were constructed by using the isocaudamer cleavage site on the vector.Then the induced cells were treated with 10 mM EDTA at 25 ? for 30 min.The enzyme activity of permeabilized whole-cell was less than that of the crude enzyme solution.and.The substrate conversion rate was up to 91%after 2.5 h when 5 mM dGMP was catalyzed at 37 °C.The substrate conversion rate was up to 95%after 1.5 h when 5 mM GMP was catalyzed at 37 ?.By fusing with the N-terminal or N-and C-terminal fusions of ice nucleation protein,we successfully displayed guanylate kinase and acetate kinase on the surface of E.coli cells.A large amount of soluble target protein can be obtained when induced with 0.2 mM IPTG at 25 ?for 30 h.The conversion of dGMP was up to 91%when catalyzed by the surface display enzyme at 37 ? for 4 h.And the conversion of GMP was up to 95%after 3 h of reaction.The stability of the whole-cell catalyst at 37 ? was very good.The enzyme activity maintained above 50%after recovering 9 times.An ATP regeneration system catalyzed by ACKase provided phosphate group to the(d)GTP synthesis reaction,which greatly reduced the production cost.Our research showed that only one-twentieth of the initial substrate concentration of ATP added was sufficient to meet the reaction requirements.