Improving The Activity Of (R)-selective ?-transaminase From Burkholderia Cepacia Based On Directed Evolution

?-transaminase is a transferase that is capable of catalyzing the transformation between amino acceptor and amino among amino donor.The importance of amine transaminases for producing optically pure chiral precursors for pharmaceuticals and chemicals has substantially increased in recent years.The enzyme activity of co-transaminase from Burkholderia cepacia was enhanced by the means of directed evolution.A mutant library was obtained through adding Mg2+ and Mn2+ simultaneously to the PCR system.A positive mutant T166K/H408R has been successfully acquired,and its enzyme activity is 58.32 U/mg,1.8-fold higher than that of Pdbc.Pdbc JQ was obtained when 170 amino acids of Pdbc N-terminal was delated.The enzyme activity of Pdbc JQ was 37.64 U/mg which was higher than that of Pdbc(32.40 U/mg),which laid the foundation for our homologous modeling.A homologous modeling of Pdbc JQ has been successfully constructed.According to site saturation muragenesis,we came to realize that the residues located on the ?-pleated sheet transformation had less influence on the enzyme activity.At the same time,23 7L,23 8H and 239G have been selected for site saturation muragenesis spots,the results showed that L237Q was positive mutant,whose enzyme activity is 149.77 U/mg,3.91-fold higher than that of Pdbc JQ.Kinetic parameters of(R)-TAs Pdbc,Pdbc JQ and 237Q were studied with(R)-phenethylamine,sodium pyruvate as substrates resepectively.The catalytic efficiency of Pdbc JQ was higher than that of Pdbc for both R-phenylethylamine and sodium pyruvate.As for R-phenylethylamine,catalytic efficiency of Pdbc JQ was 4.44-fold higher than that of Pdbc JQ.As for sodium pyruvate,catalytic efficiency of Pdbc JQ was 4.05-fold higher than that of Pdbc JQ.