Aminotransferas From Thermococcus Kodakarensis Kod1:Gene Cloning,Expression And Study On Its Catalytic Performance

Chrial amines and amino acids are important building blocks for the synthesis of chiral pharmaceuticals.Transaminases provide an environment-friendly access to produce chiral amines and amino acids under mild conditions with exquisite stereoselectivity.However,the catalytic reaction with transaminases is reversible and the conversion needs to be improved.Isopropylamine is an ideal amino donor and the formed acetone can be removed easily by distillation under the condition of high temperature,which drives the reaction equilibrium toward product synthesis.Therefore,exploitation of transaminase with high activity and good thermal stability toward isopropylamine is of great significance for industrial production of chiral amines and amino acids.In this study,we mined the novel thermostable transaminases from Termococcus kodakarensis KOD1.The gene cloning,heterologous expression,purification,characterization,molecular engineering and the synthesis of L-2-aminobutyrate of the biocatalyst were investigated in details.The TK0186?TK0260?TK0275?TK1094 and TK2268 genes encoding transaminase were identified and amplified from the genomic DNA of Termococcus kodakarensis KOD1.The amplified fragments were inserted into the pET28 a expression vector.The recombinant plasmids were transformed into E.coli strain BL21(DE3)and induced by IPTG.The results showed that TK0186?TK1094?TK2268 transaminase are insoluble in E.coli,while TK0275 transaminase were mostly expressed as soluble form.As such,it was selected for further study.The recombinant TK0275 transaminase was purified by using a heat treatment procedure and one-step affinity chromatography on a Ni-NTA column,resulting in electrophoretic purity.The biochemical properties of TK0275 transaminase were investigated: it exhibited highest activity at pH 8.0,75 ?.The half-lives of TK0275 transaminase at 60 ??70??80 ? were 44.0 h ?15.5 h and 6.2 h,respectively,which indicate that it was thermostable under high temperature.The study of substrate specificity on different amino donors indicated that N2-acetyl-L-ornithine was the preferable substrate for TK0275 transaminase,whereas its activity towards isopropylamine was only 0.5% of that toward N2-acetyl-L-ornithine.TK0275 transaminase was the firstly characterized thermophilic ?-transaminase from thermophilic archaea.In order to improve the catalytic efficiency of TK0275 transaminase toward isopropyamine,the site-directed mutagenesis was carried out for the molecular modification of TK0275 transaminase.The three-dimensional homology model of TK0275 transaminase was generated by homologous modeling,and the site mutagenesis libraries were created at catalytic active site region.The variants I33 L and E174 A displayed increased activity,which was 1.23 and 1.36 fold to that of original enzyme activity respectively.The asymmetric synthesis of L-2-aminobutyrate by I33 L and E174 A were studied and the conversion yield was demonstrated to achieve 31.3% and 36.4%,respectively,after 12 h with ee >99.9%.