Directed Evolution Of Leucine Dehydrogenase And Application Research Of Its Catalytic Transamination And Deamination

Leucine dehydrogenase is widely involved in the synthesis of a variety of branched-chain amino acids in an organism.It reversibly catalyzes oxidative deamination of L-amino acids to a-keto acids.The application of leucine dehydrogenase in transamination of ?-keto acids to synthesize non-proteinogenic amino acids has attracted numerous studies and received much achievements.For example,the synthesis of L-tert-leucine and L-norvaline.However,the oxidative deamination of L-amino acids to synthesize a-keto acids by leucine dehydrogenase has been scarcely addressed,although a-keto acid is extensively used in feed,food additives,drugs and fine chemicals synthesis,and plays a crucial role in human health.In this study,leucine dehydrogenase(EDH)derived from Exiguobacterium sibiricum 255-15 was used as the research material.by applying the directed evolution approach,investigated on the increase of the deamination activity of leucine dehydrogenase.The main research contents of this paper are as follows:1.The recombinant E.coli BL21(pET28a-EDH)with high and efficient expression of leucine dehydrogenase was constructed.Afterwards,the target protein was purified and characterized.Transamination and deamination reaction showed the same optimum pH 9.5 and the optimum temperature 45? and 50? respectively.The reductive amination reaction of leucine dehydrogenase displayed higher stability on the change of pH.Km values of leucine dehydrogenase against NAD+ and L-leucine in the deamination reaction were 0.656 and 0.762 mM respectively.The catalytic ability of enzymes Vmax for NADH and TMP achieved 333.3 and 55.6?mol/(min·mg).The enzyme could only catalyze L-amino acids conversion with a broad substrate range.D-amino acids exhibited the inhibition effect on leucine dehydrogenase activity.A 50%decline of enzyme activity was observed in a reaction that contained D-leucine:L-leucine(1:1)mixture.D-leucine could enter the substrate domain like L-leucine but no reaction could take place.2.A simple and feasible high-throughput screening method for the directed evolution of leucine dehydrogenase was also developed.Using Error-prone PCR,the mutation library was designed,and a total of 1055 clones were screened.The obtained mutated leucine dehydrogenase 'MutK172R' showed 14.15 U/mg enzyme activity which is about 45.9%higher than the wild type.The MutK172R enzyme exhibited better affinity on L-leucine and coenzyme NAD+ for the values of Km were 0.313 and 0.5 mM respectively.and improved product yield.In 24 hours preliminary catalytic experiments to synthesize ?-ketoisocaproic acid,the yield by MutK172R reached 7.22 mM which is?29.02%higher than the wild type enzyme(6.59 mM).Sadly,the mutation leading to oxidative deamination of D-amino acid could not be achieved.3.Furthermore,the construction of the coenzyme regeneration cycles coupling leucine dehydrogenase with glucose dehydrogenase for L-tert-leucine synthesis was conducted and some reaction conditions were optimized for the double plasmid expression system E.coli BL21(pET28a-GDH/pET20b-EDH).Results as followed the optimized molar ratio of the double substrate was 1:2,the optimized temperature and pH was 30? and pH10.0.under this optimized condition,the conversion of the substrate reached 100%,the concentration of product L-tert-leucine reached 100 mM,and its optical purity was greater than 99%(e.e.%)within 7 hours.It was also noted that compared to the BL21(pET28a-GDH)and BL21(pET28a-GDH)cocatalytic system,lower bacterial concentration and more straightforward production process could result in higher reaction efficiency.