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 α-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 55.6μmol/(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 α-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.