Cloning And Expression Of ?-Transaminase MVTA Applying In Synthsis Of Chiral ?-amino Alcohols And Vicinal Diols By Cascade Catalysis

Chiral?-amino alcohols and vicinal diols have great application value in chiral medicine,pesticides,cosmetics and multi-functional materials,and also play an significant role in asymmetric synthesis as chiral auxiliaries,separation agents and chiral ligands.Biocatalytic preparation of chiral?-amino alcohols and vicinal diols has become a hot topic in green chemistry due to its advantage of high efficiency,high regional and stereoselectivity,without sophisticated protecting groups,mild reaction conditions and environmental-friendly.The de-velopment of cascade catalysis is considered to be an important strategy for the establishment of environmentally friendly and sustainable chemical processes,compared with step-by-step synthesis,cascade reactions in organic synthesis reduces the number of purification steps,thus contributing to an improved pro-cess economy as well as to more sustainable synthetic routes.Additionally,cas-cade reactions can improve stereochemical control and substrate scope and can suppress side reactions.Lastly,the reactivity and selectivity by allowing equilib-rium reactions to proceed to nearly full conversion can be enhanced by coopera-tive effect of multiple catalysts.In this study,a variety of chiral?-amino alco-hols and chiral vicinal diols were synthesized by one-pot multistep reactions.First,a high activity and selectivity?R?-?TA?MVTA?from Mycobacterium vanbaalenii was screened by TTC method.The obtained transaminase MVTA was recloned to vector pET28a and expressed as soluble protein with high level in E.coli.the obtained target proteins were purified by nickel column and the enzyme properties were characterized by?S?-2-amino-2-phenylethanol as the substrate,The results showed that MVTA showed the best activity at pH 8.0 for de-amination of 2-amino-2-phenylethanol.and displayed the maximum activity for the de-amination reaction at 55^oC.MVTA showed residual activity of above90%over a period of 24 h at pH 7.0 and 8.0;>95%residual activity activity of MVTA were measured after incubation of the enzyme below 30^oC for 24 h;Ki-netic resolution of a set of racemic?-amino alcohols by MVTA was demon-strated.The results show that MVTA displayed better catalytic activity and en-antioselectivity to racemic?-amino alcohols,the conversions rates were up to50-62%,the ee values were up to>99%.Asymmetric reductive amination of three?-hydroxy ketones?10-300 mM?by MVTA was conducted,?S?-?-amino alcohols were obtained with>99%ee and 80-99%conversion.Preparation ex-periment for the reductive amination of 00 mM 2-hydroxyacetophenone by the resting cells of recombinant E.coli?MVTA?was proceeded smoothly and prod-uct?S?-2-amino-2-phenylethanol was obtained with 71%isolated yield,>99%ee.The current research proved that the MVTA is a potential enzyme for the prepa-ration of chiral?-amino alcohol.Second,cascade biocatalysis via intracellular transamination and reduction was developed as a green and efficient method for simultaneous preparation of both enantiomer of?-amino alcohol and vicinal diol in high ee and high yield,By a modular combination of two transaminases and two carbonyl reductases,all four types of enantiopure?-amino alcohol and vicinal diol can be produced in a flexible way.The generality of the cascade biocatalysis was demonstrated with the whole-cell approaches to convert 10-60 mM?R?,?S?-?-amino alcohol to?R?,?S?-diol and?R?,?S?-?-amino alcohol in 90-99%ee with 50-52%conversion.Preparative biotransformation was demonstrated at a 50 mL scale with mixed recombinant cells to give both?R?,?S?-2-amino-2-phenylethanol and?R?,?S?-1-phenyl-1,2-ethanediol in>99%ee and 40–42%isolated yield from race-mic 2-amino-2-phenylethanol.Last,a hybrid organo-and biocatalytic system for the asymmetricconver-sion of L-?-Amino Acids into chiral vicinal diol was developed.Combining a simple organic catalytic process?NaBH₄-H₂SO₄ system?and recombinant Esch-erichia coli that was engineered to coexpress three enzymes?transaminase,car-bonyl reductase and glucose dehydrogenase?to catalyze 3-step reactions in one pot,was successfully applied to six conventional natural L-?-Amino Acids in aqueous medium in high conversion?70-90%?and high ee values?91-99%?.The current research highlights the enormous potential of engineering cas-cade biocatalysis or cascade chemo-biocatalysis for one-pot synthesis of crucial non-natural chiral compounds in a safe,green,sustainable,enantioselective,and efficient ways.