Exploitation Of New ?-transaminases And Their Applications In The Synthesis Of Chiral Amines And Vicinal Amino Alcohols

Chiral amines and chiral amino alcohols have been widely used in chiral pharmaceutical industry,organic ligands,chiral auxiliaries,separation agents and optical display materials.Biocatalytic preparation of chiral amines and chiral vicinal amino alcohols is a more attractive and competitive method than traditional chemical catalysis method due to its advantages of high efficiency,high selectivity,mild reaction conditions and environmental-friendly.In addition,the reaction mechanism without the redox cofactor recycling of?-transaminase has attracted the attentions of many researchers.In this study,we established a high throughput screening method to screen the target strain containing transaminases.Four ?-transaminases from the target strain were cloned by genome mining.The enzymes were purified and characterizated.First,an efficient,rapid and sensitive screening method for?-transaminase was established.The 2-hydroxyacetophenone was oxidized in the presence of the 2,3,5-triphenyltetrazolium chloride?TTC?and the reaction generated the red triphenylformazan?TPF?.It was referred as a color reaction.TPF was quantified at a 510 nm wavelength.The best pH range of the colorreaction was 6.0-10.0,and the optimal temperature range was 20-30 oC.The color reaction could be completed in about 3-5 min.The assay demonstrated a high sensitivity with visible color changes at conversion levels from 1.6% to32.5% when using phenylglycine as the substrate.And this method had good applicability to the whole cell of bacteria.In this study,Pseudomonas putida NBRC 14164 with high selectivity and selectivity to phenylglycine was successfully screened.Second,four uncharacterized ?-transaminases Pp21050,PpbauA,Pp36420 and PpspuC from Pseudomonas putida NBRC 14164 with a sequence identity of 37%,35%,56% and 58% respectively to CV2025 from Chromobacterium violaceum were selected by genome mining.The genes were successfully soluble expression in E.coli by the construction of expression vector.In order to further study their enzymatic properties,the target proteins were purified by nickel column to obtain electrophoretic purified transaminases.Third,the enzymatic properties of purified ?-transaminases were characterized by using racemic phenethylamine as substrate.The results showed that optimum pH were 9.0,10.0,8.0 and 9.0 and the optimum temperatures were 35 oC,50o C,35 oC and 35 oC of four ?-transaminases Pp21050,PpbauA,Pp36420 and Ppspu C,respectively.PpbauA was more stable compared to other three enzymes,it still retained about 70% of the relative enzyme activity after being incubated at 60 oC for 2 h.While Pp21050,Pp36420 and Ppspu C were sensitive to temperature and had poor thermal stability.The kinetic parameters of Pp21050,PpbauA,Pp36420 and Ppspu C were determined by using racemic phenylethylamine as the substrate.KM of four ?-transaminases were 161.3 mM?Pp21050?,136.7 mM?PpbauA?,398mM?Pp36420?and 130.9 mM?PpspuC?with Vmax of 2.8 U/mg,0.5 U/mg,5.6U/mg and 3.6 U/mg and yielding a catalytic efficiency kcat/KM of 0.015 mM^-1s^-1,0.003 mM^-1s^-1,0.012 mM^-1s^-1,and 0.023 mM^-1s^-1.Finally,three different racemic vicinal amino alcohols and four racemic amines were selected for the substrate-specific study of ?-transaminases.The results showed that the four ?-transaminases showed different catalytic activity and stereoselectivity for the vicinal amino alcohols and amines.In the process of kinetic resolution of racemic amines,all of these transaminases only showed highly?S?-selective towards amines.Among these enemzys,Pp21050,Pp36420 and PpspuC showed good catalytic ability to racemic amines with perfect conversions?48-50%?and ee?98-99%?.In the process of kinetic resolution of racemic vicinal amino alcohols,four ?-transaminases had catalytic activity only to the?R?-selective towards the vicinal amino alcohols.Among these enemzys,Pp21050,Pp36420 and PpspuC showed better catalytic ability to racemic phenylglycinol and the conversions rates were 50.2%,49.4%and 49.3%,the ee values were up to 99%,97% and 99%,respectively.Besides,PpbauA with excellent catalytic ability could completely convert the substrate of racemic 2-amino-1-butanol and valinol to optically pure products?ee>99?.Futhermore,?S?-phenylglycinol was successfully prepared by PpspuC kinetic resolution of racemic phenylglycinol in the larger reaction system and the yield was 45%.In conclusion,the high activity,stereoselectivity and large substrate spectra of the four new distinct ?-transaminases had a potential application value in the preparation of chiral amines and chiral vicinal alcohols.