Cloning, Expression, And Application In Synthesis Of Chiral Drugs Of (R)-Selective ?-Transaminases

?-transaminases (?-TAs) are the significant transferase that catalyze the transformation between amino and carbonyl. Due to excellent optical purity, strong stereoselectivity, mild reaction conditions, and friendly environment, the ?-TAs had been used as an attractive approach for biosynthesis of optically pure amines. Nowadays, compared with (S)-selective transaminases ((S)-TAs), (R)-selective transaminases ((R)-TAs) studied less and received more attention with the development of chiral amine drugs, which makes (R)-TAs have more research and application value.Two (R)-TAs were discovered through genome mining from Burkholderia cepacia (pdBC) and Fusarium oxysporum (hpFO), respectively. The (R)-TAs pdBC, hpFO were cloned into pET22b, overexpressed in Escherichia coli, purified with the molecular mass of approximately 54 kDa and 36 kDa, respectively.Characterizations of (R)-TAs pdBC, hpFO were studied with (R)-phenethylamine, sodium pyruvate as model substrates, it turned out that the optimal temperature of pdBC was 35? and the optimum pH value is 10. The (R)-TA pdBC was activated by Fe3+, Fe2+ and strongly inhibited by Cu2+ Mn2+. The activity of pdBC was lost at 35 ?. The pdBC Km for (R)-phenethylamine was 31.238 mM, the Km for sodium pyruvate was 26.793 mM. The (R)-TA hpFO optimum temperature was 25? and the optimum pH was 7. The activity of hpFO was lost at 25?. The Km of hpFO for (R)-phenethylamine was 11.031 mM, the Km for sodium pyruvate was 14.692 mM. The (R)-TA hpFO was inhibited by Mg2+and activated by Mn2+, Zn+. In addition, their reactivities toward a series of amino donors and amino acceptors were determined. Generally speaking, (R)-TAs pdBC, hpFO exhibited specific activity toward phenethylamine and their analogues.In the synthesis of sitagliptin and sitagliptin intermediates (R)-3-amino-4-(2,4,5-trifluoro-phenyl)-butyric acid ethyl ester with the (R)-TA ATA-117, ATA-117 was expressed in Escherichia coli and Pichia pastoris, respectively. Escherichia coli Transetta (DE3) was selected as a better host because of simple operation, short fermentation period. ATA-117 was applicated in the synthesis of sitagliptin intermediates with (R)-phenethylamine as amino donor in 81.35% enantiomeric excess (ee) with the percent conversion 70.47%, which provided a new method for the synthesis of sitagliptin.