Cloning And Expression Of Transaminase Nec Applying For The Synthesis Of Sitagliptin Chiral Intermediate

Sitagliptin can play an effective role in the treatment of diabetes. The method of enzyme catalysis for the preparation of Sitagliptin is now recognized as an efficient mean. This paper designs the Sitagliptin intermediates and biological catalysis route.First of all, this paper established a new analysis method for the chiral intermediate. The method of gene mining was applied, taking the ATA117 amino acid sequence as the template. Five potential genes fragments in gene database were found. Then they were constructed in pETDuet-1 and pET-30a(+) plasmid vector, and imported into the E.coli BL21(DE3). Through HPLC, five transaminases were measured, where only the pETDuet-1-Nec has weak activity of 0.08 U/L and the specific activity is 0.007 U/mg.In order to improve the catalytic activity, this paper mainly focuses on the saturated mutation of the transaminase’s small pocket area, choosing three sites including V60, F113 and A275. A total of 57 mutations were gotten and compared. The best sites were selected to do the combined mutation. Eventually the combination of V60T and F113M has the highest specific activity, which reached 0.0095 U/mg, up 36% compared with the original enzyme.Finally, this paper carried on the enzymology properties characterization. Results show that the optimum reaction temperature is 30℃ and optimum pH value is 8.5.