Study On Biocatalytic Preparation Of Chiral Amine Drugs Sitagliptin

Sitagliptin is a chiral amine drug,which can be used in cure of type 2 diabetes with well curative and small side effects,and has a good market prospects.Compared with the organic synthesis,the biocatalytic preparation of sitagliptin has the advantages of green environmental protection and high optical purity.In this paper,the omega-aminotransferase used in the biocatalytic preparation of sitagliptin was modified and the preparation process was optimized.In this paper,we first obtained the sequence of the omega-transaminase ATA-117 gene published in Gene Bank and constructed the E.coli BL21/pET28a-ATA-117 engineering strain to verify its transamination activity.The feasibility of chromophore conversion reaction on plate medium was verified by using o-xylylenediamine dihydrochloride as the color-selective amino donor,and a high-throughput screening method for plate coloration was constructed.Amino acids at positions 124,126,150,and 152 of the ATA-117 template were fixed for saturation mutation to obtain the target mutations.Five favorable mutations were obtained,which were T126L(41.0%),T126N(31.1%),S150Y(23.6%),T126F(20.5%),S1521(10.7%).Based on T126L,the superposition effect of the other mutations was studied.The optimal fermentation conditions of the mutant strain T126L were explored.Under the optimum fermentation conditions,the activity of the lyophilized cells was 6321U/g.Followed by high-density fermentation in 30L fermenter,and OD600 reached 115.432 at the end of fermentation.The mutant strain T126L was used to replace ATA-117 to optimize the biocatalytic preparation of sitagliptin.The optimized reaction conditions were as follows:cosolvent DMSO concentration 50%,pH 9.0,reaction temperature 50℃.Under these conditions,and the substrate concentration of 200g/L,the conversion rate reached 98%or more after.The product was then separated and purified,with a final yield of 66.9%and ee>99.99%.