Transaminase and application thereof in preparation of sitagliptin

Abstract

The invention discloses transaminase and application thereof in preparation of sitagliptin. The amino acid sequence of the transaminase is shown as SEQ ID NO: 1, or the amino acid sequence of the transaminase is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the SEQ ID NO: 1. The transaminase can asymmetrically catalyze sitagliptin precursor ketone (2Z)-4-oxo-4-[3- (trifluoromethyl)-5, 6-dihydro-[1, 2, 4] triazolo [4, 3-a] pyrazine-7-(8H)-yl]-1-(2, 4, 5-trifluorophenyl) butyl-2-ketone to generate sitagliptin intermediate (3R)-3-amino-1-[3-(trifluoromethyl)-5, 6, 7, 8-tetrahydro-1, 2, 4-triazolo [4, 3-a] pyrazine-7-yl]-4- (2, 4, 5-trifluorophenyl) butyl-1-ketone, the enzyme activity of the transaminase can reach 1100U / g, and when an enzymatic reaction is carried out for 2h, 50 microliters of transaminase liquid can make the substrate conversion rate reach 55%. Compared with existing transaminase for catalyzing the same reaction, the transaminase has the advantages of high activity, ideal stability and good organic solvent tolerance.

Description

Technical field [0001] The present application relates to enzyme engineering and biopharmaceutical areas, and therefore referring to a transaminase and its application in the preparation of rising littins. Background technique [0002] Sitagliptin is a valid drug for treating type II diabetes, a total known (3R) -3-amino-1- [3- (trifluoromethyl) -5,6-dihydro-1, 2 , 4-triazole [4,3-A] pyrazine -7 (8h) -yl] -4- (2,4,5-trifluorophenyl) butyl-1-ketone is a dipeptide The base peptidase-4 (DPP-4) inhibitor is mainly controlled by protecting endogenous intestinal hypoglycetin and enhancing its effects, with better safety and tolerance. [0003] In the prior art, the method of preparing the Western lipstin mainly has chemical synthesis and biosynthesis, and chemical synthesis methods are mature, but there are many process steps, reagents (such as metal catalysts, etc.) are expensive, partial reagents (Such as metal catalysts, etc.) have disadvantages such as toxic effect on the environme...

AI Technical Summary

Problems solved by technology

Transaminases can carry out the catalytic reaction of asymmetric transamination of sitagliptin precursor ketone to form sitagliptin intermediates, but most of the wild-type transaminases that exist in nature have no catalytic activity or low catalytic activity for the above catalytic reactions, so it is necessary to Carry out directional transformation to it to improve the conversion rate and product yield of the catalytic reaction, such as Codexis company transforms a kind of omega-transaminase derived from Arthrobacter sp. Gliptin proketone 1-[3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,2,4-triazolo[4,3-a]pyrazin-7-yl ]-1,3-Butanedione has high catalytic activity

[0004] However, there are still many defects in the existing biosynthesis method, for example: (1) due to the low activity of the existing transaminase, a large amount of enzyme solution needs to be added in the reaction system, so that a large amount of free protein remains in the reaction system after the reaction ends , which is not conducive to the separation and purification of the product; (2) the stability of free transaminase is relatively poor, and the storage environment and transportation operation requirements of the enzyme are high; (3) the tolerance of transaminase to organic solvents is poor, and it is difficult to produce in the reaction solution containing organic solvents. for prolonged response

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