Transaminase-coenzyme co-immobilization engineering bacteria and application

Abstract

The invention provides a method for catalytic synthesis of a sitagliptin chiral intermediate (3R)-3-amino-1-[3-(trifluoromethyl)-5,6,7,8-teralin-1,2,4-triazol[4,3-a]pyrazine-1-yl]-4-(2,4,5-trifluoro-phenyl)buta-1-one by means of engineering bacterium cells containing transaminase-coenzyme pyridoxal phosphate co-immobilization Escherichia coli, E.coli). The method comprises the steps of culturing engineering bacteria containing transaminase Escherichia coli, preparing co-immobilization cells, and synthesizing the immobilization cell asymmetric catalysis itagliptin chiral intermediate. The transaminase-PLP co-immobilization cells are adopted as a catalyst, the stability is good, the service life is long, the organic solvent is good in tolerance, repeated usage can be performed, no expensiveexogenous coenzyme needs to be added in the reaction process, and the production cost is greatly lowered. The method is simple in technology, low in cost and high in product yield and purity, and hasthe quite high application value in the industrial production of the sitagliptin chiral intermediate.

Description

[0001] (1) Technical field [0002] The invention belongs to the technical field of bioengineering, and specifically relates to a method for immobilizing transaminase-coenzyme co-immobilized microbial cells and using co-immobilized cells as a catalyst to synthesize sitagliptin drug intermediate (3R)-3-amino-1-[ 3-(Trifluoromethyl)-5,6,7,8-tetrahydro-1,2,4-triazolo[4,3-a]pyrazin-7-yl]-4-(2,4 , 5-trifluorophenyl) butan-1-one method. [0003] (2) Background technology [0004] Diabetes mellitus is a chronic metabolic disease characterized by hyperglycemia caused by defects in insulin secretion or insulin action. Diabetes can be divided into insulin-dependent diabetes mellitus (Insulin-dependent diabetes mellitus, IDDM, also known as type I diabetes) and non-insulin-dependent diabetes mellitus (NIDDM, also known as type II diabetes mellitus). Among them, type Ⅱ diabetes accounts for 90% of the total incidence. Diabetes can also cause a variety of complications, including: visual...

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Problems solved by technology

[0007] In 2013, Rehn et al. immobilized transaminase cells on carrier materials such as calcium alginate, titanium trioxide, and chitosan. Although the activity of the immobilized enzyme was still greater than 90% after 8 consecutive reactions, the reaction scale was small and batches Fewer cycles, the reaction needs to add additional coenzymes, and the conversion rate is relatively low (2013, Rehn G, et al., Journal Biotechnology)

In 2014, Andrade et al. co-immobilized transaminase Escherichia coli and coenzymes on methacrylic acid particles, e.e. >99%, and could react in the organic phase without adding additional coenzyme, and the reaction could last up to 10 days, but this method The conversion rate is low, and the enzyme activity drops significantly after the batch reaction (2014Andrade L H, et al., Organic Letters)

[0008] At present, there are few studies on the synthesis of chiral intermediates of sitagliptin drug catalyzed by immobilized cells at home and abroad. The related researches have low recovery rate of total enzyme activity, high preparation cost of immobilized enzyme, unsatisfactory catalytic activity, poor stability, low However, problems such as low concentration of substances and poor tolerance to organic solvents seriously limit the application of immobilized biocatalysts in the production of key intermediates of statin drugs.

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