Research On Fermentation,Immobilization Of Aminotransferases And Preparation Of Sitagliptin By Enzymatic Method

Sitagliptin is a chiral amine drug,which can be used in cure of type ? diabetes.Compared with other drugs for treating diabetes,it has good safety and tolerance with few side effects,and has a good market prospect and rising space.Compared with chemical synthesis,the enzymatic synthesis of sitaliptin has the advantages of high catalytic efficiency,mild reaction conditions and friendly environment.Therefore,studying the technology of aminotransferase production by microbial fermentation,improving the stability of immobilized aminotransferase and enzymatic synthesis of sitagliptin have positive practical significance.This article uses the recombinant Escherichia coli for experiment strains,The fermentation conditions for aminotransferase production,amplification of fermentation process and batch fermentation kinetics by 10 L fermentation cylinder were studied,the immobilization process of aminotransferase was determined and the reaction conditions for the catalytic synthesis of sitagliptin by immobilized aminotransferase were studied.The results are as follows:Optimization of shake flask fermentation conditions for recombinant Escherichia coli producing aminotransferase The optimum initial pH of the fermentation was determined by a single factor test of 7.0.meanwhile the optimal peptone concentration,optimal IPTG concentration and optimum speed of shaking table range were determined.Based on single factor,box-behnken test design and response surface analysis were used to predict the best fermentation conditions:peptone concentration 16 g/L,IPTG concentration 0.8mM,rotating speed of the shaking table 214 rpm.Under this condition,the average activity of aminotransferase was 245.7 U/mL by verification test.Study on fermentation kinetics of aminotransferase production by Escherichia coli The fermentation was magnified in fermenter of 10 L.Using the Origin2018 software,the Logistic equation and the Luedeking-piret equation were used to simulate the dynamic process of fermentation,the non-linear fitting analysis of the experimental data of aminotransferase production by recombinant Escherichia coli KX-3 was carried out,and the fermentation kinetic equation of aminotransferase was established.The models are as follows:(1)The kinetic model of cell growth(?)(2)The kinetic model of synthesis of aminotransferase(?)(3)The kinetic model of substrate consumption(?)Study on process conditions of aminotransferase immobilization After selecting domestic epoxy resin NBW-1000EP(F)as the immobilization carrier,The optimal immobilization time was determined as 28h by single factor test,at the same time,the range of optimal buffer pH,optimal immobilization temperature and optimal carrier dosage was determined.Based on single factor,box-behnken test design and response surface analysis were used to predict the Optimal conditions for aminotransferase immobilization:Buffer pH8.0,immobilization temperature 26.5?,carrier dosage 0.092 g/mL.Under this condition,the average activity of immobilized aminotransferase was 2537.7 U/mL,the enzyme activity recovery rate reached 68.4%by verification test..Establishment of reaction system for the catalytic synthesis of sitagliptin by immobilized aminotransferase The most suitable cosoluent was isopropanol through the cosoluent selection test,the optimal reaction system was obtained as follows:isopropanol concentration 80%,substrate concentration 20 g/L,conversion temperature 45?,and enzyme dosage 250 U/mL.Under this reaction system,self-made immobilized aminotransferase was recovered and 65 batches were tested repeatedly.The results showed that the average reaction time of all batches was 21.5 h and the average conversion rate was 95.2%.The results showed that the effect of immobilized aminotransferase on synthesis of sitagliptin was good.