Study On Biological Production Process Of (S)-indoline-2-carboxylic Acid

(S)-indoline-2-carboxylic acid is a key chiral intermediate for perindopril,a treatment for hypertension.Its production cost and purity affect the price and quality of drugs.The method of obtaining optically pure（S）-indoline-2-carboxylic acid in the industry is mainly based on chemical resolution.The disadvantages of such methods are that the resolving reagents are expensive,the recycling operation is complicated,and the low recycling of resolving reagents,which increases the production cost and also causes great pressure on the environment.It has also been reported that the（S）-indoline-2-carboxylic acid is directly synthesized by asymmetric synthesis or chiral source synthesis,but the problem of too many reaction steps and low yield also hinders the industrial application greatly.There are few studies on the preparation of（S）-indoline-2-carboxylic acid by biological methods around the world,and the process of selectively hydrolyzing the racemic substrate to directly obtain the S configuration acid has not been reported.Therefore,the establishment of a biological method to prepare high optical purity（S）-indoline-2-carboxylic acid in one step can not only provide a new production idea,but also meet the environmentally-friendly and efficient industrial development trend.This research mainly carried out from three aspects,to determines the appropriate separation conditions and optimizes its process route.and optimizes its process route:1.The lipase-producing strain enriched from the soil sample and the laboratory-preserved freeze-dried powder were screened using（R,S）-ethyl indoline-2-carboxylate as substrate.A strain with high stereoselective hydrolysis activity on the substrate was screened,and identified by colony morphology observation,Gram staining,16S rDNA alignment and BIOLOG physiological and biochemical analysis.The strain was identified as Bacillus aryabhattai and named Bacillus aryabhattai B8W22.Finally,entrust the Center of Chinese Type Culture Collection to preserve the strains,deposit number:CCTCC NO:M 2017621.2.A single factor experiment was designed to optimize the fermentation conditions of Bacillus aryabhattai B8W22.The experimental results show that glucose is the best carbon source and yeast extract is the best nitrogen source.The optimized fermentation medium formulation was follow（g/L）:Glucose 15,yeast extract 20,MgSO₄?7H₂O 1.0,NaCl 0.5,initial pH is 7.2.After incubation at 35°C,180 rpm for 24 hours,Bacillus aryabhattai B8W22 had the largest amount of bacteria and enzyme activity.The amount of bacteria is about 8 g/L,enzyme activity is 4.5U/mL.3.Bacillus aryabhattai B8W22 freeze-dried powder prepared by vacuum freeze-drying and used as catalyst to study the resolution of（R,S）-ethyl indoline-2-carboxylate under different reaction conditions.The suitable resolution conditions were determined:0.05 g Bacillus aryabhattai B8W22 freeze-dried powder and 0.025 g（R,S）-ethyl indoline-2-carboxylate were added to the 1.5 mL reaction system,the cosolvent is n-hexane,and the addition amount is 20%（V/V）.The reaction is maintained at pH 7.0 and 35℃for 1.5 h,the e.e._p of product was 96%,and the conversion was 33%.The structure of the product was characterized.The results of NMR and mass spectrometry showed that the hydrolyzate conformed to the structural characteristics of（S）-indoline-2-carboxylic acid.This indicates that the process of preparing（S）-indoline-2-carboxylic acid by one-step resolution of the（R,S）-ethyl indoline-2-carboxylate is feasible.