Immobilization Of Lipase And Enzymatic Resolution To Prepare(R)-2-Tetrahydrofuroate Methyl Ester

Faropenem sodium is a wide-spectrum atypicalβ-lactam antibiotic that can be taken orally and injected.It is mainly used for various anti-infective treatments.It shows antibacterial activity against aerobic gram-positive bacteria,gram-negative bacteria and anaerobes.It has antibacterial activity,which is better than the existing cephalosporin oral antibiotics.Among them,(R)-2-tetrahydrofuroate methyl ester is a key intermediate for the synthesis of faropenem sodium.And lipase has substrate specificity and is a typical biocatalyst for the resolution of chiral compounds.It has the advantages of mild conditions and environmental friendliness.Therefore,it is of great significance to study the efficient and green biocatalytic synthesis of(R)-2-tetrahydrofuroate methyl ester.The main research contents of this paper are as follows:(1)The enantiomers of 2-tetrahydrofuroate methyl ester were resolved by HPLC.Through the chiral stationary phase and mobile phase screening experiments,the influence of the polar modifier ratio,flow rate,column temperature and other parameters on the separation was investigated,and the following optimal conditions were established:Chiralcel OD-H chiral column,the mobile phase is n-hexane:Isopropanol(90:10,V/V),the flow rate is 1.0 m L/min,temperature is 25°C,and the detection wavelength is 220 nm,the separation effect is best,the analysis time is within 10 minutes,and the enantiomers resolution was 3.33.Investigate the linear relationship,repeatability and stability of the experiment,the enantiomeric concentration of methyl tetrahydrofuroate has a good linear relationship within the range of 0.2～8 mg/m L,R~2 is 0.9991,and the detection limit of the S-configuration is 0.246 mg/m L and the detection limit of R-configuration is 0.183 mg/m L.It has good repeatability and stability.This method is fast,mild,and high-resolution,and provides a simple and efficient detection method for the synthesis and quality control of the enantiomer.(2)Lipase screening and enzymatic resolution to prepare(R)-2-tetrahydrofuroate methyl ester.By screening eight kinds of lipases stored in the laboratory,it is found that Lipase AS can be hydrolyzed to obtain the product(R)-2-tetrahydrofuroate methyl ester with high selectivity,and PPL can be hydrolyzed to obtain(S)-2-tetrahydrofuroate methyl ester with high selectivity,and the conditions for Lipase AS catalytic hydrolysis reaction were screened,and the following optimal catalytic conditions were obtained:substrate concentration was 100 mmol/L,temperature was 30°C,enzyme amount was 20 g/L,buffer p H was 7.0,and the buffer salt concentration was 200 mmol/L.Under the optimal conditions,the reaction was carried out for 12 h,the best extractant is n-hexane,the extraction temperature was 30℃,the extraction time was 30 min,and the extraction times reached 2times.yield was 42.8%and ee>99.9%.The method has mild conditions,high catalytic activity and high enantioselectivity,and is environmentally friendly and pollution-free.(3)Screening of immobilized carrier and optimization of immobilization conditions.11different types of resin carriers were screened based on protein adsorption rate and enzyme activity recovery rate,and it was determined that the anion exchange resin 1000-HA immobilized Lipase AS with the best effect.The optimal immobilization process conditions were determined as follows:the optimal protein load of the carrier was 56.5 mg/g,the immobilization time was 8 h,the buffer concentration was 500 mmol/L,the p H was 8.0,and the temperature was 30°C.The final specific enzyme activity of the immobilized enzyme was 75.4 U/g,and the recovery rate of enzyme activity reached 92.5%.At the same time,the immobilized enzyme AS@1000HA has been studied on the properties of acid-base and heat tolerance.The experimental results show that compared with the free enzyme,AS@1000HA exhibits a wider p H range,in the range of 6.5～8.The residual enzyme activity is higher than90.5%.AS@1000HA can still reach more than 80.3%enzyme activity at a temperature of45℃.The immobilized enzyme has better acid-base stability and thermal stability.(4)Preparation of(R)-2-tetrahydrofuroate methyl ester and optimization of conditions through immobilized enzyme-catalyzed resolution.The immobilized enzyme AS@1000HA was used to catalyze the preparation of(R)-2-tetrahydrofuroate methyl ester and the conditions were optimized.The optimal reaction conditions were determined as follows:temperature was 40℃,amount of immobilized enzyme was 0.4 g,buffer p H was 8.0,The rotation speed was 150 rpm.Under this reaction condition,the activity of the immobilized enzyme reaches the highest after 12 hours of reaction.yield was 38.8%and ee>99.9%..Finally,the storage stability of the immobilized enzyme and the stability of repeated batch operations were investigated.It was found that 94.3%of the residual enzyme activity was retained after 15 days of storage in the refrigerator at 4℃,and 80.2%of the original enzyme activity was maintained after repeated use for 10 times,Showing good storage stability and operational stability.