Based On Medium Engineering For Enzymatic Prepration Of Cilastatin Key Chiral Intermediate

Tienam(Cilastatin and Imipenem),is widely accepted as a clinical drug of first choice for mixed infection and found to have a broad spectrum of antimicrobial activity.S-(+)-2,2-dimethylcyclopropa-necarboxylic acid(DMCPA)is an important chiral building block for the production of Cilastatin.Nowadays,biocatalytic hydrolysis of ethyl-2,2-dimethylcyclopropanecarboxylate(DMCPE)to DMCPA has attracted more attentions due to its high stereoselectivity,mild reaction condition and environmental friendliness.It is found however,like most lipase-catalyzed processes,the detachment of lipase from binding site and poor water-soluble of DMCPE are adverse to enzyme activity and hindered mass transfer of substrate and product,thus limiting the catalytic efficiency.In order to improve the solubility of the substrate/product and the stability of the enzyme,various reaction systems are established,and their effects on the hydrolytic reaction are also investigated in subsequent experiments.Firstly,effects of five kinds of hydrophilic organic solvents and five kinds of hydrophobic organic solvents on the biohydrolysis are investigated.The results demonstrated that N,N-Dimethylformamide(DMF)can increase the solubility of substrate and is employed as the best organic solvent.Furthermore,some key reaction conditions are optimized,and the optimal reaction conditions are as follows:40 g/L DMCPE,16 g/L Novozyme 435,5%DMF,1 mol/L phosphate buffer,pH 7.2,reaction at 30? for 144 h.Under above conditions,40.2%of yield and 99.0%of enantiomeric excess(ee)are obtained.The yield is increased from 33.4%to 40.2%in contast to that reacted in the momophasic aqueous system and the substrate solubility is increased from 113.3 mg/L to 366.3 mg/L.Secondly,effects of twenty-four kinds of hydrophilic ionic liquids and twelve kinds of hydrophobic ionic liquids are examined on the biocatalytic separation of DMCPE.The results showed that the highest catalytic efficiency was obtained in the hydrophilic ionic liquid[BMIM][OTF]-containing system.After optimization,the optimum reaction conditions are found to be as follows:60 g/L DMCPE,16 g/L Novozyme 435,7%[BMIM][OTF],1 mol/L phosphate buffer,pH 7.2,reaction at 30? for 144 h.Under above conditions,the maximum yield of 42.8%and 99.2%of enantiomeric excess of product are obtained.Compared with the monophasic aqueous system,the yield is increased from 31.4%to 42.8%in[BMIM][OTF]-containing system and the substrate solubility is increased from 113.3 mg/L to 371.5 mg/L.Thirdly,to understand the effects of the mixed system containing surfactant and ionic liquid on the reaction,[BMIM][OTF]associated with eight kinds of surfactant(three type:anionic,cationic,non-ionic)are studied.The results showed that bis(2-ethylhexyl)sulfosuccinate sodium salt(AOT)exert positive effect,and the optimum loading proportion was 3%(w/v)AOT and 4%(v/v)[BMIM][OTF].The optimum reaction conditions for the preparation of S-(+)-DMCPA in the mixed medium are found to be as follows:60 g/L DMCPE,16 g/L Novozyme 435,1 mol/L phosphate buffer,pH 7.2,reaction at 30? for 120 h.Under above optimal conditions,a best yield of 45.1%and 99.2%of product ee are obtained.The yield is increased from 31.4%to 45.1%in contast to that reacted in the momophasic aqueous system,and the reaction time is also shortened by 24 h,the substrate solubility is increased from 113.3 mg/L to 490.9 mg/L.Finally,several media are screened out based on the solubility of substrate,the structure of immobilized enzyme and reuse times of enzyme.[BMIM][OTF]associated with AOT is selected as the optimal reaction system,a best yield of 43.2%and 99.1%of ee value are obtained within 120 h in 5L fermenter.The catalytic efficiency achieved in fermenter was the same as that reacted in shake flasks,with remarkable success.It provides a foundation for industrial production.