Enzymatic Synthesis Of Ethyl(R)-4-cyano-3-hydroxybutyrate

Ethyl(R)-4-cyano-3-hydroxybutanoate(HN)is an important chiral synthon for side chain of the cholesterol-lowering drug atorvastatin.HN is also used as a synthon in the production of L-carnitine,and(R)-4-amino-3-hydroxybutanoic acid.The synthetic strategies of optically active HN are divided into chemical synthesis utilizing epichlorohydrin or L-malic acid as substrate,and biosynthesis utilizing carbonyl reductase,halohydrin dehalogenase,nitrilase and lipase as catalysts.Biosynthesis of HN has received extensive attentions due to its mild conditions,environmentally friendly feature,high stereo-selectivity and less reaction steps,which are consistent with the requirements of "Green Chemistry".This paper was focused on the biotransformation of ethyl-4-chloroacetoacetate(COBE)to HN via ethyl(S)-4-chloro-3-hydroxybutanoate((S)-CHBE)as intermediate.It investigated in terms of the screening,preparation,application of biocatalysts and separation of the product in details.The bioconversion process of COBE to(S)-CHBE was studied firstly.A strain ZJB09244 producing carbonyl reductase(CRD)which had high activity and stereo-selectivity toward COBE,and a strain ZJB09251 producing glucose dehydrogenase(GDH)which was used in the cofactor regeneration,were isolated from laboratory's culture collection.The pH-and thermal-stability of two crude enzymes were investigated.The CRD was stable under neutral or alkaline conditions,while GDH was stable under neutral conditions.The half-lives of CRD and GDH at 30,40 and 50? were 40.3,13.9,8 h,and 14.4 h,4.4 h,0.08 h,respectively.The influences of reaction conditions of biotransformation were also evaluated.According to the modes of addition of the substrate,the optimal conditions were different.In single-batch mode,the concentration of COBE,glucose and NADP+ were 100,120 mM and 25 ?mol/L,respectively.The amount of CRD and GDH were both 3 g/L.The optimal temperature was 35 ?.The pH was kept at 6.3 using 2 M K2HPO4 as titrating agent.Under these conditions,(S)-CHBE with a yield of 98%was obtained after a 30 min reaction.In fed-batch mode,the initial concentration of COBE was 100 mM,and 33.3 mM of COBE was added into the reaction mixture at 30,40,60 and 90min,respectively.The temperature and pH were 35? and 6.3,respectively.Under these conditions,(S)-CHBE with a yield of 92.1%and 99%e.e.was obtained.COBE showed poor water solubility and strong inhibitory effect on the enzyme activity.In order to resolve these problems,the water-organic solvent biphasic system was constructed.Butyl acetate was selected as the organic phase for the asymmetric reduction of COBE to(S)-CHBE.The reaction conditions were optimized.(a)In single-batch mode,the optimum conditions were as follows:volumetric phase ratio 1:1,shaking speed 250 rpm,COBE 500 mM,Glucose 600 mM,CRD 9 g/L,GDH 9 g/L,NADP+25 ?mol/L,35 ?,pH 6.3.Under these conditions,(S)-CHBE in ee>99%was synthesized with a yield of 95%.(b)In fed-batch mode,volumetric phase ratio and shaking speed were 1:1 and 250 rpm,respectively.The initial concentration of COBE was 300 mM,and 100 mM of COBE was added into the reaction mixture at 30,40,60,90,120,150 and 180min,respectively.The initial amount of CRD was 9 g/L,and 3 g/L CRD was added into the reaction solution at 40 and 90 min.The amount of GDH was 3 times the amount of CRD.The temperature and pH were 35? and 6.3,respectively.Under these conditions,the yield of(S)-CHBE reached 95.3%.After extraction and vacuum distillation,309.6 g product(yield of 92.9%)was obtained from 2 L reaction mixture.On the basis of the successful synthesis of(S)-CHBE,the enzymatic synthesis of HN was investigated.A strain ZJB09256 producing halohydrin dehalogenases(HHDH)was selected to catalyze the conversion of(S)-CHBE to HN.The enzyme was found to have good pH stability under neutral or slightly acidic conditions and thermal stability.The half-lives of HHDH at 30,40,50,60 and 70 ? were 57.7,23.1,12.8 h,9.6 h and 0.24 h,respectively.The reaction conditions were also studied and the optimum reaction conditions were obtained as follows:(S)-CHBE 100 g/L,resting cells 100 mg/gsubstrate(10 g/L),45 ?.The pH was adjusted to 7.7 by adding 30%NaCN during reaction.Under these conditions,the yield of HN reached above 85%.Further,the extraction process of HN was carried out.Cells were removed by filtration after flocculation using CaCl2 as flocculant.The flocculation conditions were set as follows:CaCl2 3 g/L,neutral pH,30-40?,rapid mixing(120 rpm)1 min,slow stirring(80 rpm)5 min and stand for 30 min.Then the samples with clarity of 97%were obtained by vacuum filtration.By using ethyl acetate as extractant,HN was extracted from the reaction solution.The extraction process was optimized.pH was adjusted to 7.0 or less.The volume ratio of ethyl acetate and reaction solution was 0.75.The mixture was stirred for 10 min at 30 ?,and then the organic phase was separated.The total extraction rate reached 94%after extraction for three times.By the optimal flocculation and extraction operation,62 g product(yield of 82.1%)was obtained from 800 mL reaction mixture.One-pot three-enzyme synthesis of HN was preliminarily studied.The control parameters were tuned as follows:The carbonyl reduction reaction with CRD and GDH was took place firstly under the condition of pH 6.3 and 35 ?.When the substrate COBE was converted completely,the reaction temperature was increased to 45 ?,pH was adjusted to 7.7 by 30%NaCN,and then HHDH was added into the reaction solution.It was found that the HHDH activities were inhibited by COBE.When the concentrations of COBE were less than 1 mM,the enzyme activity was rapidly decreased.HHDH was completely inactivated in the presence of 5 mM of COBE.Therefore,complete conversion of COBE was the key factor to achieve the one-pot synthesis.Fed-batch mode was used to increase the substrate concentration,in which the final concentration of COBE was increased to 200 mM,and at the end of the reaction,the yield of HN reached 77.9%.The effects of different organic solvents to the HHDH activities were also investigated.Dimethyl phthalate was selected to construct a two-phase system for the one-pot three-enzyme reaction.In the two-phase system,165 mM of HN was obtained from 300 mM of COBE.