Asymmetry Production Of (R)-1,3-butanediol By Recombinant Carbonyl Reductase

(R)-1,3-butanediol(BDO)is an optically alcohol which can be used as an intermediate for the production of pharmaceuticals such as azetidinone derivatives.As reported,the chemical methodology has many disadvantages,such as:poor stereoselectivity and serious environmental pollution.So,more and more attention was focused on efficient production of(R)-1,3-BDO.There was one effective way of synthesis of(R)-1,3-BDO by asymmetric reduction pro-chiral 4-Hydroxy-2-butanone(4H2B)to produce optically active(R)-1,3-BDO.In this study,we focued on the construction of recombinant E.coli expressed carbonyl reductase to asymmetric reduction of 4-Hydroxy-2-butanone to produce(R)-1,3-BDO.The gene encoding carbonyl reductase(CR)was cloned from genomic DNA of Leifsonia aquatica(ZJB-09230).The gene contains an open reading frame consisting of 756 nucleotides corresponding to 251 amino acid residues.The discrimination of protein secondary structure showed that the CR has 7 ?-helixes and 7 ?-sheets,the subunit molecular weight and the isoelectric point was calculated to be 25 KDa and 4.58.Based on the cloning of cr coding sequence,the recombinant plasmid(pET-28b-cr)was constructed,and then transformed into E.coli BL21(DE3).Recombinant protein with the molecular mass was about 25 kDa determined by polyacrylamide gel electrophoresis.The transformation product was analyzed by GC-MS and chial GC,the results indicated that the product was(R)-1,3-BDO with e.e.99.9%.In this study,the medium components and cultivation conditions of recombinant E.coli BL21(DE3)/pET-28b-cr were optimized.The results showed that the optimal medium components were as follows(g/L):glycerol 10.0,yeast extract 6.0,peptone 7.0,K2HPO4 2.0,MgSO4 2.0,NaCl 1.0,pH 7.0.The induction conditions were as follows:induction concentration of lactose is 7.5 g/L,induced temperature was 28?,induced time was 12 h,with enzyme activity 891.9 U/L,increasing 146.7%compared with original cultivation conditions.We also studied the properties of CR.It shows that the optimal pH is 6.5,and is stable at pH 6.0?8.0.The optimal temperature is 35 ?,and is stable at 40?at most.CR prefers NADH to NADPH as cofactor.The maximum reaction rate(Vmax)and apparent Michaelis-Menten constant(Km)for 4H2B and NADH were calculated to be 1.74 ?mol/min·mg and 1.16 mmol/L,4.87?mol/min·mg and 2.96 mmol/L,respectively.The CR showed catalyze activity to a number of short-chain ketones and aldehydes,furthermore,the relactive activity of 2,3-butanedione and 2-hexanone reached 2262.4%,2056.8%,respectively.The CR activity could be activated by Ca2+,and strongly inhibited by Cu2+ and Ag+.DTT and PMSF pretend CR from oxidating.The CR also can stand high concertration of methanol,ethanol and dimethylamine.Simultaneously,the asymmetric reduction of 4H2B by resting cells was optimized.When the assay mixture contained 0.2 g/mL E.coli BL21(DE3)/pET-28b-cr,0.1 M of potassium phosphate buffer(pH 8.0),1.0 mM of NAD+,15%of isopropanol(v/v),0.5 M of 4H2B,and the reaction was monitored at 40 ?,CR showed the highest activity.After 24 hour-reaction under the above conditions,the convertion of 4H2B and the e.e.value,reached 77.1%,99.9%,respectively.