Resolution Of Racemic Metalaxyl By Biocatalyst And Production Of R-metalaxyl

Metalaxyl is a chiral phenylamide fungicide widely used in the control of plant diseases caused by pathogens of fungus in several crops.It has two enantiomers,R-?-?and S-?+?,but the fungicidal activity mostly comes from the former.In this paper,an microorganism containing metalaxyl enantioselective hydrolyzing esterase was screened from soil samples.The esterase was purified and characterized.Using the microorganism cells as resolution catalyst,A new process for producing R-metalaxyl was designed.The strain with the enantioselective esterase activity was isolated from soils and identified as Albibacters by physiological and biochemical identification and 16S rDNA sequence analysis.It was named Albibacters sp.zjut528.The cells were disrupted by ultrasound and the intracellular container was treated by ammonium sulfate precipitation,followed by ion-exchange chromatography and gel filtration chromatography.Finally,a SDS-PAGE electrophoresis–pure esterase protein was obtained.It was a monomeric protein with a molecular mass of about 40 kDa.Its activity recovery rate achieved 10.78%and its fold purification was 9.52.The N-terminal amino acid sequence of the protein was NH₂-Ala-Ala-Lys-Ala-Pro-Leu-Arg-Leu-Lys-Glu.The optimum temperature and pH for the esterase were about 40?and pH 9.0.The enzyme was stable at 20⁴0?and kept more than90%of its initial activity after 4 h incubation at these temperatures.It was stable at pH 5.0¹0.0 and remained more than 90%of its initial activity after incubation for 48h?at 4??at these pHs.2 mM of Fe²⁺,Mn²⁺,Zn²⁺slightly improved its activity,but Fe³⁺slightly inhibited it.The activity of the esterase was improved when some organic solvents was added into its solution?20%w/v?,such as acetonitrile,isopropanol,acetone,isopropyl ether,cyclohexane and n-hexane.The Michaelis-Menten kinetics parameters for the esterase was:V_m=0.05g/?L·min?,K_m=0.64 g/L,K_cat=0.85 min^-1.A process was designed to produce R-matalaxyl.First,the racemic Matalaxyl was hydrolyzed to produce R-acid and S-metalaxyl by the cells of Albibacters sp.zjut528.The substrate concentration was 5%?m/v?.The cell concentration was 1.5%?m/v?.20%of isopropanol was added as cosolvent.The conversion rate of the substrate was 47.1%after 28 h.The ee_p of R-acid was more than 99%.The R-acid and the unreacted S-matalaxyl was separated.Next,R-acid was esterized with methanol to synthesize R-metalaxyl with the yield of 80.1%and ee_p of 99.4%.S-metalaxyl?ee=99.4%?was racemized by heating and ee was reduced to 4.2%after 7 h and the conversion rate reached 47.9%.