( R )-N-(2,6-dimethylphenyl) aminopropionic acid methyl ester (( R )-DMPM) is an important chiral intermediate of the fungicide N-(2,6-Dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester (( R )-Metalaxyl). In this study, (1) D3520 (macroporous acrylic anion resin), selected from the ten resins, was used to immobilize the esterase from Pseudochrobactrum asaccharolyticum WZZ003 (PAE07) for resoluting the ( R,S )-DMPM to obtain ( R )-DMPM. (2) Up to 20 g/L PAE07 could be immobilized onto D3520 with a high enzymatic activity of 32.4 U/g. Moreover, the Km and Vmax values of 19.1 mM and 2.8 mM/min for D3520-immobilized PAE07 indicated its high activity and stereoselectivity. (3) The optimal temperature and pH for the immobilized PAE07 were 40 ℃ and 8.0, and substrate concentration was up to 0.35 M. After 15 h reaction, the conversion rate from ( R,S )-DMPM to ( R )-DMPM was 48.0% and the e.e.p and E values were 99.5% and 1393.0, respectively. In scale-up resolution, 200 g/L substrate and 12.5 g immobilized esterase PAE07 condition, a conversion rate from substrate to product of 48.1% and a product e.e.p of 98% were obtained within 12 h, with the activity of immobilized PAE07 retained 80.2% after 5 cycles of reactions. These results indicated that the D3520-immobilized esterase PAE07 had great potential for enzymatic resolution of ( R,S )-DMPM to prepare ( R )-Metalaxyl.
Feng Cheng,Feifei Cheng,Jianyong Zheng,Guanzhong Wu,Yinjun Zhang,Zhao Wang
Only a few esterases have been used for the synthesis of optically pure fungicide. For example, ( R )-metalaxyl synthesized using esterase-involved bioreaction displays fungicide activity, whereas ( S )-enantiomer is redundant. However, the biosynthesis of ( R )-metalaxyl is currently hampered by the lower activity, selectivity and thermostability of esterase. Therefore, to obtain a better biocatalyst, several esterase genes were cloned from Pseudochrobactrum asaccharolyticum WZZ003. The esterase PAE07, among eight enzymes, was selected because it exhibited the highest hydrolysis activity toward ( R , S )-DMPM. The DNA and amino acid sequence analysis suggested that PAE07 is a new member of lipolytic enzyme family V. The enzymatic properties of PAE07 toward ( R , S )-DMPM and model substrate ( p- nitrophenyl acetate) were investigated. PAE07 was found to be a highly active esterase with excellent enantioselectivity . The reaction conditions including temperatureand pH were optimized, and the effects of metal ions, organic solvents and detergents were also investigated. Results indicated that PAE07 is a competitive candidate for ( R )-metalaxyl manufacturing.
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