Abstract
(R)-N-(2,6-dimethylphenyl) alanine ((R)-MAP-acid) is an important chiral intermediate of the Fungicide (R)-Metalaxyl. In this study, ten kinds of immobilized resins(XAD1180N, H103, HAD7HP, D3520, NKA, D101 , DM11,850 JinKai, Primary amino resin and 850 synthetic resin) were used to adsorption-covalent crosslinked esterase PAE07 for splitting (R, S)-DMPM. The resin D3520 with porous structure and hydrophobic polystyrene was selected for immobilization as the carrier, after optimization of the immobilization conditions, the enzyme load is 20:1 (mg/g), the adsorption time is 4h, and the adsorption buffer pH is 7.0 . The Km and Vmax of the free esterases were 35.66 mM and 4.46 mM/mg·min, respectively, The Km and Vmax of the immobilized PAE07 were 19.05 mM and 2.84 mM/mg·min. The SEM analysis showed that the immobilized esterase PAE07 had higher thermal stability, pH stability and substrate specifity than those from the free esterase. Under the optimal conditions,the reaction was carried out at 35°C and 200 rpm for resolution of 350 mM substrate for 14 hours, the conversion rate reached 48%, and the e.e.p was 99.5%.The repeatability of immobilized esterase PAE07 was evaluated by continuous catalytic resolution of (R, S)-DMPM. The results showed that after 15 times of repeated use, 86.2% of the relative enzyme activity was retained. These results proved that immobilized esterase PAE07 as a new catalyst had great potential for the application and industrial enzymatic resolution of (R, S)-DMPM to prepare (R)-metalaxyl.
The effects of macromolecular crowding and surface charge on the properties of an immobilized enzyme: activity, thermal stability, catalytic efficiency and reusability
