Optical Resolution Of N-acetyl-DL-Methionine By Coupled Reator Filled With Immobilized Aminoacylase And Immobilized Aspergillus Oryzae

The coupled reator (BSTR and PBR) was used to optical resolution of N-Ac-DL-Methionine for the first time. BSTR was filled with immobilized Aspergillus oryzae pellets, while PBR was filled with immobilized aminoacylase. The fluid was first treated by immobilized Aspergillus oryzae pellets, and then the enzymatic deacylation was treated by immobilized aminoacylase with high rate of conversion.The immobilization of aminoacylase was performed via ionic adsorption onto several kinds of weakly basic macroporous anion resin. Diethylaminoethyl divinyl benzene (DEAE-D/H-Ⅲ) was selected as the best immobilization carrier, while the specific activity was up to 1356.41U/g, which was much higher than the others. The appropriate concentration of free enzyme solution in immobilization was investigated. The result showed that the optimal concentration of free enzyme solution was 115U/ml; the activity yield was up to 59.49%. When it was used for the optical resolution of N-acetyl-DL-methionine, the optimal pH, temperature and the concentration of Co2+ were 6.5, 65℃and 5×10-4mol/L. Moreover, the enzyme stability was significantly improved, the immobilized enzyme maintain 72.6% initial activity after 30 days of continuous operation. After enzyme inactivation, the protein could be fully desorbed from resin, and then the resin can be reused, the specific activity of aminoacylase immobilized on regenerative DEAE-D/H was up to 1380.64U/g. Therefore this had good industrial potentiality.Aspergillus oryzae was cultured in liquid culture medium, the effect factors referred to others. The activity of dried pellets is was up to 2039 U/g, while after the pellet immobilized by formalin and gelatin, the activity of it was 1708.68U/g, the yield of immobilization was 83.76%. When it was used for the optical resolution of N-acetyl-DL-methionine, the optimal pH, temperature and the concentration of Co2+ were 8.0, 65℃and 5×10-4mol/L.To determine the catalysis property of the enzyme reaction, experiments were carried out. There was substrate inhibition, competitive inhibition of L-Met for immobilized pellets, and there was substrate inhibition and non-production inhibition for immobilized enzyme. The concentration of substrate inhibition of immobilized pellets and immobilized enzyme were 0.2mol/L and 0.4mol/L. when the concentration...