Production of the Apoptotic Cellular Mediator 4-Methylthio-2-oxobutyric Acid by Using an Enzymatic Stirred Tank Reactor with in Situ Product Removal

摘要

D-Amino acid oxidase(DAAO)was used to study the oxidative deamination of racemic mixtures of D,L-methionine in its soluble and immobilized forms and thus obtain the corresponding a-keto acid.The soluble enzyme form was obtained from a Trigonopsis variabilis CBS 4095 extract,free of L-amino acid oxidase,and was co-immobilized with a 200-fold excess of catalase to avoid the undesirable side reaction of H2O2 with the a-keto acid,which would otherwise render its corresponding decarboxylated acid,the 3-methylthiopropionic acid(MTPA).With this biocatalyst,quantitative conversion(>98%)of D-methionine into the a-keto acid 4-methylthio-2-oxobutyric acid(MTOB)and into MTPA was achieved using 5 mg·mL~(-1)of biocatalyst at pH 8.0,25 °C,and pure oxygen at 3 vvm.A stirred tank reactor with in situ product removal(STR-ISPR)was developed to avoid conversion of MTOB into MTPA.The reaction medium was re-circulated through a strong anion exchange column(Amberlite IRA-400).This resulted in the complete removal of MTOB from the reaction medium.After the reaction,the reaction products were eluted sequentially with water(L-methionine),10 mM HC1(MTPA),and 0.5 M HC1(MTOB).After elution,MTOB was crystallized to its sodium salt.