Production of the apoptotic cellular mediator 4-methylthio-2-oxobutyric acid by using an enzymatic stirred tank reactor with in situ product removal

Abstract

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 α-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 α-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 α-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 HCl (MTPA), and 0.5 M HCl (MTOB). After elution, MTOB was crystallized to its sodium salt. © 2008 American Chemical Society and American Institute of Chemical Engineers.