A Bi-enzymatic System Catalyzing Deracemization Of Mandelic Acid

(R)-Mandelic acid is an important drug intermediate, and can be obtained through deracemization of racemic mandelic acid. FMN-dependent (S)-mandelate dehydrogenase (SMDH) can catalyze the oxidation of (S)-mandelic acid to benzoylformic acid, resulting in the reduction of FMN. FMN can be regenerated by potassium ferricyanide in vitro. Laccase can catalyze the conversion of ferrocyanide to ferricyanide meanwhile oxygen is reduced to water. SMDH and laccase are coupled by potassium ferricyanide and continuously catalyze deracemization of mandelic acid to produce (R)-mandelic acid.The SMDH and laccase are immobilized on chitosan beads prepared with glutaraldehyde as the crosslinking solution through Schiff base linkage. The pH and temperature optimum of free SMDH are found to be pH6.4and30℃, and the pH and temperature optimum of immobilized SMDH are pH3.4and45℃. The Km values of free and immobilized SMDH for mandelic acid are0.92mM and0.27mM respectively, and those for potassium ferricyanide are3.87mM and0.77mM respectively. The Km values of immobilized SMDH are lower than those of the free enzyme, which indicates that the immobilized SMDH has an apparent higher affinity for its substrate than the free enzyme does. The pH and temperature optimum of free laccase are pH3.0and30℃, and those of immobilized laccase are pH6.0and55℃. The Km values of free and immobilized laccase for potassium ferrocyanide are0.12mM and0.99mM respectively. The Km value of immobilized laccase is higher than that of free laccase, which indicates that the immobilized laccase has an apparent lower affinity for its substrate than free laccase dose. It is observed that thermal and storage stabilities of SMDH and laccase are increased after immobilization.The free and immobilized bi-enzymatic system coupling SMDH and laccase are used to catalyze deracemization of mandelic acid. Compared with the free bi-enzymatic system, it is found that the substrate concentration of the immobilized bi-enzymatic system increases2fold and the production efficiency increases about14fold. In addition, the enantiomeric purity of the bi-enzymatically produced (R)-mandelic acid is detected by chiral HPLC and exceeds99%.