Immobilization of (S)-mandelate dehydrogenase and its catalytic performance on stereoselective transformation of mandelic acid

•(R)-Mandelic acid was isolated from a chiral mixture by a stereoselective transformation.•(S)-Mandelate dehydrogenase catalyzes the stereoselective transformation.•FMN was regenerated continuously by low-cost ferricyanide.•The stability of immobilized (S)-mandelate dehydrogenase was improved.•The purity of (R)-mandelic acid as a production exceeded 99%.

(S)-Mandelate dehydrogenase (SMDH) is a FMN-dependent enzyme and catalyzes the oxidation of (S)-mandelic acid to benzoylformic acid, resulting in the reduction of FMN. SMDH was immobilized on chitosan beads through glutaraldehyde by Schiff base linkage, and its immobilization yield was 78% and activity was 12.8 U/g. Some characteristics of immobilized SMDH were evaluated. It was found that its optimal pH was 3.4 and optimal temperature was 45 °C. The Km value of immobilized SMDH for racemic mandelic acid was 0.27 mM and that for ferricyanide was 0.75 mM. Compared with immobilized SMDH, the Km values of soluble SMDH for racemic mandelic acid (0.92 mM) and ferricyanide (3.8 mM) were higher. The Vmax was 0.49 μmol/min. The immobilized SMDH showed some improvement on thermal stability and storage stability, with a 5-fold reduction of the deactivation rate for 45 °C heat treatment and that for 4 °C storage was about 1.4-fold. The stereoselective biotransformation of mandelic acid was studied using immobilized SMDH, with the cofactor FMN regenerated by low-cost ferricyanide. The ee value of (R)-mandelic acid in the final reaction mixture was above 99%.