Discovery And Application Of Novel D-Mandelate Dehydrogenase In Multienzymatic Synthesis Of L-Phenylglycine

Z-phenylglycine and its derivatives are an important class of chiral unnatural amino acids, which can be used as chiral building blocks for pharmaceuticals, chemicals and pesticides. The most important application was found in the antibiotics and anti-platelet drugs. Due to the higher environmental friendliness, catalytic efficiency and excellent regio-and enantioselecitivity, biosynthesis of L-phenylglycine becomes a promising strategy among all the synthesis routes.In this study, using the genome database mining approach, five NAD+dependent D-mandelate dehydrogenases (DMDHs) were discovered from protein database. Through systematic screening, a DMDH from Lactobacillus brevis(LbDMDH) was identified with the best catalytic performance in the asymmetric oxidation of D-mandelic acid. The LbDMDH was purified to homogeneity and characterized. The optimum reduction activity was observed at pH7.0, while the optimum dehydrogenation activity was observed at pH10.0. The highest activity was found around40℃, which indicated that the LbDMDH belonged to mesophilic enzymes. LbDMDH was relative stabile at30℃. When the temperature was higher than40℃. the enzyme was easily inactivated. The Km value of LbDMDH on D-mandelic acid was1.14±0.12mM, kcal was355±11s-1, Vmax was574±18μmol min-1·mg-1·protein, and the Km value for benzoylformate was0.18±0.04mM, correspondingkcat was2262±142s-1, Vmax was3659±230μmol·min-1·mg-1·protein.Using the LbDMDH, mandelic acid racemase (MRM, Pseudomonas putida ATCC12336) and leucine dehydrogenase (EsLeuDH, Exiguobacterium sibiricum), a three-enzyme-cascade reaction system was constructed to produce chiral L-phenylglycine from rac-mandelic acid. After optimization on ratio of three enzymes, substrate/catalyst ratio, cofactor amount and agitation speed, as much as30g/L rac-mandelic acid could be converted into L-phenylglycine, which was5times higher than the highest record. At1-L scale, containing5kU/L MRM,10kU/L LbDMDH and10kU/L EsLeuDH,3mM MgCl2and0.1mM NAD+,200mM rac-mandelic acid can be substantially converted into L-phenylglycine (>94%analytical yield) within10hours. This works provides a much more efficient strategy for the preparation of L-phenylglycine with internal cofactor recycling, no intermediates accumulating, and the eliminating the need for separation and extraction steps of intermediates.