Preparation Of (R)-Mandelic Acid With Microorganism Whole-cell As Biocatalyst

Optical mandelic acid is a versatile chiral synthon used for the synthesis of various pharmaceuticals. The applications of optical mandelic acid have grown markedly in recent years and are likely to expand in the coming years. Therefore, the studies on the preparation methods of optically active mandelic acid and screening of biocatalysts are very important both for the knowledge on biocatalytic mechanism and its application.In this paper, two different methods to prepare (R)-mandelic acid were studied by whole-cell bioconversion with racemic mandelic acids and benzoylformic acid as substrates respectively. Among various microorganisms including bacteria, yeasts and molds stored in the lab, two strains were selected. Brevibacterium sp. was able to convert the racemates to (R)-mandelic acid and Saccharomyces ellipsoideus could reduce benzoylformic acid to (R)-mandelic acid, and the bioconversion results were 97.5% enantiomeric excess(e.e.), 46.3% yield and 99.3%e.e., 74.2% yield, respectively.The conversion path by Brevibacterium sp. was investigated, and the process of bioconversion showed that the microorganism asymmetricly degraded (S)-mandelic acid from the racemates to energy source while (R)-mandelic acid was maintained. After the optimization of reaction conditions, the product was obtained with 99% e.e. and 48.7% yield. When some hydroxyl acids or metabolic intermediates were supplied to the medium, the whole-cell catalysis activity of Brevibacterium sp. was increased clearly, moreover, cell biomass and conversion efficiency were also promoted. Some other nutriments such as carbon source, nitrogen source and metal ions did not affect the conversion.Using the rac-mandelic acid as the sole carbon source in the medium, Brevibacterium sp. could also grow with the (S)-mandelate degraded and (R)-mandelate left. Even when rac-mandelic acid was added to the broth incubating for 24 h, the strain could also convert the substrate to (R)-mandelate. Trough above two ways, the e.e. value both reached 99% finally. The cell of Brevibacterium sp. could repeatedly be employed three times to perform efficiently, and fed-batch addition of the substrate to reaction system for three times, the final concentration of (R)-mandelic acid could cumulate to three folds.After the effect of culture condition on growth and conversion of Saccharomyces ellipsoideus was investigated, the optimal conditions were decided, i.e. pH 7.0, 30℃, incubating 24 h and flask volume 50 mL. The microorganism could reduce benzoylformic acid to (R)-mandelic acid with 99%e.e. and 95% yield in optimal reaction conditions. Operational stability of S. ellipsoideus whole cell catalyzing the reducation reaction was investigated, an exciting result was seen that the yield of product kept 80% and e.e. value maintained 98% after continuous reaction for 20 times, 960 h.Effect of metal ions in the reaction system on bioreduction catalyzed by S. ellipsoideus was studied. The result showed that on the one hand some ions decreased the conversion e.g. Zn2+, Cu2+,...