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Synthesis Of Optically Active α-hydroxy Acid By Yeast Mediated Bioreduction And Properties Of The Dehydrogenase

Posted on:2011-10-28Degree:DoctorType:Dissertation
Country:ChinaCandidate:J L GuoFull Text:PDF
GTID:1101330332971154Subject:Fermentation engineering
Abstract/Summary:PDF Full Text Request
Biocatalysis has been an important route for the asymmetric synthesis of chiral compounds due to its remarkable enantio-, regio- and chemoselectivity. Optically active alpha-hydroxy acids are key intermediates of pharmaceuticals and fine chemicals. (R)-Mandelic acids are the most important representatives from the commercial point of view, which are not only the essential building blocks for drugs such as semi-synthetic penicillins, cephalosporins, antitumor and antiobesity agents but also the versatile resolving agents in chiral resolution processes. Special attention has been paid for the production of these compounds. Biocatalytic asymmetric reduction is an attractive alternative because of the high theoretical yield and the convenient operation conditions. However, weaknesses frequently met are the poor stability of biocatalysts, permeability issues of the whole cells and the low product concentration. In this study, (R)-mandelic acid was chosen as the model of alpha-hydroxy acids. The screening of new biocatalysts with desired properties, the systematically study of the process factors affecting the asymmetric reduction, the modification of the substrate, the application of in situ product removal technique and the purification of the key dehydrogenase were studied herein. The main results were shown as follows:(1) Benzoylformic acid was chosen as the model of alpha-keto acid. By gradually increased substrate concentration in successive batch mode, Saccharomyces ellipsoideus GIM 2.105 was selected as an effective biocatalyst with remarkable operational stability. After twenty cycles of reuse or storage for five weeks at 4 oC, whole-cells of S. ellipsoideus maintained their activity and no obvious decrease in conversion as well as the enantiomeric excess (ee) were observed. In addition, four substituted aromatic (R)-alpha-hydroxy acids were prepared in high ee (95– >99%) and good conversion (>90%).(2) The D-mandelate dehydrogenase from S. ellipsoideus was purified by 46-fold to homogeneity through ammonium sulphate precipitation, Phenyl-Sepharose FF and Superdex 75 gel filtration. The subunit size was about 44 kDa estimated by SDS-PAGE and it required NADPH as a cofactor, which were different from other D-mandelate dehydrogenases. Its optimal reaction pH and temperature were pH 5.5 and 30 oC. The enzyme was stable in the pH range of pH 5.5– pH 8.0 and the temperature below 30 oC.(3) Effect of various reaction parameters on the asymmetric reduction with S. ellipsoideus was studied. The optimal conditions were determined as follows: limited oxygen supply, pH 7.5, 35 oC and 10 g/L glucose as the cosubstrate. Under the optimal conditions, yield and ee of the product attained to above 90% and 99% after 48 h. By determining the activity of intracellular mandelate dehydrogenase, studying the effect of cosubstrate and permeability issue of the whole cells, it was determined to be the permeability issue of the cell membrane to the substrate/product leading to the long reaction time. It could be shorten sharply by the modification of the substrate.(4) Methyl benzoylformate was chosen as the model substrate of alpha-keto ester to shorten the reaction time. It was found that the decomposition of the substrate and the product was the key reaction-specific constraints (constraints dependent on the nature of the substrate and product) that limited the efficiency of this reaction. Under the preliminary selected reaction parameters that minimize these constraints, an effective whole cell biocatalyst (S. cerevisiae AS2.1392) was obtained for the asymmetric reduction of methyl benzoylformate. Under further optimized conditions, the yield and ee of the product attained to 92.4% and 95% within 1.5 h.(5) The inhibition effect of substrate and product on the asymmetric reduction was evaluated by determining the initial reaction rate, and adsorbent resins were applied to alleviate the substrate/product inhibition. Due to the high adsorbent capacity and the promoting effect on the reaction, resin NKA-II was selected as the adsorbent material. Under the optimum resin amount and the proper adding mode, the product concentration achieved to 128.2 mmol/L.
Keywords/Search Tags:biocatalyst, asymmetric reduction, alpha-hydroxy acid, (R)-mandelic acid, permeability issue, substrate modification, in situ product removal, D-mandelate dehydrogenase
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