Purification And Characterization Of Alcohol Dehydrogenases From Candida Parapsilosis CCTCCM 203011

The importance of optically active compounds has been increasingly recognized in the pharmaceutical, food, fine chemistry, and agrochemical industry. For the preparation of biologically active compounds, chiral building blocks are valuable synthons, which can be obtained by resolution of racemic mixtures or by enantioselective conversions of prochiral compounds. To produce chiral alcohols, deracemization by microorganisms or enzymes is a convenient method compared to chemical methods. Optically active 1-phenyl-1, 2-ethanediol is a versatile group as chiral building block. To discovery the mechanism of the stereoinversion catalyzed by Candida parapsilosis CCTCCM203011, and to obtain information for improving the practical process, purification and characterization of the enzyme involved in the stereoinversion is very important. In this research, Candida parapsilosis CCTCCM203011 was chosen as raw material. The French pressure cell was effective and easy to handle for releasing the enzyme from the microorganisms, comparing with other methods to disrupt yeast cell. The enzyme was extracted with phosphate buffer ,one unit of enzyme activity was defined as the amount of enzyme catalyzing reduction 1μmol NADP+ per minute under the assay conditions. The purification procedure of alcohol dehydrogenase I involved alcohol precipitation and followed by ion-exchange chromatography on DEAE-sepharose, gel filter chromatography on Sephadex G-75 , and affinity chromatography on Blue sepharose Fast flow. A NADP+-dependent dehydrogenase was purified to homogeneity on SDS-PAGE , and the molecular mass of subunit is approximately 43KD.The alcohol dehydrogenase II co-purified from Candida parapsilosis CCTCCM203011 was purified to homogeneity by following three steps such as alcohol precipitation, affinity chromatography and gel filter chromatography. One unit of enzyme activity was defined as the amount of enzyme catalyzing oxydation 1μmol NADH per minute under the assay conditions. The subunit of enzyme was estimated to 40KD on SDS-PAGE. The native molecular weight of the two enzymes (alcohol dehydrogenase I and II) analyzed by HPLC gel filter chromatography were found to be 45KD and 40 KD respectively, what shows that the two enzymes were monomers. The use of Shiff reagent to dye the gel after electrophoresis, showed no colored band. The enzymatic characterization showed that alcohol dehydrogenase I catalyzed the oxidation of diol in the presence of NADP+, with the maximal activity at 45℃ and at optimal pH 8.0. Whereas alcohol dehydrogenase II showed its maximal activity at 50℃ and at optimal pH 6.0 with the use of hypnone as the substrate. On the other hand, the presence of Mg2+ showed to increase the enzyme activity. In the resolution reaction, the alcohol dehydrogenase I showed the high selectivity of (R)-1-phenyl-1, 2-ethanediol from racemate. However, the deracemization can not be completely catalyzed by a single purified enzyme. The use of two enzymes(alcohol dehydrogenase I and II ) in the resolution system, (S)- enantiomer was found in production, showing that the alcohol dehydrogenase II is another key enzyme in the deracemization procedure.