Purification And Characterization Of Carbonyl Enantioselective Reductase From Morganella Morganii J-8

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 discovery the mechanism of the action catalyzed by enzyme, and to obtain information for improving the practical process, purification and characterization of the enzyme involved in the stereoinversion is very important.Morganella morganii J-8 which was screened in our laboratory produced d-pseudoephedrine from 1-phenyl-2-methylamine-acetone (MAK). Studies on the fermentation conditions, purification and characterization of carbonyl enantioselective reductase were carried out in this dissertation. The main reports were as following:After fermentation condition was optimized, the optimal fermentation medium was as follows: glucose 3 %, peptone 2.5 %, yeast 0.5 %, K2HPO4 0.3 %, MgSO4·7H2O 0.2 %,NaCl 0.2 %. The fermentation condition was: initial pH 7.5; seed fermentation period 24 hr; fermentation time 36 hr and temperature 30℃.The enzymatic characterization of crude enzyme showed that the enzyme is stable between pH 5 and 8, and the optimum pH is 7.5. The optimum temperature is 30℃and the enzyme is stable when the temperature is below 35℃.Cells were disintegrated by ultrasonic. Then the carbonyl enantioselective reductase was purified with a combination of ammonium precipitation, Phenyl Superose hydrophobic chromatography, DEAE anion exchange and PAGE electrophoresis. The subunit of purified enzyme was estimated to 42.5 kD on SDS-PAGE.The native molecular weight of the enzyme analyzed by HPLC gel filter chromatography was found to be 84.1 kD, what shows that the enzyme was a dimmer.The enzymatic characterization showed that the partially purified carbonyl enantioselective reductase activity was higher in the presence of NADH than NADPH as coenzyme.The maximal activity at 30℃and at optimal pH 6.5. The enzyme shows good stability below 35℃and between pH 6 and 8, but is inhibited by heavy metal ions, while activated by Mg²⁺ and Mn²⁺.The purified enzyme was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MADLI-TOF-MS). Retrieval showed what one had gained or acquired peptide section for one kind of leucine dehydrogenase.