Key Enzyme In Glycerol Metabolism Separation, Purification And Characterization Of,

This paper presents a research on the production, isolation and characterization of the key enzymes (glycerol dehydrogenase, 1,3-propanediol oxidoreductase and glycerol dehydrate) for glycerol fermentation by Klebsiellapneumoniae.To obtain these intracellular enzymes ultrasonic disintegrator is used for cell disruption. The effect of the ultrasonic time and amplitude on enzyme activity is studied. The optimum pH and temperature for enzyme assays were determined. Thus sensitive and reliable enzyme assay methods are established which can facilitate the following purification procedure.Several carbon sources, organic nutrients, nitrogen sources and salts were examined for their effect on enzyme formation. Appling uniform design, combined with artificial neural network and genetic algorithm, the optimum medium ingredients are (g/L): glycerol 30, KC1 1.6, NH4C1 6.7, CaCl2 0.28, yeast extract 2.8. The optimum pH of the medium, cultivation temperature, inoculate ratio, rotate speed and harvest time are investigated under above condition. The maximal activity is attained at pH 7.0 ,37 C, 200 rpm and 5 % inoculate ratio. The activity level of glycerol dehydrogenase, 1,3-propanediol oxidoreductase and glycerol dehydratase are 2.24, 1.59 and 1.50 times higher respectively compared to conditions before optimization.Glycerol dehydrogenase and 1,3-propanediol oxidoreductase are separated and purified by ion exchange chromatography on Q Sepharose Fast Flow and affinity chromatography on Blue Sepharose CL-6B. The purification of glycerol dehydrogenase is 12 fold, the specific activity and recovery are 49U/mg and 66 % respectively. The specific activity of 1,3-propanediol oxidoreductase is increased by 5 fold to 31 U/mg, with the recovery of 12 %.Kinetic studies on the two isolated enzymes indicated that Km of glycerol dehydrogenase's is 0.13±10-3 M for NAD and 0.79±10-3 M for glycerol. As to 1,3-propanediol oxidoreductase, the Km value for NAD and 1,3-propanediol are 0.15 ±10-3 M and 0.97±10-3 M respectively. Furthermore, other properties of the two separated enzymes such as substrate specificity, cation effect, optimum temperature and pH are characterized. These works can contribute to establish an enzyme catalyze system for 1, 3-propanediol production.