Enzymze Reaction Kinetic Of 1,3-Propanediol Xidoreductase

1,3-propanediol oxidoreductase was an important enzyme in the biosynthesis of 1,3-propanediol. 1,3-propanediol oxidoreductase catalyzed the reaction of 3-hydroxypropionaldehyde convert to 1,3-popanediol, which was a reversible reaction with double substrates and double products.1,3-propanediol oxidoreductase was purified from the lysate of a constructed E. coli, which contain a dhaT over-expressing plasmid. The dhaT gene was cloning from Klebsiella pneumoniae, a 1,3-propanediol producing microorganism. The 1,3-propanediol oxidoreductase accounting for 39.8% of total soluble protein in cytoplasm. The lysate of E.coli DH5α(pDK-dhaT) was purified by ammonium sulfate condensation, DE23 Cellulose ion-exchange chromatography and Sephadex G-200 gel filtration.Finally PDOR soulution obtained have a 3.94-fold purification, and the recovery ratio was 15.7%.3-hydroxypropionaldehyde was synthesized form the hydrolyz of acrolein. A gas chromatography method was established for analyzed the reaction products. There were the conditions such as temperature 50℃, sulfuric acid 0.25mol/L, acrolein 2mol/L and the reaction time was 4 h. Under the condition, the convert ratio was 73.42%.The model of enzyme reaction kinetic of 1,3-propanediol oxidoreductase was construed followed the Ordered Bi-Bi model. There were 9 parameters in the model such as K_mA, K_mB, K_f, K_mP, K_mQ, K_ia, K_eq, K_ia and K_iq. The approximate values of the first six were identified by linear plotting with a simplified model, in this step the data used was the initial ratio of the reaction. Then fixed the 6 parameters in the model, the later 3 parameters were identified by nonlinear regression; the data used was the reaction progress. Finally, all the preliminary value of the 9 parameter were used as guesses for the final parameters estimation, using the data of reaction progress via nonlinear regression, the accurate value of the parameters of K_mA, K_mB, K_f, K_mP, K_mQ, K_r, K_eq, K_ia and K_iq were: 8.47μmol/L, 904.31μmol/L, 358565.18μmol/(L.min), 487.12μmol/L, 461.69μmol/L, 769.67μmol/(L.min), 1261.10, 2107975.42μmol/L and 7810.24μmol/L, respectively.