Research On Key Enzymze Of 1,3-propanediol Biosynthetic Pathway

The kinety model of 1,3-propanediol bio-synthese pathway was investigated in thispaper, the main work done in this paper is modelding the glycerol dehydrogenaseKlebsiella pneumoniae glycerol dehydrogenase gene was colone and overexpressed inE.coli. The cell culture process was optimizaed, it was found that the maximal specificactivity of glycerol dehydrogenase in E. coli was obtained with 0.8 mM IPTG added to themedium after 6 hours induction. Cell lysate was obtained by ultrasonic crush from the cellmush, Glycerol dehydrogenase was purified by ammonium sulfate salting-out,SephadexG-200 gel filtration and DE-23 cellulose column chromatography. Afterpurification the specific activity of the product is 946U/mg, the final enzyme obtained hasa 2.58-fold purification with the recovery ratio of 37%. The subunit molecular weight ofenzyme was 39KDa shown by SDS-PAGE. The activitie of glycerol dehydrogenase atdifferent pH buffer was tested; it was shown the activity of the enzyme is increase with pHincrease. GDH activated in a broad range of temperature, the maximal activity wasobserved at 50℃.The model of enzyme reaction kinetic of glycerol dehydrogenase was construedfollowed the Ordered Bi-Bi model. There were 11 parameters in the model such as KmNAD,KmGLY, KiNAD, Vf, KmDHA, KmNADH, KiNADH, Vr, Keq, KiGLYand KiDHA. The approximatevalues of the first eight were identified by linear plotting with a simplified model, in thisstep the data used was the initial ratio of the reaction. Then fixed the eight parameters inthe model, the later three parameters were identified by nonlinear regression; the data usedwas the reaction progress. Finally, all the preliminary value of the 11 parameter were usedas guesses for the final parameters estimation, using the data of reaction progress vianonlinear regression, the accurate value of the parameters of KmNAD, KmGLY, KiNAD, Vf,KmDHA, KmNADH, KiNADH, Vr, Keq, KiGLYand KiDHAwere: 1924.47μmol/L, 11300.10μmol/L, 294.00μmol/L, 262.5μmol/(L.min), 332.50μmol/L, 95.00μmol/L,383.52μmol/L, 3092.27μmol/(L.min), 0.00095, 27768.37μmol/L, 12.57μmol/Lrespectively. Dihydroxyacetone kinase genes DHAKI and DHAKII were cloned from theKlebsiella pneumonia TUAC01 to by PCR, the size of the two genes were 1.86kb and3.18kb. The gene of glycerol dehydratase was cloned from the Klebsiella pneumoniaTUAC01 ues the same method, and the size of thegene was 2.76kb. The three genes wereconnected whin PDK6 plasmid to construct express plasmid PDK6- DHAKI,PDK6-DHAKII and PDK6-GDHt.