Recombinant glycerol dehydratase from Klebsiella pneumoniae XJPD-Li: induction optimization, purification and characterization.
Glycerol dehydratase (GDHt) is the rate limiting enzyme in the biosynthesis of 1,3-propanediol from glycerol. The optimization of inducting process for recombinant GDHt from Klebsiella pneumoniae XJPD-Li carried out to increase specific activity and ratio of soluble form. The optimum condition was inducing under the isopropyl-beta-D-thiogalactoside concentration of 0.8 mM and the temperature of 20 degrees C for 3 h. Homogeneity of GDHt then was obtained by affinity chromatography, resulted in 2.11-fold purification and an overall yield of 47.5%. The optimum pH and reaction temperature of GDHt were pH 8.0 and 45 degrees C, respectively. The K(m) for glycerol, 1,2-propanediol, 1,2-ethanediol and coenzyme B12 were 0.48, 1.43, 3.07 mM, and 10.03 nM, respectively. The GDHt showed relatively stable even under temperature of 40 degrees C and a bit blunt to oxygen. The thermo-inactivation kinetic models were fit linear under different temperatures.
School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China. xuxl@shzu.edu.cn
X L
XL
Xu
G L
GL
Zhang
B
B
Lv
Y J
YJ
Yuan
C
C
Li
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D005990:Glycerol / Q000378:metabolism*; D006836:Hydro-Lyases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics; D011409:Propylene Glycols / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D011409:Propylene Glycols; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; C041787:1,3-propanediol; D006836:Hydro-Lyases; C020671:glycerol dehydratase; D005990:Glycerol