| 1,3-propanediol(1,3-PDO) is one of the most important chemicals, used as a monomer to produce the desired polyester such as polytrimethylene terephthalate. Glycerol dehydratase (GDHt) is the rate limiting enzyme in the biosynthesis of 1,3- PDO, which could dehydrate glycerol into 3-hydroxylpropionaldehyde. In provious study, Klebsiella pneumoniae XJPD-Li, screened by our group, was found to consume glycerol fast and produce 1.3-PDO with high productivity and molar yield of (1, 3-PDO to glycerol) So the properties of catalysis, structure and reacticvation of glycerol dehydratase (GDHt) from K. pneumoniae XJPD-Li were investigated in this research..Firstly, the gene of GDHt was cloned from K. pneumoniae XJPD-Li genome and the following sequence alignment with GDHt (U30903) showed the differences of non-active site amino acid residue at No.193, 407 inαsubunit and No.47, 189 inβsubunit. To study the special properties of GDHt from K. pneumoniae XJPD-Li, the overexpression system was successfully constructed and the optimization of inducing process for recombinant GDHt were carried out. The optimum inducing conditions were the IPTG concentration of 0.8 mmol·L-1, the temperature of 20℃and inducing time of 3 h. The ratio of soluble form increased to 1.432 under the optimum condition.Purification was carried out by affinity chromatography. Homogeneity of recombinant GDHt was obtained conveniently resulted in 2.11-fold purification and an overall yield of 47.5%. The optimum pH and reaction temperature of putified recombinant GDHt were pH 8.0 and 45℃, respectively. The Km of GDHt for different substrates showed that glycerol was the optimum substance and the affinity was strong between coenzyme B12 and GDHt. It showed that the recombinant GDHt was relative thermostability, and a bit blunt to oxygen. The thermo inactivation kinetics fit the first order kinetics. Oxygen and coenzyme analogue inactivation kinetics was agreed with exponential regression.Analyzing results by biosoftware described the typicalα/βstructure of K. pneumoniae GDHt,including multiple soft regions. The No. 407 ofαsubunit was sited in soft region, whose hydrophobic character was different with that ofαsubunit in GDHt (U30903). The mutant E407A of GDHt showed lower special activity and higher Km than that of previous mutation. The circle dichromism (CD) spectra of GDHt showed the second structure of GDHt has little change, while fluorescence spectra some disturbances before and after mutation. That is the No 407 ofαsubunit could influence the conformation and activity of GDHt. Molecular Dynamic Simulation was applied to determine the relationship between structure and function. The RMSD,Hydrogen bonds inter and intro moleculars were changed obviously. It is proved that non-active site amino acid residues, No.193, 407 inαsubunit and No.47, 189 inβsubunit, were important to the stability of recombinant GDHt, especially its active site, which provided the structure of GDHt more compacted.The reactivation properties of recombinant GDHt was determined by GDHt reactivation factor (GDHt-Rf) from K. pneumoniae XJPD-Li, which was founded existing mainly in the form of inclusion body. Renaturation was carried out by the method of one step purification and renaturation. The renaturation ratios and special activity of GDHt-Rf were 20.7%, 60.6 min-1?mg-1. Addition of GSH to GSSG ( 6:1 ) and ATP in the renaturation solution was suitable to correctly renaturate the inclusion body of GDHt-Rf. The in vitro reactivation kinetic model was established according to Pingpang BiBi mechanism and the parameters were calculated. The simulated values can fit the experimental values. The reactivation life of GDHt in vitro was doubled when the reactivation system was modified. |
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