The Molecular Modification Of Glycerol Dehydratase From Klebsiella Pneumoniae By Site-Saturation Mutagenesis

Glycerol dehydratase (EC 4.2.1.30) is a key and rate-limited enzyme in the metabolic pathway of producing 1,3-propanediol, which is a useful material in chemical industry. By site-saturation mutagenesis, the effects of different amino acids in the conservative site on the characteristics of enzymes were investigated. The research provided the theories basis for the further molecular modification of glycerol dehydratase.By using the 3D structures (PDB ID is liwp in the PDB database) which has the most homology with the glycerol dehydratase from K. pneumoniae as a template, the 3D structures of corresponding enzymes were successfully constructed by homology modeling on the internet using swissmodel. The models can be used to analyse the relationships between the 3D structures of the mutant enzymes and their properties. ProSa 2003, a software of energy analysis, was taken use of analyzing the energy of each amino acid. The results of analysis revealed that the energy reduced when the Ile in the wild enzyme was taken place of Val, but increased when substituted to others. In order to find out the effects of different amino acid on the site 498 of a subunit on the properties of glycerol dehydratase, this research mutated the amino acid on the site 498 of a subunit to other 19 amino acids by site-saturation mutagenesis. Then the mutated genes were expressed by induction of IPTG in E.coli, respectively. After being purified, the mutated enzymes were used for property analysis. By analyzing the property of the mutant enzymes, we found that when the amino acid in 498 of a subunit was substituted to phenylalanine or glycine the enzyme activity disappeared; while the activity was enhanced using glycerol as a substrate when substituted to alanine, methionine or valine; and the activity increased using 1,2-propanediol as a substrate when the amino acid was substituted to alanine, cystine, methionine, asparagine, serine or valine. The optimal temperature of most mutant enzyme were reduced to 42℃, and the optimal pH all changed.