title: Enhancement of pH stability and activity of glycerol dehydratase from Klebsiella pneumoniae by rational design.
abstract: Glycerol dehydratase (GDHt) is a key and rate-limiting enzyme in the pathway of 1,3-propanediol (1,3-PD) synthesis. The improvement of GDHt's stability and enzymatic activity is desirable for the biosynthesis of 1,3-PD. The gldABC gene encoding GDHt of Klebsiella pneumoniae was cloned and expressed in Escherichia coli XL10-Gold, and the mutation sites of GDHt were obtained through prediction by PoPMuSiC program. Consequently, two mutants (KpG60 and KpG525) were developed by rational design through site-mutagenesis based on 3D structure which was constructed from homology modeling. Analyses of enzymatic properties showed that pH stability of the mutants was about 1.25-2 times higher than that of the wild type, and specific activity, V(max) and K(cat)/K(m) of KpG525 were about 1.5-2 times higher than those of the wild type. This work presented a simple and useful measure to improve the performance of industrial enzyme.
authors: School of Food and Biological Engineering, Jiangsu University, Zhenjiang, 212013, China. qxh@ujs.edu.cn
authors: Xianghui
authors: X
authors: Qi
authors: Qi
authors: Q
authors: Guo
authors: Yuotuo
authors: Y
authors: Wei
authors: Hong
authors: H
authors: Xu
authors: Ribo
authors: R
authors: Huang
mesh_terms: D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011409:Propylene Glycols / Q000378:metabolism; D011487:Protein Conformation; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial; D050505:Mutant Proteins; D011409:Propylene Glycols; C041787:1,3-propanediol; D006836:Hydro-Lyases; C020671:glycerol dehydratase