Improving the thermostability of N-carbamyl-D-amino acid amidohydrolase by error-prone PCR.
To facilitate the easier production of D-amino acids using N-carbamyl-D-amino acid amidohydrolase (DCase) in an immobilized form, we improved the enzymatic thermostability of highly soluble DCase-M3 of Ralstonia pickettii using directed mutagenesis. Six novel mutation sites were identified in this study, apart from several thermostability-related amino acid sites reported previously. The most thermostable mutant, in which the 12th amino acid had been changed from glutamine to leucine, showed a 7 degrees C increase in thermostability. Comparative characterization of the parental and mutant DCases showed that although there was a slight reduction in the oxidative stability of the mutants, their kinetic properties and high solubility were not affected. The mutated enzymes are expected to be applied to the development of a fully enzymatic process for the industrial production of D-amino acids.
Yu
Hong
H
Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
Li
Jian
J
Zhang
Dalong
D
Yang
Yunliu
Y
Jiang
Weihong
W
Yang
Sheng
S
D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D032621:DNA Shuffling; D004795:Enzyme Stability; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods*; D015202:Protein Engineering / Q000379:methods*; D049920:Ralstonia pickettii / Q000201:enzymology*; D013696:Temperature
D001426:Bacterial Proteins; D000581:Amidohydrolases; C112401:N-carbamyl-D-amino acid amidohydrolase