Journal
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
Volume 82, Issue 2, Pages 279-285Publisher
SPRINGER
DOI: 10.1007/s00253-008-1748-z
Keywords
N-carbamyl-D-amino acid amidohydrolase; Thermostability; D-amino acid; Error-prone PCR
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Funding
- 863 Hi-Tech Program [2007AA02Z205]
- Knowledge Innovation Program of the Chinese Academy of Sciences [KSCX2-YW-G-018]
- Knowledge Innovation Program of Shanghai Institute for Biological Sciences, Chinese Academy of Sciences [2007KIP102]
- DuPont Young Professor Award
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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 A 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.
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