Improving the thermostability of <Emphasis Type="Italic">N</Emphasis>-carbamyl-<Emphasis Type="SmallCaps">d</Emphasis>-amino acid amidohydrolase by error-prone PCR |
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Authors: | Hong Yu Jian Li Dalong Zhang Yunliu Yang Weihong Jiang Sheng Yang |
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Institution: | (1) Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China;(2) Graduate University of the Chinese Academy of Sciences, Beijing, 100080, China;(3) Huzhou Research center of Industrial Biotechnolgy, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Huzhou, 313000, China;(4) Shanghai Research and Development Center of Industrial Biotechnology, Shanghai, 201201, China; |
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Abstract: | 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 °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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Keywords: | d-amino acid amidohydrolase" target="_blank">N-carbamyl-d-amino acid amidohydrolase Thermostability d-amino acid" target="_blank">d-amino acid Error-prone PCR |
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