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Improving the thermostability of N-carbamyl-d-amino acid amidohydrolase by error-prone PCR

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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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Acknowledgments

This work was funded by the 863 Hi-Tech Program (no. 2007AA02Z205), the Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-YW-G-018), and the Knowledge Innovation Program of Shanghai Institute for Biological Sciences, Chinese Academy of Sciences (no. 2007KIP102). Sheng Yang was funded by the DuPont Young Professor Award. We wish to thank Miss J. Zhou and Miss Y. Yuan (Hunan Flag Biotech Co., Ltd, China) for their assistance during the library screening. We are also grateful to Mr. Jun Chen (Institute of Plant Physiology and Ecology, Chinese Academy of Sciences) for his technical assistance in the HPLC analysis. We extend many thanks to the anonymous referees for their helpful comments.

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Correspondence to Sheng Yang.

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Yu, H., Li, J., Zhang, D. et al. Improving the thermostability of N-carbamyl-d-amino acid amidohydrolase by error-prone PCR. Appl Microbiol Biotechnol 82, 279–285 (2009). https://doi.org/10.1007/s00253-008-1748-z

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Keywords

  • N-carbamyl-d-amino acid amidohydrolase
  • Thermostability
  • d-amino acid
  • Error-prone PCR