Isolation of Agrobacterium sp. strain KNK712 that produces N-carbamyl-D-amino acid amidohydrolase, cloning of the gene for this enzyme, and properties of the enzyme

Bioscience, Biotechnology, and Biochemistry
H NanbaS Takahashi

Abstract

Agrobacterium sp. strain KNK712, which produced N-carbamyl-D-amino acid amidohydrolase (DCase) was isolated from soil. The bacterium had D-specific hydantoinase activity also. Both enzymes are suitable for use in the production of D-amino acids. The DCase gene from Agrobacterium sp. strain KNK712 was cloned into Escherichia coli. The cloned DNA fragment contained one open reading frame, predicted to encode a peptide of 304 amino acids, with a calculated molecular weight of 34,285. The DCase gene was overexpressed under the control of the lac promoter, and DCase accounted for 50% of the soluble protein in the cells. The enzyme was purified and some properties were investigated. Both the optimum pH and the pH that gave greatest stability were about pH 7.0. The optimum temperature was 65 degrees C, and the enzyme was stable at 55 degrees C. The enzyme had strict specificity toward N-carbamyl-D-amino acids, and was inhibited by thiol reagents, Cu2+, Hg2+, Ag+, and ammonia.

References

Dec 1, 1977·Proceedings of the National Academy of Sciences of the United States of America·F SangerA R Coulson
Nov 24, 1979·Nucleic Acids Research·H C Birnboim, J Doly

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Citations

Jun 15, 2007·European Biophysics Journal : EBJ·C EtchebestA G de Brevern
Oct 20, 2005·Journal of Bioscience and Bioengineering·H NanbaS Takahashi
Oct 16, 2009·Applied Microbiology and Biotechnology·Sergio Martínez-RodríguezFrancisco Javier Las Heras-Vázquez
Apr 3, 2003·Biochemical and Biophysical Research Communications·Chao-Hung Kao, Wen-Hwei Hsu
May 15, 2007·Microbiological Research·Yanzhen MeiPingkai Ouyang
May 21, 2013·Biotechnology and Applied Biochemistry·Hemraj S NandanwarGurinder S Hoondal
Nov 28, 2001·Protein Expression and Purification·D SareenR M Vohra

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