Abstract
An amidase produced by Pseudomonas chlororaphis B23 was purified and characterized. The purification procedure used included ammonium sulfate precipitation and hydrophobic, anion-exchange, gel filtration, and ceramic hydroxyapatite chromatography steps. This amidase has a native molecular mass of about 105 kDa and is a homodimer whose subunits have a molecular mass of 54 kDa. The enzyme exhibited maximal activity at 50(deg)C and at pH values ranging from 7.0 to 8.6. We found no evidence that metal ions were required, and the enzyme was inhibited by several thiol reagents. This amidase exhibited activity against a broad range of aliphatic and aromatic amides and exhibited enantioselectivity for several aromatic amides, including 2-phenylpropionamide (enantiomeric excess [ee] = 100%), phenylalaninamide (ee = 55%), and 2-(4-chlorophenyl)-3-methylbutyramide (ee = 96%), but not 2-(6-methoxy-2-naphthyl)propionamide (the amide form of naproxen) (ee = 0%). The characteristics of the P. chlororaphis B23 amidase are the same as the characteristics of enantioselective amidases described by Mayaux et al. (J. F. Mayaux, E. Cerbelaud, F. Soubrier, D. Faucher, and D. Petre, J. Bacteriol. 172:6764-6773, 1990; J. F. Mayaux, E. Cerbelaud, F. Soubrier, P. Yeh, F. Blanche, and D. Petre, J. Bacteriol. 173:6694-6704, 1991) and Kobayashi et al. (M. Kobayashi, H. Komeda, T. Nagasawa, M. Nishiyama, S. Horinouchi, T. Beppu, H. Yamada, and S. Shimizu, Eur. J. Biochem. 217:327-336, 1993).
Full Text
The Full Text of this article is available as a PDF (229.2 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Ambler R. P., Auffret A. D., Clarke P. H. The amino acid sequence of the aliphatic amidase from Pseudomonas aeruginosa. FEBS Lett. 1987 May 11;215(2):285–290. doi: 10.1016/0014-5793(87)80163-1. [DOI] [PubMed] [Google Scholar]
- Gilligan T., Yamada H., Nagasawa T. Production of S-(+)-2-phenylpropionic acid from (R,S)-2-phenylpropionitrile by the combination of nitrile hydratase and stereoselective amidase in Rhodococcus equi TG328. Appl Microbiol Biotechnol. 1993 Aug;39(6):720–725. doi: 10.1007/BF00164456. [DOI] [PubMed] [Google Scholar]
- Kobayashi M., Komeda H., Nagasawa T., Nishiyama M., Horinouchi S., Beppu T., Yamada H., Shimizu S. Amidase coupled with low-molecular-mass nitrile hydratase from Rhodococcus rhodochrous J1. Sequencing and expression of the gene and purification and characterization of the gene product. Eur J Biochem. 1993 Oct 1;217(1):327–336. doi: 10.1111/j.1432-1033.1993.tb18250.x. [DOI] [PubMed] [Google Scholar]
- Kobayashi M., Nagasawa T., Yamada H. Enzymatic synthesis of acrylamide: a success story not yet over. Trends Biotechnol. 1992 Nov;10(11):402–408. doi: 10.1016/0167-7799(92)90283-2. [DOI] [PubMed] [Google Scholar]
- Mayaux J. F., Cerbelaud E., Soubrier F., Yeh P., Blanche F., Pétré D. Purification, cloning, and primary structure of a new enantiomer-selective amidase from a Rhodococcus strain: structural evidence for a conserved genetic coupling with nitrile hydratase. J Bacteriol. 1991 Nov;173(21):6694–6704. doi: 10.1128/jb.173.21.6694-6704.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mayaux J. F., Cerebelaud E., Soubrier F., Faucher D., Pétré D. Purification, cloning, and primary structure of an enantiomer-selective amidase from Brevibacterium sp. strain R312: structural evidence for genetic coupling with nitrile hydratase. J Bacteriol. 1990 Dec;172(12):6764–6773. doi: 10.1128/jb.172.12.6764-6773.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nishiyama M., Horinouchi S., Kobayashi M., Nagasawa T., Yamada H., Beppu T. Cloning and characterization of genes responsible for metabolism of nitrile compounds from Pseudomonas chlororaphis B23. J Bacteriol. 1991 Apr;173(8):2465–2472. doi: 10.1128/jb.173.8.2465-2472.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
- Soubrier F., Lévy-Schil S., Mayaux J. F., Pétré D., Arnaud A., Crouzet J. Cloning and primary structure of the wide-spectrum amidase from Brevibacterium sp. R312: high homology to the amiE product from Pseudomonas aeruginosa. Gene. 1992 Jul 1;116(1):99–104. doi: 10.1016/0378-1119(92)90635-3. [DOI] [PubMed] [Google Scholar]