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Applied and Environmental Microbiology, December 2005, p. 7961-7973, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.7961-7973.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

L-Selective Amidase with Extremely Broad Substrate Specificity from Ochrobactrum anthropi NCIMB 40321

DSM Pharma Chemicals—Advanced Synthesis, Catalysis and Development, P.O. Box 18, 6160 MD Geleen,¹ Van't Hoff Institute for Molecular Sciences, University of Amsterdam, Nieuwe Achtergracht 129, 1018 WS Amsterdam, The Netherlands²

Received 21 April 2005/ Accepted 26 August 2005

An industrially attractive L-specific amidase was purified to homogeneity from Ochrobactrum anthropi NCIMB 40321 wild-type cells. The purified amidase displayed maximum initial activity between pH 6 and 8.5 and was fully stable for at least 1 h up to 60°C. The purified enzyme was strongly inhibited by the metal-chelating compounds EDTA and 1,10-phenanthroline. The activity of the EDTA-treated enzyme could be restored by the addition of Zn²⁺ (to 80%), Mn²⁺ (to 400%), and Mg²⁺ (to 560%). Serine and cysteine protease inhibitors did not influence the purified amidase. This enzyme displayed activity toward a broad range of substrates consisting of -hydrogen- and (bulky) ,-disubstituted -amino acid amides, -hydroxy acid amides, and -N-hydroxyamino acid amides. In all cases, only the L-enantiomer was hydrolyzed, resulting in E values of more than 150. Simple aliphatic amides, ß-amino and ß-hydroxy acid amides, and dipeptides were not converted. The gene encoding this L-amidase was cloned via reverse genetics. It encodes a polypeptide of 314 amino acids with a calculated molecular weight of 33,870. Since the native enzyme has a molecular mass of about 66 kDa, it most likely has a homodimeric structure. The deduced amino acid sequence showed homology to a few other stereoselective amidases and the acetamidase/formamidase family of proteins (Pfam FmdA_AmdA). Subcloning of the gene in expression vector pTrc99A enabled efficient heterologous expression in Escherichia coli. Altogether, this amidase has a unique set of properties for application in the fine-chemicals industry.