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

Author:
Sonke, Theo, Ernste, Sandra, Tandler, Renate F., Kaptein, Bernard, Peeters, Wilco P. H., Assema, Friso B. J. van, Wubbolts, Marcel G., Schoemaker, Hans E.
Source:
Applied and environmental microbiology 2005 v.71 no.12 pp. 7961-7973
ISSN:
0099-2240
Subject:
EDTA (chelating agent), Escherichia coli, Ochrobactrum anthropi, amidase, amides, amino acid sequences, cysteine proteinase inhibitors, dipeptides, enzyme activity, gene expression, genes, molecular weight, pH, proteins, sequence homology, serine, substrate specificity
Abstract:
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 [alpha]-hydrogen- and (bulky) [alpha],[alpha]-disubstituted [alpha]-amino acid amides, [alpha]-hydroxy acid amides, and [alpha]-N-hydroxyamino acid amides. In all cases, only the L-enantiomer was hydrolyzed, resulting in E values of more than 150. Simple aliphatic amides, {szligbeta}-amino and {szligbeta}-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.
Agid:
587275