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 α-hydrogen- and (bulky) α,α-disubstituted α-amino acid amides, α-hydroxy acid amides, and α-N-hydroxyamino acid amides. In all cases, only the l-enantiomer was hydrolyzed, resulting in E values of more than 150. Simple aliphatic amides, β-amino and β-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.
Studies on the Utilization of the α-Hydroxy Acid of Methionine by Chicks Fed Crystalline Amino Acid Diets