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 Zn 2؉ (to 80%), Mn 2؉ (to 400%), and Mg 2؉ (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, ␤-amino and ␤-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.Enantiomerically pure ␣-amino acids, both natural and unnatural, form an important class of chiral building blocks for the pharmaceutical and agrochemical industries. Examples of important ␣-hydrogen-␣-amino acids are D-phenylglycine (DPhg) and D-p-hydroxyphenylglycine, which are produced as side chains for the manufacture of semisynthetic ␤-lactam antibiotics such as ampicillin, amoxicillin, and cephalexin (14, 53), and D-valine, an intermediate for the pyrethroid insecticide fluvalinate (28). Another frequently used ␣-hydrogen-␣-amino acid is L-tert-leucine, which is used as a building block for a variety of antiviral (e.g., anti-human immunodeficiency virus), antiarthritis, and anticancer drugs under development and as a chiral auxiliary in chemical asymmetric synthesis (5, 10). Besides ␣-hydrogen-␣-amino acids, ␣,␣-disubstituted ␣-amino acids also constitute a group of compounds of increasing importance, as exemplified by the use of L-␣-methyl-3,4-dihydroxyphenylalanine (L-␣-methyldopa) as an antihypertensive drug (34, 42), ␣-methylvaline as an intermediate for the herbicide Arsenal and related herbicides (47, 49), and L-␣-methylphenylglycine as a building block for the new fungicide fenamidone (27).Enantiomerically pure ␣-amino acids can be produced on a commercial scale by a number of different processes, both chemical and enzymatic. Biocatalytic processes that have been commercialized include the acylase process operated by Degussa (8) a...

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