title: L-selective amidase with extremely broad substrate specificity from Ochrobactrum anthropi NCIMB 40321.
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 degrees 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, beta-amino and beta-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.
authors.affiliation: DSM Pharma Chemicals-Advanced Synthesis, Catalysis and Development, P.O. Box 18, 6160 MD Geleen, The Netherlands. theo.sonke@dsm.com
authors.forename: Theo
authors.initials: T
authors.lastname: Sonke
authors.forename: Sandra
authors.initials: S
authors.lastname: Ernste
authors.forename: Renate F
authors.initials: RF
authors.lastname: Tandler
authors.forename: Bernard
authors.initials: B
authors.lastname: Kaptein
authors.forename: Wilco P H
authors.initials: WP
authors.lastname: Peeters
authors.forename: Friso B J
authors.initials: FB
authors.lastname: van Assema
authors.forename: Marcel G
authors.initials: MG
authors.lastname: Wubbolts
authors.forename: Hans E
authors.initials: HE
authors.lastname: Schoemaker
mesh_terms: D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D016384:Consensus Sequence; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D020621:Ochrobactrum anthropi / Q000201:enzymology* / Q000254:growth & development; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D010446:Peptide Fragments; D000581:Amidohydrolases; C019665:amidase