Screening for Amidases: Isolation and Characterization of a Novel D-Amidase from Variovorax paradoxus

• Published in: Advanced synthesis & catalysis Vol. 344; no. 9; pp. 965 - 973
• Main Authors: Krieg, Lutz, Ansorge-Schumacher, Marion B., Kula, Maria-Regina
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 01.10.2002; WILEY‐VCH Verlag; Wiley
• Subjects: Chemistry; Chemistry, Applied; Chemistry, Organic; D-amidase; D-amino acids; enzyme screening; Physical Sciences; Science & Technology; tert-leucine; Variovorax paradoxus; tert-leucine; enzyme screening; ASYMMETRIC-SYNTHESIS; Variovorax paradoxus; OCHROBACTRUM-ANTHROPI; D-amino acids; VERSATILE; D-amidase; PURIFICATION; PEPTIDE AMIDASE; AMINOPEPTIDASE; PROTEINS; CATALYSTS; COMAMONAS-ACIDOVORANS KPO-2771-4; AMINO-ACID AMIDASE
• ISSN: 1615-4150; 1615-4169
• DOI: 10.1002/1615-4169(200210)344:9<965::AID-ADSC965>3.0.CO;2-Z

Using racemic tert‐leucine amide as sole nitrogen source in minimal medium, 162 strains were isolated by enrichment techniques and shown to contain amidase activity. Among these isolates three D‐amidase producers were found and identified as Variovorax paradoxus (two strains) and Klebsiella spec. The D‐amidase from Variovorax paradoxus was purified to homogeneity by three chromatographic steps. With dl‐Tle‐amide as substrate Michaelis Menten kinetics were observed with a KM of 0.74 mM, a KI of 640 mM and a Vmax of 1.4 U/mg. The amidase has a broad pH‐optimum between 7 and 9.5 and a temperature optimum at 47–49 °C. The amidase hydrolyzed amino acid amides as well as carboxamides and 2‐hydroxy acid amides. The stereoselectivity of the reaction was variable, however. Hydrolyzing dl‐Tle‐amide the enantiomeric ratio E was >200 resulting in D‐Tle with an ee of >99% and up to 47% conversion. Similar results were obtained with dl‐Leu‐amide and dl‐Val‐amide while dl‐Phe‐amide was hydrolyzed with an enantiomeric ratio E of only 5.