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Screening for Amidases: Isolation and Characterization of a Novel D ‐Amidase from Variovorax paradoxus
Author(s) -
Krieg Lutz,
AnsorgeSchumacher Marion B.,
Kula MariaRegina
Publication year - 2002
Publication title -
advanced synthesis and catalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.541
H-Index - 155
eISSN - 1615-4169
pISSN - 1615-4150
DOI - 10.1002/1615-4169(200210)344:9<965::aid-adsc965>3.0.co;2-z
Subject(s) - amidase , chemistry , amide , nitrilase , hydrolysis , enantiomer , enantiomeric excess , enzyme kinetics , stereochemistry , organic chemistry , enantioselective synthesis , active site , enzyme , catalysis
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 K M of 0.74 mM, a K I of 640 mM and a V max 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.