Purification and characterization of a novel enzyme, arylalkyl acylamidase, from Pseudomonas putida Sc2

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Abstract
A novel enzyme, arylalkyl acylamidase, which shows a strict specificity for N-acetyl arylalkyl-amines, but not acetanilide derivatives, was purified from the culture broth of Pseudomonas putida Sc2. The purified enzyme appeared to be homogeneous, as judged by native and SDS/PAGE. The enzyme has a molecular mass of approximately 150 kDa and consists of four identical subunits. The purified enzyme catalyzed the hydrolysis of N-acetyl-2-phenylethylamine to 2-phenylethylamine and acetic acid at the rate of 6.25 μmol · min−1· mg−1 at 30°C. It also catalyzed the hydrolysis of various N-acetyl arylalkylamines containing a benzene or indole ring, and acetic acid arylalkyl esters. The enzyme did not hydrolyze acetanilide, N-acetyl aliphatic amines, N-acetyl amino acids, N-acetyl amino sugars or acylthiocholine. The apparent Km for N-acetylbenzylamine, N-acetyl-2-phenyl-ethylamine and N-acetyl-3-phenylpropylamine are 41 mM, 0.31 mM and 1.6 mM, respectively. The purified enzyme was sensitive to thiol reagents such as Ag2SO4, HgCl2 and P-chloromercuribenzoic acid, and its activity was enhanced by divalent metal ions such as Zn2+, Mg2+ and Mn2+.