A new aryl acylamidase from Rhodococcus sp. strain Oct1 acting on ω-lactams: Its characterization and gene expression in Escherichia coli
Rhodococcus sp. strain Oct1 utilizing ω-octalactam as a sole source of carbon and nitrogen was isolated from soil. ω-Octalactam hydrolyzing enzyme was purified to homogeneity. The purified enzyme has a molecular weight of approximately 48,100 by SDS polyacrylamide gel electrophoresis and 99,100 by gel filtration, indicating that the enzyme consists of 2 subunits. The purified enzyme catalyzed the hydrolysis of ω-octalactam to form 8-aminooctanoic acid at a rate of 3.95 U/mg. The purified enzyme also acted on ω-heptalactam, ω-laurolactam, nitroacetoanilide substitutions, and various aliphatic amides. The most suitable substrate was o-nitroacetanilide for the enzyme (11.6 U/mg). The enzyme belongs to aryl acylamidase. The gene for the enzyme was cloned and the deduced amino acid sequence showed similarity to ω-laurolactam hydrolase from Rhodococcus sp. U224 (51%) and putative aryl acylamidase from Nocardia farcinica IFM 10152 (98%), and N-terminal amino acid sequence (28 residues) of aryl acylamidase from Nocardia globerula IFO 13510 (92%). Aryl acylamidases and 6-aminohexanoate-cyclic-dimer hydrolases are in the same phylogenic lineage. These enzymes were mostly active toward non-natural amides. From phylogenic analysis, these enzymes were classified into amidase signature family. The enzyme was produced in a soluble form as a fusion protein (extension of 13 amino acids at C-terminal) in Escherichia coli.
Journal: Enzyme and Microbial Technology - Volume 46, Issues 3–4, 5 March 2010, Pages 237–245