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Báo cáo khoa học: A novel R-stereoselective amidase from Pseudomonas sp. MCI3434 acting on piperazine-2-tert-butylcarboxamide

A novel amidase acting on (R,S)-piperazine-2-tert-butyl-carboxamide was purified fromPseudomonassp. MCI3434 and characterized. The enzyme acted R-stereoselectively on (R,S)-piperazine-2-tert-butylcarboxamide to yield (R)-piperazine-2-carboxylic acid, and was tentatively named R-amidase. The N-terminal amino acid sequence of the enzyme showed high sequence identity with that deduced from a gene named PA3598 encoding a hypothetical hydrolase in Pseudomonas aeruginosaPAO1. | Eur. J. Biochem. 271 1580-1590 2004 FEBS 2004 doi 10.1111 j.1432-1033.2004.04069.x A novel -stereoselective amidase from Pseudomonas sp. MCI3434 acting on piperazine-2-fert-butylcarboxamide Hidenobu Komeda1 Hiroyuki Harada1 Shingo Washika1 Takeshi Sakamoto2 Makoto Ueda2 and Yasuhisa Asano1 1 Biotechnology Research Center Toyama Prefectural University Kurokawa Kosugi Toyama Japan 2Mitsubishi Chemical Group Science and Technology Research Center Inc. Aoba-ku Yokohama Kanagawa Japan A novel amidase acting on R S -piperazine-2-tert-butyl-carboxamide was purified from Pseudomonas sp. MCI3434 and characterized. The enzyme acted R-stereoselectively on R S -piperazine-2-tert-butylcarboxamide to yield R -piperazine-2-carboxylic acid and was tentatively named R-amidase. The N-terminal amino acid sequence of the enzyme showed high sequence identity with that deduced from a gene named PA3598 encoding a hypothetical hydrolase in Pseudomonas aeruginosa PAO1. The gene encoding R-amidase was cloned from the genomic DNA of Pseudomonas sp. MCI3434 and sequenced. Analysis of 1332 bp of the genomic DNA revealed the presence of one open reading frame ramA which encodes the R-amidase. This enzyme RamA is composed of274 amino acid residues molecular mass 30 128 Da and the deduced amino acid sequence exhibits homology to a carbon-nitrogen hydrolase protein PP3846 from Pseudomonas putida strain KT2440 72.6 identity and PA3598 protein from P. aeruginosa strain PAO1 65.6 identity and may be classified into a new subfamily in the carbon-nitrogen hydrolase family consisting of aliphatic amidase b-ureidopropionase carbamylase nitrilase and so on. The amount of R-amidase in the supernatant of the sonicated cell-free extract of an Escherichia coli transformant overexpressing the ramA gene was about 30 000 times higher than that of Pseudomonas sp. MCI3434. The intact cells of the E. coli transformant could be used for the R-stereoselective hydrolysis of racemic pip-erazine-2-tert-butylcarboxamide.