TAILIEUCHUNG - Báo cáo Y học: Identification and characterization of a new gene from Variovorax paradoxus Iso1 encoding N -acyl-D-amino acid amidohydrolase responsible for D-amino acid production

AnN-acyl-D-amino acid amidohydrolase (N-D-AAase) was identified in cell extracts of a strain, Iso1, isolated from an environment containing N-acetyl-D-methionine. The bac-terium was classified as Variovorax paradoxusby phylo-genetic analysis. The gene was cloned and sequenced. The gene consisted of a 1467-bpORF encoding a polypeptide of 488 amino acids. TheV. paradoxus N-D-AAase showed significant amino acid similarity to theN-acyl-D-amino acid amidohydrolases of the two eubacteriaAlcaligenes xylo-soxydans A-6 (44–56% identity),Alcaligenes facelisDA1 (54% identity) and the hyperthermophilic archaeon Pyro-coccus abyssi(42% identity) | Eur. J. Biochem. 269 4868-4878 2002 FEBS 2002 doi Identification and characterization of a new gene from Variovorax paradoxus Isol encoding -acyl-D-amino acid amidohydrolase responsible for D-amino acid production Pei-Hsun Lin1 Shiun-Cheng Su1 Ying-Chieh Tsai2 and Chia-Yin Lee1 1Graduate Institute of Agricultural Chemistry National Taiwan University Taipei Taiwan 2Graduate Institute of Biochemistry Yang-Ming University Taipei Taiwan An N-acyl-D-amino acid amidohydrolase N-D-AAase was identified in cell extracts of a strain Iso1 isolated from an environment containing N-acetyl-D-methionine. The bacterium was classified as Variovorax paradoxus by phylogenetic analysis. The gene was cloned and sequenced. The gene consisted of a 1467-bp ORF encoding a polypeptide of 488 amino acids. The V. paradoxus N-D-AAase showed significant amino acid similarity to the N-acyl-D-amino acid amidohydrolases of the two eubacteria Alcaligenes xylo-soxydans A-6 44-56 identity Alcaligenes facelis DA1 54 identity and the hyperthermophilic archaeon Pyrococcus abyssi 42 identity . After over-expression of the N-D-AAase protein in Escherichia coli the enzyme was purified by multistep chromatography. The native molecular mass was kDa which agreed with the predicted molecular mass of 52 798 Da and the enzyme appeared to be a monomer protein by gel-filtration chromatography. A homogenous protein with a specific activity of 516 U-mg-1 was finally obtained. After peptide sequencing by LC MS MS the results were in agreement with the deduced amino acid sequence of the N-D-AAase. The pl of the enzyme was and it had an optimal pH and temperature of and 50 C respectively. After 30 min heat treatment at 45 C between pH 6 and pH 8 80 activity remained. The N-D-AAase had higher hydrolysing activity against N-ace-tyl-D-amino acid derivates containing D-methionine D-leucine and D-alanine and against N-chloroacetyl-D-phe-nylalanine. Importantly the enzyme does .

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