Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/69785
標題: Characterization of a bifunctional aminoacylase/carboxypeptidase from radioresistant bacterium Deinococcus radiodurans R1
作者: Lin, L.L.
Chen, M.H.
Chien, H.C.R.
Kan, S.C.
Chen, C.C.
Hu, H.Y.
Hsu, W.H.
關鍵字: aminoacylase
carboxypeptidase
Deinococcus radiodurans
gene
expression
biochemical characterization
complete genome sequence
archaebacterium sulfolobus-solfataricus
fine
chemicals
l-aminoacylase
amino-acids
purification
carboxypeptidase
archaeon
gene
pharmaceuticals
期刊/報告no:: Journal of Biotechnology, Volume 128, Issue 2, Page(s) 322-334.
摘要: The gene encoding a Deinococcus radiodurans R1 bifunctional aminoacylase/carboxypeptidase (DR_ACY/CP) was amplified by polymerase chain reaction and cloned into pQE-30 to generate pQE-DRAC. The cloned gene consists of an open reading frame of 1197 bp encoding a protein with a molecular mass of 42,729 Da. The predicted amino acid sequence shows high homology with those of Geobacillus kaustophilus aminoacylase, Geobacillus stearothermophilus aminoacylase, Pyrococcus horikoshii carboxypeptidase/aminoacylase and Thermoanaerobacter tengcongensis aminoacylase/carboxypeptidase. The expressed enzyme was purified from the crude extract of IPTG-induced Escherichia coli M15 (pQE-DRAC) to homogeneity by nickel-chelate chromatography. The molecular mass of the purified enzyme was determined to be 43 kDa by SDS-PAGE. Maximal aminoacylase activity with N-acetyl-methionine as the substrate occurred at pH 8.0 and 40 degrees C in the sodium phosphate buffer. The aminoacylase activity was strongly inhibited by metal-chelating agents, and was largely restored by divalent cations, such as Co2+, Mn2+ and Ni2+. The purified enzyme had broad specificity toward N-acetylated L-amino acids as well as N-CBZ-peptides. Carboxypeptidase activity of DR_ACY/CP to N-CBZ-Gly-Ala exhibited K-m and k(cat) values of 4.3 mM and 28 s(-1), respectively. The enzyme also had activity toward the cell wall-related substrates, D-Ala-Gly, D-Ala-Gly-Gly and L-Orn-L-Ala. (c) 2006 Elsevier B.V. All rights reserved.
URI: http://hdl.handle.net/11455/69785
ISSN: 0168-1656
文章連結: http://dx.doi.org/10.1016/j.jbiotec.2006.10.011
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