Isolation and characterization of a new family 42 beta-galactosidase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius: Identification of the active site residues (Articolo in rivista)

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  • Isolation and characterization of a new family 42 beta-galactosidase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius: Identification of the active site residues (Articolo in rivista) (literal)
Anno
  • 2008-01-01T00:00:00+01:00 (literal)
Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#doi
  • 10.1016/j.bbapap.2007.10.013 (literal)
Alternative label
  • Di Lauro B.; Strazzulli A.; Perugino G.; La Cara F.; Bedini E.; Corsaro M.M.; Rossi M.; Moracci M. (2008)
    Isolation and characterization of a new family 42 beta-galactosidase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius: Identification of the active site residues
    in Biochimica et biophysica acta. Proteins and proteomics; Elsevier, Amsterdam (Paesi Bassi)
    (literal)
Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#autori
  • Di Lauro B.; Strazzulli A.; Perugino G.; La Cara F.; Bedini E.; Corsaro M.M.; Rossi M.; Moracci M. (literal)
Pagina inizio
  • 292 (literal)
Pagina fine
  • 301 (literal)
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  • 1784 (literal)
Rivista
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  • 2 (literal)
Note
  • ISI Web of Science (WOS) (literal)
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  • Institute of Protein Biochemistry, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131, Naples, Italy Dipartimento di Chimica Organica e Biochimica, Università di Napoli \"Federico II\", Complesso Universitario di Monte S. Angelo, Via Cinthia 4, 80126 Naples, Italy Dipartimento di Biologia Strutturale e Funzionale, Università di Napoli \"Federico II\", Complesso Universitario di Monte S. Angelo, Via Cinthia 4, 80126 Naples, Italy (literal)
Titolo
  • Isolation and characterization of a new family 42 beta-galactosidase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius: Identification of the active site residues (literal)
Abstract
  • The thermoacidophilic bacterium Alicyclobacillus acidocaldarius is a rich source of glycoside hydrolases enabling its growth on several di-and polysaccharides. We report here the purification and the characterization of a beta-galactosidase from this source, the cloning of its gene, and the expression and the characterization of the recombinant enzyme (Aa beta-gal).The enzyme was purified 46-fold from A. acidocaldarius extracts; the gene for Aa beta-gal encoded a new member of the glycoside hydrolase family 42 (GH42) and it is flanked by a putative AraC/XylS regulator, however, the two genes were transcribed independently. The recombinant Aa beta-gal was characterized in detail revealing that it is optimally active and stable at 65 degrees C. Aa beta-gal is very specific for glycosides with an axial C4-OH at their non-reducing end, with k(cat)/K-M values of 484, 186, and 332 s(-1) mM(-1) for 2-nitrophenyl-beta-D-galactoside, -fucoside, and 4-nitropbenyl-alpha-L-arabinoside, respectively. Finally, the characterization of the site-directed mutants Glu157Gly and Glu313Gly confirmed the latter as the nucleophile of the reaction and gave experimental evidence, for the first time in GH42, of the role of Glu157 as the acid/base of the catalyzed reaction. (literal)
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