Molecular cloning and characterization of a novel glucocerebrosidase of Paenibacillus sp. TS12

Tomomi Sumida, Noriyuki Sueyoshi, Makoto Ito

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13 Citations (Scopus)

Abstract

We report here the molecular cloning and characterization of a glucocerebrosidase [EC 3.2.1.45] from Paenibacillus sp. TS12. The open reading frame of the glucocerebrosidase gene consisted of 2,493 bp nucleotides and encoded 831 amino acid residues. The enzyme exhibited no sequence similarity with a classical glucocerebrosidase belonging to glycoside hydrolase (GH) family 30, but rather showed significant similarity with GH family 3 β-glucosidases from Clostridium thermocellum, Ruminococcus albus, and Aspergillus aculeateus. The recombinant enzyme, expressed in Escherichia coli BL21-(DE3)pLysS, had a molecular weight of 90.7 kDa and hydrolyzed NBD-labeled glucosylceramide, but not galactosylceramide, GM1a or sphingomyelin. The enzyme was most active at pH 6.5, and its apparent Km and Vmax values for NBD-labeled glucosylceramide and p-nitrophenyl-β-glucopyranoside were 223 μM and 1.60 μmol/min/mg of protein, and 593 μM and 112 μmol/min/mg of protein, respectively. Site-directed mutagenesis indicated that Asp-223 is an essential amino acid for the catalytic reaction and possibly functions a catalytic nucleophile, as in GH family 3 β-glucosidases. This is the first report of the molecular cloning and characterization of a glucocerebrosidase from a procaryote.

Original languageEnglish
Pages (from-to)237-243
Number of pages7
JournalJournal of biochemistry
Volume132
Issue number2
DOIs
Publication statusPublished - Aug 2002

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

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