We report here the molecular cloning and characterization of a glucocerebrosidase [EC 188.8.131.52] 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.
|Number of pages||7|
|Journal||Journal of biochemistry|
|Publication status||Published - Aug 2002|
All Science Journal Classification (ASJC) codes
- Molecular Biology