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Cloning, expression, characterization, and nucleophile identification of family 3, Aspergillus niger beta-glucosidase

TitleCloning, expression, characterization, and nucleophile identification of family 3, Aspergillus niger beta-glucosidase
Publication TypeJournal Article
Year of Publication2000
AuthorsDan, S, Marton, I, Dekel, M, Bravdo, BA, He, SM, Withers, SG, Shoseyov, O
JournalJOURNAL OF BIOLOGICAL CHEMISTRY
Volume275
Pagination4973-4980
Date PublishedFEB 18
ISSN0021-9258
Abstract

{The beta-glucosidase from Aspergillus niger (CMI. CC 324262) was purified, and an N-terminal sequence and two internal sequences were determined. BglI genomic gene and the cDNA were cloned from a genomic library and by reverse transcriptase-polymerase chain reaction, respectively. The cDNA was successfully expressed in Saccharomyces cerevisiae and Pichia pastoris. Sequence analysis revealed that the gene encodes a 92-kDa enzyme that is a member of glycosidase family 3. H-1-NMR analysis of the reaction catalyzed by this enzyme confirmed that, in common with other family 3 glycosidases, this enzyme hydrolyzes with net retention of anomeric configuration. Accordingly, the enzyme was inactivated by a-deoxy-a-fluoro beta-glucosyl fluoride, with kinetic parameters of k(i) = 4.5 min(-1)

DOI10.1074/jbc.275.7.4973