Characterization and kinetic analysis of a thermostable GH3 ß-glucosidase from Penicillium brasilianum

Kristian Bertel Rømer Krogh, P.V. Harris, C.L. Olsen, K.S. Johansen, Jesper Juul Højer-Pedersen, J. Borjesson, Lisbeth Olsson

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    A GH3 beta-glucosidase (BGL) from Penicillium brasilianum was purified to homogeneity after cultivation on a cellulose and xylan rich medium. The BGL was identified in a genomic library, and it was successfully expressed in Aspergillus oryzae. The BGL had excellent stability at elevated temperatures with no loss in activity after 24 h of incubation at 60A degrees C at pH 4-6, and the BGL was shown to have significantly higher stability at these conditions in comparison to Novozym 188 and to other fungal GH3 BGLs reported in the literature. The BGL had significant lower affinity for cellobiose compared with the artificial substrate para-nitrophenyl-beta-d-glucopyranoside (pNP-Glc) and further, pronounced substrate inhibition using pNP-Glc. Kinetic studies demonstrated the high importance of using cellobiose as substrate and glucose as inhibitor to describe the inhibition kinetics of BGL taking place during cellulose hydrolysis. A novel assay was developed to characterize this glucose inhibition on cellobiose hydrolysis. The assay uses labelled glucose-C-13(6) as inhibitor and subsequent mass spectrometry analysis to quantify the hydrolysis rates.
    Original languageEnglish
    JournalApplied Microbiology and Biotechnology
    Volume86
    Issue number1
    Pages (from-to)143-154
    ISSN0175-7598
    DOIs
    Publication statusPublished - 2010

    Keywords

    • Fungal expression
    • Kinetics
    • Purification
    • Cellulose hydrolysis
    • Glucose inhibition

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