Characterization and kinetic analysis of a thermostable GH3 beta-glucosidase from Penicillium brasilianum.
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 60 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-13C6 as inhibitor and subsequent mass spectrometry analysis to quantify the hydrolysis rates.
Krogh
Kristian B R M
KB
Center for Microbial Biotechnology, Technical University of Denmark, Building 223, 2800 Lyngby, Denmark.
Harris
Paul V
PV
Olsen
Carsten L
CL
Johansen
Katja S
KS
Hojer-Pedersen
Jesper
J
Borjesson
Johan
J
Olsson
Lisbeth
L
D001236:Aspergillus oryzae / Q000201:enzymology / Q000235:genetics; D001709:Biotechnology / Q000379:methods; D002482:Cellulose / Q000737:chemistry / Q000378:metabolism*; D003470:Culture Media; D004795:Enzyme Stability; D015723:Gene Library; D005947:Glucose / Q000737:chemistry / Q000494:pharmacology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014990:Xylans / Q000378:metabolism; D001617:beta-Glucosidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D003470:Culture Media; D011994:Recombinant Proteins; D014990:Xylans; D002482:Cellulose; D001617:beta-Glucosidase; D005947:Glucose