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

Krogh, Kristian B. R. M.; Harris, Paul V.; Olsen, Carsten L.; Johansen, Katja S.; Hojer-Pedersen, Jesper; Börjesson, Johan LU and Olsson, Lisbeth (2010) In Applied Microbiology and Biotechnology 86(1). p.143-154
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.... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Fungal, Purification, Cellulose hydrolysis, Glucose inhibition, expression, Kinetics
in
Applied Microbiology and Biotechnology
volume
86
issue
1
pages
143 - 154
publisher
Springer
external identifiers
  • wos:000274543200015
  • scopus:77149128346
ISSN
1432-0614
DOI
10.1007/s00253-009-2181-7
language
English
LU publication?
yes
id
bf417611-2d38-438f-96a9-4ab1a291f9a9 (old id 1568986)
date added to LUP
2010-03-17 10:07:57
date last changed
2018-05-29 10:33:32
@article{bf417611-2d38-438f-96a9-4ab1a291f9a9,
  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.},
  author       = {Krogh, Kristian B. R. M. and Harris, Paul V. and Olsen, Carsten L. and Johansen, Katja S. and Hojer-Pedersen, Jesper and Börjesson, Johan and Olsson, Lisbeth},
  issn         = {1432-0614},
  keyword      = {Fungal,Purification,Cellulose hydrolysis,Glucose inhibition,expression,Kinetics},
  language     = {eng},
  number       = {1},
  pages        = {143--154},
  publisher    = {Springer},
  series       = {Applied Microbiology and Biotechnology},
  title        = {Characterization and kinetic analysis of a thermostable GH3 beta-glucosidase from Penicillium brasilianum},
  url          = {http://dx.doi.org/10.1007/s00253-009-2181-7},
  volume       = {86},
  year         = {2010},
}