The cellulosome produced by Piromyces sp. strain E2 during growth on filter paper was purified by using an optimized cellulose-affinity method consisting of steps of EDTA washing of the cellulose-bound protein followed by elution with water. Three dominant proteins were identified in the cellulosome preparation, with molecular masses of 55, 80 and 90kDa. Treatment of cellulose-bound cellulosome with a number of denaturing agents was also tested. Incubation with 0.5% (w/v) SDS or 8M urea released most cellulosomal proteins, while leaving the greater fraction of the 80, 90 and 170kDa components. To investigate the major 90kDa cellulosome protein further, the corresponding gene, cel9A, was isolated, using immunoscreening and N-terminal sequencing. Inspection of the cel9A genomic organization revealed the presence of four introns, allowing the construction of a consensus for introns in anaerobic fungi. The 2800bp cDNA clone contained an open reading frame of 2334bp encoding a 757-residue extracellular protein. Cel9A includes a 445-residue glycoside hydrolase family 9 catalytic domain, and so is the first fungal representative of this large family. Both modelling of the catalytic domain as well as the activity measured with low level expression in Escherichia coli indicated that Cel9A is an endoglucanase. The catalytic domain is succeeded by a putative β-sheet module of 160 amino acids with unknown function, followed by a threonine-rich linker and three fungal docking domains. Homology modelling of the Cel9A dockerins suggested that the cysteine residues present are all involved in disulphide bridges. The results presented here are used to discuss evolution of glycoside hydrolase family 9 enzymes.
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July 2002
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Research Article|
July 01 2002
An intron-containing glycoside hydrolase family 9 cellulase gene encodes the dominant 90 kDa component of the cellulosome of the anaerobic fungus Piromyces sp. strain E2
Peter J.M. STEENBAKKERS;
Peter J.M. STEENBAKKERS
∗Department of Microbiology, Faculty of Science, University of Nijmegen, Toernooiveld 1, NL-6525 ED Nijmegen, The Netherlands
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Wimal UBHAYASEKERA;
Wimal UBHAYASEKERA
†Department of Molecular Biology, Swedish University of Agricultural Sciences, Biomedical Centre, P.O. Box 590, SE-751 24 Uppsala, Sweden
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Harry J.A.M. GOOSSEN;
Harry J.A.M. GOOSSEN
∗Department of Microbiology, Faculty of Science, University of Nijmegen, Toernooiveld 1, NL-6525 ED Nijmegen, The Netherlands
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Erik M.H.M. van LIEROP;
Erik M.H.M. van LIEROP
∗Department of Microbiology, Faculty of Science, University of Nijmegen, Toernooiveld 1, NL-6525 ED Nijmegen, The Netherlands
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Chris van der DRIFT;
Chris van der DRIFT
∗Department of Microbiology, Faculty of Science, University of Nijmegen, Toernooiveld 1, NL-6525 ED Nijmegen, The Netherlands
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Godfried D. VOGELS;
Godfried D. VOGELS
∗Department of Microbiology, Faculty of Science, University of Nijmegen, Toernooiveld 1, NL-6525 ED Nijmegen, The Netherlands
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Sherry L. MOWBRAY;
Sherry L. MOWBRAY
†Department of Molecular Biology, Swedish University of Agricultural Sciences, Biomedical Centre, P.O. Box 590, SE-751 24 Uppsala, Sweden
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Huub J.M. Op den CAMP
Huub J.M. Op den CAMP
1
∗Department of Microbiology, Faculty of Science, University of Nijmegen, Toernooiveld 1, NL-6525 ED Nijmegen, The Netherlands
1To whom correspondence should be addressed (e-mail huubcamp@sci.kun.nl).
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Publisher: Portland Press Ltd
Received:
December 20 2001
Revision Received:
March 20 2002
Accepted:
April 16 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2002
2002
Biochem J (2002) 365 (1): 193–204.
Article history
Received:
December 20 2001
Revision Received:
March 20 2002
Accepted:
April 16 2002
Citation
Peter J.M. STEENBAKKERS, Wimal UBHAYASEKERA, Harry J.A.M. GOOSSEN, Erik M.H.M. van LIEROP, Chris van der DRIFT, Godfried D. VOGELS, Sherry L. MOWBRAY, Huub J.M. Op den CAMP; An intron-containing glycoside hydrolase family 9 cellulase gene encodes the dominant 90 kDa component of the cellulosome of the anaerobic fungus Piromyces sp. strain E2. Biochem J 1 July 2002; 365 (1): 193–204. doi: https://doi.org/10.1042/bj20011866
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