A highly expressed family 1 beta-glucosidase with transglycosylation capacity from the anaerobic fungus Piromyces sp. E2.
Anaerobic fungi have very high cellulolytic activities and thus degrade cellulose very efficiently. In cellulose hydrolysis, beta-glucosidases play an important role in prevention of product inhibition because they convert oligosaccharides to glucose. A beta-glucosidase gene (cel1A) was isolated from a cDNA library of the anaerobic fungus Piromyces sp. E2. Sequence analysis revealed that the gene encodes a modular protein with a calculated mass of 75800 Da and a pI of 5.05. A secretion signal was followed by a negatively charged domain with unknown function. This domain was coupled with a short linker to a catalytic domain that showed high homology with glycosyl hydrolases belonging to family 1. Southern blot analysis revealed the multiplicity of the gene in the genome. Northern analysis showed that growth on fructose resulted in a high expression of cel1A. The cel1A gene was successfully expressed in Pichia pastoris. The purified heterologously expressed protein was shown to be encoded by the cel1A gene by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis of a tryptic digest. Purified heterologous Cel1A was active towards several artificial and natural substrates with beta-1-4 linked glucose molecules with a remarkably high activity on cellodextrins. The enzyme was strongly inhibited by D-glucono-1,5-delta-lactone (K(i)=22 microM), but inhibition by glucose was much less (K(i)=9.5 mM). pH and temperature optimum were 6 and 39 degrees C, respectively. The enzyme was fairly stable, retaining more than 75% of its activity when incubated at 37 degrees C for 5 weeks. Transglycosylation activity could be demonstrated by MALDI-TOF MS analysis of products formed during degradation of cellopentaose.
Harhangi
Harry R
HR
Department of Microbiology, Faculty of Science, University of Nijmegen, Toernooiveld 1, NL-6525 ED, Nijmegen, The Netherlands.
Steenbakkers
Peter J M
PJ
Akhmanova
Anna
A
Jetten
Mike S M
MS
van der Drift
Chris
C
Op den Camp
Huub J M
HJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D002482:Cellulose / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018628:Gene Dosage; D015723:Gene Library; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D010802:Phylogeny; D010843:Pichia / Q000378:metabolism; D020089:Piromyces / Q000201:enzymology / Q000235:genetics*; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D005656:Fungal Proteins; D009844:Oligosaccharides; C019193:maltopentaose; D002482:Cellulose; D001617:beta-Glucosidase