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Title: Trichoderma .beta.-glucosidase

Abstract

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl3, and the corresponding BGL3 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL3, recombinant BGL3 proteins and methods for producing the same.

Inventors:
; ; ;
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1175603
Patent Number(s):
6,982,159
Application Number:
09/957,880
Assignee:
Genencor International, Inc. (Palo Alto, CA) OSTI
DOE Contract Number:
AC36-99GO10337
Resource Type:
Patent
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Dunn-Coleman, Nigel, Goedegebuur, Frits, Ward, Michael, and Yao, Jian. Trichoderma .beta.-glucosidase. United States: N. p., 2006. Web.
Dunn-Coleman, Nigel, Goedegebuur, Frits, Ward, Michael, & Yao, Jian. Trichoderma .beta.-glucosidase. United States.
Dunn-Coleman, Nigel, Goedegebuur, Frits, Ward, Michael, and Yao, Jian. Tue . "Trichoderma .beta.-glucosidase". United States. doi:. https://www.osti.gov/servlets/purl/1175603.
@article{osti_1175603,
title = {Trichoderma .beta.-glucosidase},
author = {Dunn-Coleman, Nigel and Goedegebuur, Frits and Ward, Michael and Yao, Jian},
abstractNote = {The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl3, and the corresponding BGL3 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL3, recombinant BGL3 proteins and methods for producing the same.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {Tue Jan 03 00:00:00 EST 2006},
month = {Tue Jan 03 00:00:00 EST 2006}
}

Patent:

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  • We have demonstrated the induction by sophorose of ..beta..-glucosidase and other cellulases in Trichoderma cultures, isolated large quantities of intact RNA from induced and noninduced cultures, isolated large quantities of high molecular weight DNA free from contaminating polysaccarides and obtained about 3000 recombinant clones containing inserts, presumably of restriction enzyme cleaved Trichoderma DNA. Additionally we have examined the effect of sophorose on the ..beta..-glucosidase of Sporotrichum pulverentum, established an in vitro translation system, and prepared enzymes from the bacterium Oerskovia which will aid in attempts to insert ethanol producing genes into Trichoderma. 28 refs., 2 figs.
  • Our project was to isolate and characterize the enzyme ..beta..-glucosidase and to clone and characterize the ..beta..-glucosidase gene; our goal is to clone and characterize each of the cellulase genes from Trichoderma. The induction of the Trichoderma reesei cellulase complex by cellulose and by the soluble inducer, sophorose, has been demonstrated. Although the induction of the cellulase complex has previously been well documented, the induction of ..beta..-glucosidase had been questioned. 49 refs., 6 figs., 2 tabs.
  • The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.
  • A simple technique, using an esculin-ferric salt visualization system, for selective isolation of mutants of Trichoderma reesei was employed. The ..beta..-glucosidase crude enzyme complexes of the 17 mutants isolated from some 66,000 colonies screened were characterized. Type of inhibition (competitive, noncompetitive) by glucose, and kinetic parameters K/sub m/ (mM pNPG), V/sub max/ (units/mg extracellular protein), and K/sub i/ slopes (mM glucose) were determined for the mutants using p-nitrophenyl ..beta..-D-glucoside (pNPG) as substrate. All the isolates were inhibited competitively by glucose, but certain of them were less sensitive than parent and wild-type to inhibition by glucose. 5 figures, 1 table.
  • The inhibition of /beta/-glucosidase in Trichoderma reesei C30 cellulase by D-glucose, its isomers, and derivatives was studied using cellobiose and p-nitrophenyl-/beta/-glucoside as substrates for determining enzyme activity. The enzymatic hydrolysis of both substrates was inhibited competitively by glucose. This inhibition by glucose was maximal at pH 4.8 and no inhibition was observed at pH 6.5 and above. The /alpha/anomer of glucose inhibited /beta/-glucosidase to a greater extent than did the /beta/form. Compared with D-glucose, L-glucose, D-glucose-6-phosphate, and D-glucose-1-phosphate inhibited the enzyme to a much lesser extent, unlike D-glucose-L-cysteine which was almost as inhibitory as glucose itself when cellobiose was usedmore » as substrate. Fructose (2-100mM) was found to be a poor inhibitor of the enzyme. It is suggested that high rates of cellobiose hydrolysis catalyzed by /beta/-glucosidase may be prolonged by converting the reaction product glucose to fructose using a suitable preparation of glucose isomerase. 20 refs.« less