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"Free Enzyme and Cellulosome Preparations for Cellulose Hydrolysis" in Patent Application Approval Process

February 21, 2014

By a News Reporter-Staff News Editor at Energy Weekly News -- A patent application by the inventors RESCH, Michael (Golden, CO); BAKER, John O. (Golden, CO); QI, Xu (Golden, CO); ADNEY, William S. (Golden, CO); DECKER, Steven R. (Golden, CO); HIMMEL, Michael E. (Golden, CO); DONOHOE, Bryon (Golden, CO), filed on July 29, 2013, was made available online on February 6, 2014, according to news reporting originating from Washington, D.C., by VerticalNews correspondents.

This patent application is assigned to Alliance for Sustainable Energy, LLC.

The following quote was obtained by the news editors from the background information supplied by the inventors: "Plant cell walls represent a vast, renewable carbon source in the biosphere. Biofuels derived from plant cell wall material is a promising renewable energy technology in part because of the large amount and low cost of the biomass feedstock. Efficient action of cellulases to release fermentable sugars from biomass cellulose is an important step in making this conversion economically viable.

"Nature has evolved multiple enzymatic strategies for the degradation of plant cell wall polysaccharides that are central to carbon and nitrogen flux in the biosphere and an integral part of renewable biofuels development. Many biomass-degrading organisms secrete cocktails of individual enzymes with one or a few catalytic domains per enzyme, whereas some bacteria synthesize large multi-enzyme complexes, termed cellulosomes, which may contain 50-60 catalytic units per complex. Both enzyme systems employ similar catalytic chemistry, but the physical mechanisms by which these enzyme systems degrade polysaccharides have not been compared directly.

"These enzymatic strategies largely rely on glycoside hydrolases, oxidative enzymes, and other accessory proteins. Secreted free enzyme cocktails typically contain various proteins that diffuse independently of one another and, via different substrate specificities, work together to degrade biomass. These free enzymes range from systems in which the enzymes contain one catalytic unit to systems in which there may be several catalytic units per protein. In particular, the fungus Hypocrea jecorina (formally Trichoderma reesei) secretes a potent cocktail of free carbohydrate-active enzymes to degrade cellulose and hemicellulose. The T. reesei enzyme cocktail and related systems typically secrete enzymes with only one catalytic unit per protein.

"Sorting through the vast array of bacterial and fungal enzymes to determine the optimal combination of activities for cellulose degradation is a continuing challenge to the biofuels industry. Discovering enzyme cocktails that work together to increase the efficiency of sugar release from biomass sources is essential to making biofuels economically competitive with petroleum-based fuels.

"The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the drawings."

In addition to the background information obtained for this patent application, VerticalNews journalists also obtained the inventors' summary information for this patent application: "The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools and methods that are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other improvements.

"Exemplary embodiments provide methods for degrading cellulose or lignocellulosic biomass by contacting a cellulose containing material or lignocellulosic biomass with an enzyme cocktail comprising at least one fungal cellulase and at least one cellulosome complex.

"In certain embodiments, the cellulosome complex is a high molecular weight (HMW) cellulosome complex. In some embodiments, the cellulosome complex is from a bacterium of the genus Clostridium, such as the bacterium C. thermocellum.

"In some embodiments, the fungal cellulase comprises a Family 7 cellobiohydrolase such as Cel7A. In certain embodiments, the Family 7 cellobiohydrolase is from a fungus of the genus Hypocrea, such as the fungus H. jecorina.

"In further embodiments, the enzyme cocktail further comprises a .beta.-glucosidase, a hemicellulase, or an oxidoreductase. In some embodiments, the cellulase comprises a commercial enzyme preparation such as CTec2.

"In certain embodiments, the contacting a cellulose containing material or lignocellulosic biomass is carried out at a temperature of between C. or at C.

"Also provided are enzyme cocktails comprising at least one HMW cellulosome complex and a Family 7 cellobiohydrolase.

"Further provided are methods for producing a biofuel from lignocellulosic biomass by contacting the lignocellulosic biomass with an enzyme cocktail described herein and converting the sugars to a biofuel by fermentation.

"In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following descriptions.


"Exemplary embodiments are illustrated in referenced figures of the drawings. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than limiting.

"FIG. 1 shows a comparison of cellulosomes and free enzymes in the digestion of cellulose and biomass substrates Avicel (A), Whatman filter paper (B), dilute acid pretreated switchgrass (C) and dilute acid pretreated poplar (D).

"FIG. 2 shows TEM micrographs of Avicel particles digested with free enzymes or cellulosomes. Scale bars A-C, F, G=200 nm, D, H=500 nm, E=100 nm.

"FIG. 3 shows TEM micrographs of immuno-labeled dilute acid pretreated switchgrass samples digested with free enzymes (A, A') or cellulosomes (B, B') for 24 hours. Scale bars=0.5 .mu.m.

"FIG. 4 illustrates the synergistic effects of free enzymes and cellulosomes on Avicel examined by activity assays and TEM imaging. Scale bars=500 nm.

"FIG. 5 shows an illustration of the mechanisms by which free enzymes (left) and cellulosomes (right) differ in their action on cellulose microfibril bundles.

"FIG. 6A illustrates size exclusion chromatography (SEC) separation and pooling of fractions containing the HMW cellulosomes. FIG. 6B shows results from native PAGE analysis used to identify the fractions that contained HMW cellulosomes.

"FIG. 7 illustrates enhanced cellulase activity of chromatographically-selected cellulosome fraction.

"FIG. 8 shows an optimization of cellulosome enzymatic activity conditions on Avicel as a function of aerobic or anaerobic conditions, .beta.-glucosidase presence, and the presence of chemical protectants.

"FIG. 9 shows a comparison of cellulosome and free enzyme (CTec2) hydrolytic activity on phosphoric acid swollen cellulose (PASC).

"FIG. 10 shows the effect of adding hemicellulase enzymes to the cell free cellulosome on pretreated (A) or untreated (B) switchgrass enzymatic digestions.

"FIG. 11 shows TEM micrographs of immuno-labeled Avicel PH101 digested with CTec2 for 120 hours (A, B) or HMW cellulosomes for 24 hours (C, D) to achieve a cellulose conversion of about 65% in each case. Scale bars=200 nm.

"FIG. 12 shows higher magnification TEM micrographs of dilute acid pretreated switchgrass samples (A, B) and enzymatic digestions of pretreated switchgrass (A', B'). Scale bar=2 .mu.m."

URL and more information on this patent application, see: RESCH, Michael; BAKER, John O.; QI, Xu; ADNEY, William S.; DECKER, Steven R.; HIMMEL, Michael E.; DONOHOE, Bryon. Free Enzyme and Cellulosome Preparations for Cellulose Hydrolysis. Filed July 29, 2013 and posted February 6, 2014. Patent URL:

Keywords for this news article include: Biotechnology, Biofuel, Cellulases, Oil and Gas, Cellulosomes, Glucosidases, Bioengineering, Glycoside Hydrolases, Enzymes and Coenzymes, Cell Surface Extensions, Alliance for Sustainable Energy LLC.

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Source: Energy Weekly News

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