WO1994029460A1 - Active heteroconjugates of cellobiohydrolase and beta-glucosidase - Google Patents

Active heteroconjugates of cellobiohydrolase and beta-glucosidase Download PDF

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Publication number
WO1994029460A1
WO1994029460A1 PCT/US1994/006528 US9406528W WO9429460A1 WO 1994029460 A1 WO1994029460 A1 WO 1994029460A1 US 9406528 W US9406528 W US 9406528W WO 9429460 A1 WO9429460 A1 WO 9429460A1
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Prior art keywords
glucosidase
cellobiohydrolase
enzyme
cellulose
enzymes
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PCT/US1994/006528
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French (fr)
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John O. Baker
Michael E. Himmel
Karel Grohmann
Steven R. Thomas
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Midwest Research Institute
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01091—Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2445—Beta-glucosidase (3.2.1.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01021—Beta-glucosidase (3.2.1.21)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates, generally, to saccharification of cellulosic biomass. Even more particularly, this invention relates to an improved saccharification method utilizing enzymes. In another aspect, the present invention relates to active conjugates of defined enzymes.
  • cellulose Although an abundant biopolymer, cellulose is unique in that it is highly crystalline, insoluble in water, and highly resistant to depolymerization. Efficient enzymatic saccharification of crystalline cellulose to fermentable sugars requires the synergistic action of at least three different types of enzymes, endoglucanases, exoglucanases (e.g., cellobiohydrolases) , and /3-glucosidases.
  • the first two types of enzymes are described as "cellulases" in that both are essential for complete solubilization of crystalline cellulose; /3-glucosidases, on the other hand, are not considered to be cellulases because hydrolysis can proceed without this activity, albeit at a slower rate (Grohmann et al . , 1992. Emerging Technologies for Materials and Chemicals from Biomass. Vol. 460, American Chemical Society: Washington, D.C, pp. 354-392) .
  • /3-1,4-endoglucanases (EC 3.2.1.4) hydrolyze internal glycosidic bonds of the polysaccharide chains to produce new chain ends on the surface of cellulose crystals.
  • Cellobiohydrolases remove successive glucose-dimer (cellobiosyl) units from the exposed non-reducing chain ends.
  • -glucosidases (EC 3.2.1.21) cleave the released cellobiose molecules to glucose, which can then be fermented to fuel ethanol.
  • the endoglucanases and the cellobiohydrolases have specialized cellulose-binding domains (CBDs) (Knowles et al. 1987. Trends Biotechnol. 5:255-261) and, therefore, tend to be removed from the solution and concentrated (adsorbed) at the site of cellulolytic action, the surface of crystalline cellulose.
  • CBDs cellulose-binding domains
  • the /?-glucosidases do not have CBDs and tend to be dispersed throughout the fluid portion of the suspension.
  • the cellobiohydrolase enzymes are particularly sensitive to product inhibition by large accumulations of cellobiose and, therefore, operate at the highest rates when the steady-state concentration of cellobiose is restricted to low levels by the presence of high concentrations of j ⁇ -glucosidase. This condition is normally achieved by adding high levels of expensive S-glucosidase enzyme to the process broth. Unlike the endoglucanases and cellobiohydrolases, the ⁇ -glucosidases do not bind to cellulose are therefore not recoverable on undigested cellulose particles after the hydrolysis process is complete.
  • U.S. Patent No. 4,822,516 (Suzuki et al .
  • U.S. Patent No. 4,956,291 treats the production of enzymes for use in the solubilization of cellulosic materials, but the patent is for the selection and culture of a new strain of mold that is claimed to produce an enhanced mixture of activities for use in saccharification. Even though some of the mixtures are analyzed, and some information is given concerning properties of individual species as analyzed, these products are intended to be used as mixtures of naturally-occurring enzymes. No mention is made of the modification, chemically or by gene-fusion, of any specific enzyme components.
  • U.S. Patent No. 4,409,329 (Silver, 1983) describes the saccharification method, but concerns itself entirely with mechanical details of the construction of a reactor vessel that provides an environment of high mechanical shear, which is supposed to constantly provide new cellulose surfaces for enzyme to attack.
  • U.S. Patent No. 4,220,721 (Emert and Blotkamp, 1980) claims a method for the recycle and reuse of the principle components of a typical cellulase mixture used in saccharification. Nowhere does Emert's patent mention either modification of individual cellulase components (whether by chemical modification or genetic engineering) or kinetic enhancement of the solubilization.
  • the active heterodimers are prepared by connecting molecules of j ⁇ -glucosidase and cellobiohydrolase with a bifunctional reagent for the covalent modification of proteins, or by genetic fusion of the two polypeptide chains.
  • the invention comprises a conjugate enzyme molecule comprising a molecule of a ⁇ -glucosidase covalently linked to a molecule of cellobiohydrolase,in such a way that the ability of the -glucosidase to catalyze the hydrolysis of cellobiose to glucose, and the abilities of the cellobiohydrolase to bind to crystalline cellulose and to catalyze the cleavage of cellobiosyl residues from the non-reducing ends of the cellulose chains, are all retained in the combined molecule.
  • the invention also includes a process for the use of this protein heterodimer, along with a suitable endoglucanase, to convert cellulosic biomass to soluble sugars fermentable to fuel ethanol.
  • Chemically crosslinking the 3-glucosidase to the cellobiohydrolase by means of commercially-available bifunctional protein-modification reagents is one embodiment. Another embodiment is to use molecular biology techniques to fuse the genes coding for the two proteins, and to express the resulting fusion protein in a suitable host in order to produce a specific active heterodimer more economically.
  • enzymatically active conjugates consisting of one molecule of a -glucosidase, such as (but not limited to) that from Aspergillus niger or the smaller /3-glucosidase produced by Microbispora bispora, and one molecule of a cellobiohydrolase such as cellobiohydrolase I (CBH I) from Trichodexma reesei .
  • a -glucosidase such as (but not limited to) that from Aspergillus niger or the smaller /3-glucosidase produced by Microbispora bispora
  • CBH I cellobiohydrolase I
  • the conjugate is believed to be superior to the separate enzymes in two important respects: 1) the close juxtaposition of the cellobiose-hydrolyzing / ⁇ -glucosidase will relieve cellobiose-inhibition of the cellobiohydrolase by reducing locally high concentrations of this product through cleavage of cellobiose to the much-less-inhibitory glucose, and 2) the j ⁇ -glucosidase will be rendered easily recyclable due to adsorption (through the cellobiohydrolase binding domain) on spent substrate.
  • FIGURE 1 is a pictorial description of the process of crosslinking the j ⁇ -glucosidase and cellobiohydrolase enzymes in such a way as to yield active heterodimers.
  • FIGURE 2 is a pictorial description of the gene fusion process for covalently linking the two enzymes to produce an active heterodimer.
  • the notation P indicates placement of promoter DNA sequence.
  • the invention described herein converts the soluble j ⁇ -glucosidase to a cellulose-bound enzyme like the endoglucanases and cellobiohydrolases, by the expedient of crosslinking the j ⁇ -glucosidase (by chemical or gene-fusion techniques) to the cellobiohydrolase.
  • all three essential components of the cellulolytic consortium will be concentrated at the site of cellulose-crystal solubilization.
  • the 3-glucosidase will be located in very close proximity to the enzyme (cellobiohydrolase) that produces the substrate for the j ⁇ -glucosidase.
  • the primary e f f e c t o f t he j ⁇ -glucosidase/cellobiohydrolase conjugation upon the kinetics of the overall reaction has to do with the fact that cellobiose, the product of the cleavage catalyzed by cellobiohydrolase, is a potent competitive inhibitor of the cellobiohydrolase (Claeyssens et al. , 1989. Biochem. J. 261:819-825), and that this inhibition is substantially relieved by subsequent j ⁇ -glucosidase-catalyzed cleavage of cellobiose to the much-less-inhibitory glucose (Wright, 1988. supra) .
  • the steady-state concentration of cellobiose can in principle be reduced essentially to zero.
  • the j ⁇ -glucosidase activity may be increased either by using a j ⁇ -glucosidase with higher intrinsic activity per enzyme molecule, or by increasing the number of molecules of a given /3-glucosidase per unit volume, or both.
  • the invention described herein takes the latter approach, producing a high local concentration of j ⁇ -glucosidase in the immediate vicinity of the cellobiohydrolase.
  • Pretreated cellulosic biomass is a porous material, with much of the primary hydrolysis (by endoglucanases and cellobiohydrolases) occurring at the surfaces inside the pores (Grethlein, 1985. Bio/Technology 3:155-160; Neuman and Walker, 1991. Biotechnol. Bioeng. 40:226-234).
  • the inhibitory cellobiose is thus not produced free in the bulk solution, as would be the case for an enzyme reaction involving conversion of a small soluble substrate molecule to one or more different small product molecules.
  • the resulting restricted diffusion of the product molecule can result in quite elevated local concentrations of the inhibitory product, if the j ⁇ -glucosidase does not "follow the cellobiohydrolase into the pore", as provided for by the present invention.
  • the invention represents the combination of two elements: (1) the advantages in terms of enhanced kinetics to be expected from placing two individual members of the cellulolytic enzyme system in close proximity to each other in a way not found in nature and (2) the accomplishment of this goal by producing a covalently-linked cellobiohydrolase / S-glucosidase heterodimer, first by in vi tro chemical crosslinking of the two enzymes in order to prove the utility of such a construct, and then in vivo by production of a fusion protein, which latter method is expected to be more economical for a scaled-up process.
  • Examples in nature are the ordered arrays of enzymes on the membranes of mitochondria (Robinson et al . 1987. supra) and the formation of complexes between enzymes catalyzing sequential steps in various synthetic pathways, such as those for tryptophan (Matchett, 1974. J. Biol. Chem. 249:4041- 4049) and cholesterol (Miziorko, et al . , supra) .
  • Examples of man-made covalently-linked combinations of different functional proteins are provided by the antibody/nerve-growth-factor dimer used by Backman and coworkers ( CIRB Newsletter, QTR 1, 1993. Colorado Institute for Research in Biotechnology: Ft. Collins, CO, p.
  • crosslinking agents or “crosslinkers”
  • crosslinkers A large array of bifunctional protein-modification reagents, commonly referred to as “crosslinking agents” or “crosslinkers” , are available commercially, and their use in producing both intermolecular and intramolecular crosslinks in proteins has been extensively documented in the scientific and technical literature (Wong et al. 1993. Biocatalvst Design for Stability and Specificity, Vol. 516, American Chemical Society: Washington, DC, pp. 266-282; Gupta 1993. Biocatalvst Design for Stability and Specificity” , Vol. 516, American Chemical Society: Washington, DC, pp. 307-324) .
  • the molecules of these reagents consist of a central linker portion of varying length, connecting two reactive chemical functionalities, each capable of combining with one or more of the types of functional groups commonly found on the surfaces of proteins (typically amine, carboxyl, sulfhydryl or guanidino groups, but including any group with active hydrogens in the case of nitrene-generating functional groups on the crosslinker) .
  • the two reactive groups of a given reagent may be identical (as in a homobifunctional reagent) or different (as in heterobifunctional reagents) .
  • the heterobifunctional reagents are further divided into one group of reagents in which both functional groups are intrinsically “armed” and active from the moment the molecules are placed into solution, and another group of reagents in which at least one of the end-groups requires very specific activation (such as, for example, exposure to light of a certain wavelength and intensity) before it becomes capable of reacting with protein surface groups.
  • These two groups of heterobifunctional reagents might be termed “simultaneous” and “sequential" crosslinkers, respectively.
  • a major advantage of the "sequential" reagents with respect to the present invention is that they allow for the reaction of one end of the reagent with one of the two proteins that are to be connected, thus producing a uniformly modified and potentially activatable derivative of the protein. Excess free reagent, which might otherwise produce undesirable further reactions, can then be removed before the second protein is introduced and the activatable crosslinker groups now borne by the first protein are activated. If the first (crosslinker-bearing) protein can be immobilized during this step, rather than being free in solution, the probability of converting the protein starting material to undesirable side-products
  • cellobiohydrolase can be immobilized on crystalline cellulose through its cellulose-binding domain, and later eluted in active form under conditions of low ionic strength and/or mild alkalinity (Gilkes et al., 1988. J. Biol. Chem. 263:10401-10407) makes the cellobiohydrolase/j ⁇ -glucosidase system an especially attractive candidate for production of active protein heterodimers by such a strategy.
  • microcrystalline cellulose is not only an absorbent but also a substrate for cellobiohydrolase, this approach also should provide for substantial protection of the cellobiohydrolase active site during crosslinking; i.e., it should favor reaction of the crosslinkers and attachment of the j ⁇ -glucosidase in regions of the cellobiohydrolase molecule other than the immediate vicinity of the active site (where such modification might either chemically modify essential active-site functionalities or result in steric blockage of the active site) or the cellulose-binding face of the CBD (where modification would be equally disruptive of the intended function of the heteroconjugate) .
  • oligomers of the proteins (which are of differing, but comparable sizes) will be substantially larger than the monomer of either will make size-exclusion chromatography a useful technique for preparative separations.
  • Affinity chromatography on cellulose will be an effective means of differentiating those oligomeric species that possess a free and functional CBD from those oligomeric species that do not.
  • the genetic fusion of two enzyme activities into a single polypeptide product differs from chemical methods in that only co-linear fusions of strings of amino acids are produced. Branched chains are not produced by normal genetic machinery. Chemical fusions commonly result from the formation of one or more covalent linkages, each connecting an amino acid located in one polypeptide chain, and an amino acid located in the other polypeptide chain. Linkages involving only the end residues of the two chains (which would produce co-linear polypeptide chains) are possible, but are statistically unlikely to dominate in the product mixture.
  • Gene fusion permits the controlled synthesis of novel, bifunctional heterodimers of cellobiohydrolase and / ⁇ -glucosidase. It requires the availability of clones for each of the desired proteins. Coding sequences can be obtained from cDNA or genomic clones, or by polymerase chain reaction (PCR) from genomic DNA, cloned DNA, or messenger RNA populations. If conveniently located restriction sites are available, the two coding sequences can be spliced together using standard molecular biology methods. In vitro mutagenesis might be employed to create such restriction sites within either or both coding sequences. PCR technology allows the production of any desired DNA sequence from a template molecule.
  • Plasmid vectors are available which permit high level, inducible expression of foreign genes in each of these host organisms.
  • the gene fusion approach to formation of protein heterodimers avoids some problems associated with chemical crosslinking methods. Firstly, because of the inherent specificity of gene fusions, only heterodimers will be produced. Furthermore, each construction will yield a unique molecular species of heterodimer. While many possible constructions could be built, the previous statements will be true for each. Thus, each of a set of different heterodimers can be analyzed in isolation from all of the others. The degree of precision possible with the gene fusion approach to formation of heteroconjugates permits control over the exact position of the covalent linkage. Armed with some knowledge about the location of the essential regions of each of the proteins, fusions affecting the function of catalytic sites and the cellulose binding domain can be avoided. Enzyme Starting Materials
  • a large array of enzymes j ⁇ -glucosidase activities have been identified, in both fungal and bacterial sources.
  • the cellobiohydrolases I and II from T. reesei are somewhat unique in kinetic and biophysical properties and would not easily be replaced by other enzymes.
  • the construction of the conjugates considered by this invention should employ one of these enzymes as the non-j ⁇ -glucosidase component.
  • Those enzyme selected for application to this invention should be available in large quantities and amenable to efficient purification schemes.
  • the 3-glucosidase from the fungus Aspergillus niger and the cellobiohydrolase I from T. reesei are ideal enzymes for application to this invention.
  • Glutaraldehyde crosslinking has been used herein to form active homodimers of A. niger / ⁇ -glucosidase.
  • This "first step" towards the ultimate goal of producing active, heterodimers of j ⁇ -glucosidase and cellobiohydrolase shows that at least one proposed enzyme component of the system, the 3-glucosidase, is amenable to crosslinking methods (i.e., not easily denatured or otherwise inactivated during the exposure to crosslinking agent) .
  • crosslinking experiments were conducted by incubating j ⁇ -glucosidase (0.2 mg/mL) with 0.35 M glutaraldehyde at 22°C for 30 minutes.

Abstract

Active heterodimers are described which are prepared by connecting molecules of beta-glucosidase and cellobiohydrolase with a bifunctional reagent (i.e., a crosslinking agent) or by genetic fusion of the two polypeptide chains. The ability of the beta-glucosidase to catalyze hydrolysis of cellobiose to glucose, and the ability of cellobiohydrolase to bind to crystalline cellulose and to catalyze the cleavage of cellobiosyl residues from the non-reducing ends of the cellulose chains, are all retained in the combined molecule.

Description

Description
Active Heteroconiugates of
Cellobiohydrolase and Beta-Glucosidase
The United States Government has rights in this invention under Contract No. DE-AC02-83CH10093 between the United States Department of Energy and the National Renewable Energy Laboratory, a Division of the Midwest Research Institute.
Technical Field The present invention relates, generally, to saccharification of cellulosic biomass. Even more particularly, this invention relates to an improved saccharification method utilizing enzymes. In another aspect, the present invention relates to active conjugates of defined enzymes.
Background Art
Although an abundant biopolymer, cellulose is unique in that it is highly crystalline, insoluble in water, and highly resistant to depolymerization. Efficient enzymatic saccharification of crystalline cellulose to fermentable sugars requires the synergistic action of at least three different types of enzymes, endoglucanases, exoglucanases (e.g., cellobiohydrolases) , and /3-glucosidases. The first two types of enzymes are described as "cellulases" in that both are essential for complete solubilization of crystalline cellulose; /3-glucosidases, on the other hand, are not considered to be cellulases because hydrolysis can proceed without this activity, albeit at a slower rate (Grohmann et al . , 1992. Emerging Technologies for Materials and Chemicals from Biomass. Vol. 460, American Chemical Society: Washington, D.C, pp. 354-392) .
1. /3-1,4-endoglucanases (EC 3.2.1.4) hydrolyze internal glycosidic bonds of the polysaccharide chains to produce new chain ends on the surface of cellulose crystals.
2. Cellobiohydrolases (EC 3.2.1.91) remove successive glucose-dimer (cellobiosyl) units from the exposed non-reducing chain ends.
3. -glucosidases (EC 3.2.1.21) cleave the released cellobiose molecules to glucose, which can then be fermented to fuel ethanol.
The endoglucanases and the cellobiohydrolases have specialized cellulose-binding domains (CBDs) (Knowles et al. 1987. Trends Biotechnol. 5:255-261) and, therefore, tend to be removed from the solution and concentrated (adsorbed) at the site of cellulolytic action, the surface of crystalline cellulose. The /?-glucosidases, on the other hand, do not have CBDs and tend to be dispersed throughout the fluid portion of the suspension.
Cost sensitivity studies have shown that the successful commercialization of cellulase-based processes, such as the conversion of cellulose to fermentable sugars, is highly dependent on the cost of enzyme production. The enhancement of all enzyme activities (in terms of kinetic rates, or lifetimes in such processes, or both) would substantially decrease the requirement for enzyme loading in the process and therefore conserve these key components. Since fungal jβ-glucosidase is the most labile enzyme in this system under process conditions, and crucial for efficient saccharification, it would be very advantageous to be able to stabilize this enzyme. There has been previously reported a procedure for the intramolecular crosslinking of purified Aspergillus niger β- glucosidase using glutaraldehyde followed by borohydride reduction (Baker et al. , 1987. Biotechnol. Lett. 10:325-330). This procedure resulted in a modified enzyme activity that was markedly more stable at 65°C. Also important for efficient saccharification of cellulose are the cellobiohydrolase (CBH) enzymes which produce cellobiose from cellulose and have the potential to bind to cellulose, thus allowing recycle of this enzyme for reuse by collection of incompletely hydrolyzed cellulose from the fermentation system. The cellobiohydrolase enzymes are particularly sensitive to product inhibition by large accumulations of cellobiose and, therefore, operate at the highest rates when the steady-state concentration of cellobiose is restricted to low levels by the presence of high concentrations of jβ-glucosidase. This condition is normally achieved by adding high levels of expensive S-glucosidase enzyme to the process broth. Unlike the endoglucanases and cellobiohydrolases, the β-glucosidases do not bind to cellulose are therefore not recoverable on undigested cellulose particles after the hydrolysis process is complete. U.S. Patent No. 4,822,516 (Suzuki et al . , 1989) concerns the selection and purification of cellulase enzymes for use in detergent formulations, but what the authors are selecting for is the exact opposite of the properties one would want in a cellulase mixture for use in solubilizing crystalline cellulose to produce fermentable sugars. The property Suzuki, et al . , select for is a high activity against non-crystalline, amorphous cellulose and a very low activity against crystalline cellulosic materials such as cotton.
U.S. Patent No. 4,956,291 (Yamanobe et al. , 1990) treats the production of enzymes for use in the solubilization of cellulosic materials, but the patent is for the selection and culture of a new strain of mold that is claimed to produce an enhanced mixture of activities for use in saccharification. Even though some of the mixtures are analyzed, and some information is given concerning properties of individual species as analyzed, these products are intended to be used as mixtures of naturally-occurring enzymes. No mention is made of the modification, chemically or by gene-fusion, of any specific enzyme components.
U.S. Patent No. 4,409,329 (Silver, 1983) describes the saccharification method, but concerns itself entirely with mechanical details of the construction of a reactor vessel that provides an environment of high mechanical shear, which is supposed to constantly provide new cellulose surfaces for enzyme to attack. U.S. Patent No. 4,220,721 (Emert and Blotkamp, 1980) claims a method for the recycle and reuse of the principle components of a typical cellulase mixture used in saccharification. Nowhere does Emert's patent mention either modification of individual cellulase components (whether by chemical modification or genetic engineering) or kinetic enhancement of the solubilization.
There has not heretofore been provided an efficient manner for utilizing -glucosidase and cellobiohydrolase enzymes in a saccharification process having the advantages and features exhibited by the present invention.
Disclosure of the Invention
It is an object of the invention to provide active heterodimers of cellobiohydrolase and /3-glucosidase enzymes. It is another object of the invention to provide heterodimers of enzymes for use in saccharification of cellulosic substrates.
It is another object of the invention to provide means for reducing enzyme requirements in the saccharification of cellulosic biomass.
Additional objects, advantages and novel features of the invention shall be set forth in part in the description that follows and in part will become apparent to those skilled in the art upon examination of the following or may be learned by the practice of the invention. The objects and the advantages of the invention may be realized and attained by means of the instrumentalities and in combinations particularly pointed out in the appended claims. To achieve the foregoing and other objects and in accordance with the purpose of the present invention, as embodied and broadly described herein, the active heterodimers are prepared by connecting molecules of jβ-glucosidase and cellobiohydrolase with a bifunctional reagent for the covalent modification of proteins, or by genetic fusion of the two polypeptide chains.
The invention comprises a conjugate enzyme molecule comprising a molecule of a β-glucosidase covalently linked to a molecule of cellobiohydrolase,in such a way that the ability of the -glucosidase to catalyze the hydrolysis of cellobiose to glucose, and the abilities of the cellobiohydrolase to bind to crystalline cellulose and to catalyze the cleavage of cellobiosyl residues from the non-reducing ends of the cellulose chains, are all retained in the combined molecule. The invention also includes a process for the use of this protein heterodimer, along with a suitable endoglucanase, to convert cellulosic biomass to soluble sugars fermentable to fuel ethanol. Chemically crosslinking the 3-glucosidase to the cellobiohydrolase by means of commercially-available bifunctional protein-modification reagents is one embodiment. Another embodiment is to use molecular biology techniques to fuse the genes coding for the two proteins, and to express the resulting fusion protein in a suitable host in order to produce a specific active heterodimer more economically.
By use of bifunctional covalent chemical modification reagents (crosslinkers) , enzymatically active conjugates will be formed, consisting of one molecule of a -glucosidase, such as (but not limited to) that from Aspergillus niger or the smaller /3-glucosidase produced by Microbispora bispora, and one molecule of a cellobiohydrolase such as cellobiohydrolase I (CBH I) from Trichodexma reesei . The conjugate is believed to be superior to the separate enzymes in two important respects: 1) the close juxtaposition of the cellobiose-hydrolyzing /β-glucosidase will relieve cellobiose-inhibition of the cellobiohydrolase by reducing locally high concentrations of this product through cleavage of cellobiose to the much-less-inhibitory glucose, and 2) the jβ-glucosidase will be rendered easily recyclable due to adsorption (through the cellobiohydrolase binding domain) on spent substrate.
The conjugates described herein for cellulase system augmentation are not available in any biological system, so far as is known.
Other advantages of the apparatus and techniques of the invention will be apparent from the following detailed description and the accompanying drawings.
Brief Description of the Drawings
The accompanying drawings, which are incorporated in and form a part of the specification, illustrate the preferred embodiments of the present invention and, with the description, explain the principles of the invention. In the drawings:
FIGURE 1 is a pictorial description of the process of crosslinking the jβ-glucosidase and cellobiohydrolase enzymes in such a way as to yield active heterodimers. FIGURE 2 is a pictorial description of the gene fusion process for covalently linking the two enzymes to produce an active heterodimer. The notation P indicates placement of promoter DNA sequence.
Detailed Description of the Invention
The invention described herein converts the soluble jβ-glucosidase to a cellulose-bound enzyme like the endoglucanases and cellobiohydrolases, by the expedient of crosslinking the jβ-glucosidase (by chemical or gene-fusion techniques) to the cellobiohydrolase. In digestion mixtures containing the linked cellobiohydrolase/jβ-glucosidase pair, all three essential components of the cellulolytic consortium will be concentrated at the site of cellulose-crystal solubilization. Furthermore, the 3-glucosidase will be located in very close proximity to the enzyme (cellobiohydrolase) that produces the substrate for the jβ-glucosidase.
This organization and relocation of enzymes can be expected to have important effects on the kinetics of the saccharification of cellulosic biomass (and therefore on the quantities of enzymes required for conversion at practical rates) . Inasmuch as the cost of enzyme production represents a major portion of the total cost of fuel alcohol production from cellulosic biomass (Wright 1988. Energy Progress 8:71-78; Lynd et al., 1991. Science 251:1318-1323), reductions in the enzyme requirements, through improved reaction kinetics, increased recycle/reuse of enzymes, or both, will have a major impact on the economics of the process.
The primary e f f e c t o f t he jβ-glucosidase/cellobiohydrolase conjugation upon the kinetics of the overall reaction has to do with the fact that cellobiose, the product of the cleavage catalyzed by cellobiohydrolase, is a potent competitive inhibitor of the cellobiohydrolase (Claeyssens et al. , 1989. Biochem. J. 261:819-825), and that this inhibition is substantially relieved by subsequent jβ-glucosidase-catalyzed cleavage of cellobiose to the much-less-inhibitory glucose (Wright, 1988. supra) . Given a sufficiently high /3-glucosidase activity in the vicinity of the cellobiohydrolase, the steady-state concentration of cellobiose can in principle be reduced essentially to zero. The jβ-glucosidase activity may be increased either by using a jβ-glucosidase with higher intrinsic activity per enzyme molecule, or by increasing the number of molecules of a given /3-glucosidase per unit volume, or both. The invention described herein takes the latter approach, producing a high local concentration of jβ-glucosidase in the immediate vicinity of the cellobiohydrolase. This approach requires far smaller absolute quantities of the jβ-glucosidase than would be required to provide the same concentration of soluble enzyme in the vicinity of the cellobiohydrolase (i.e., far less enzyme than would be required if the jβ-glucosidase were dispersed evenly throughout the liquid phase, rather than being physically attached to the cellobiohydrolase) . It should also be noted that this linkage of jβ-glucosidase and cellobiohydrolytic activities, although useful in an engineering sense, has not been found in nature.
The importance of proximity effects on the kinetics of cellulose hydrolysis is magnified by the physical form of the substrate. Pretreated cellulosic biomass is a porous material, with much of the primary hydrolysis (by endoglucanases and cellobiohydrolases) occurring at the surfaces inside the pores (Grethlein, 1985. Bio/Technology 3:155-160; Neuman and Walker, 1991. Biotechnol. Bioeng. 40:226-234). The inhibitory cellobiose is thus not produced free in the bulk solution, as would be the case for an enzyme reaction involving conversion of a small soluble substrate molecule to one or more different small product molecules. The resulting restricted diffusion of the product molecule can result in quite elevated local concentrations of the inhibitory product, if the jβ-glucosidase does not "follow the cellobiohydrolase into the pore", as provided for by the present invention.
Conceptually, the invention represents the combination of two elements: (1) the advantages in terms of enhanced kinetics to be expected from placing two individual members of the cellulolytic enzyme system in close proximity to each other in a way not found in nature and (2) the accomplishment of this goal by producing a covalently-linked cellobiohydrolase /S-glucosidase heterodimer, first by in vi tro chemical crosslinking of the two enzymes in order to prove the utility of such a construct, and then in vivo by production of a fusion protein, which latter method is expected to be more economical for a scaled-up process. The enhancement of rates of sequential enzyme reactions by close juxtaposition of the enzyme producing a given chemical species with an enzyme utilizing that species as a substrate is referred to in the literature as a "channeling" effect (Vitto, et al. 1980. Cell Compartmentation and Metabolic Channeling. VEB Gustav Fischer Verlag, Jena and Elsevier/North Holland Biomedical Press: Amsterdam, Netherlands, pp. 135-146; Robinson et al., 1987. J. Biol . Chem. 262:1786-90; Cheung et al. , 1989. J. Biol. Chem. 264:4038-4044; Miziorko et al. , 1990. J. Biol. Chem. 265:9606-9609) . Examples in nature are the ordered arrays of enzymes on the membranes of mitochondria (Robinson et al . 1987. supra) and the formation of complexes between enzymes catalyzing sequential steps in various synthetic pathways, such as those for tryptophan (Matchett, 1974. J. Biol. Chem. 249:4041- 4049) and cholesterol (Miziorko, et al . , supra) . Examples of man-made covalently-linked combinations of different functional proteins are provided by the antibody/nerve-growth-factor dimer used by Backman and coworkers ( CIRB Newsletter, QTR 1, 1993. Colorado Institute for Research in Biotechnology: Ft. Collins, CO, p. 7) to smuggle active nerve-growth factor across the blood-brain barrier (in vitro chemical crosslinking) , the use of antibody/enzyme conjugates to target cancer cells by converting prodrugs to their lethal forms in the immediate vicinity of the cancer cells (Senter, 1990. FASEB Journal 4:188-193: Senter et al., 1991. Bioconiugate Chem. 2:447-451) ( in vi tro c r o s s l i nk i ng ) , a n d t h e a c t i v e jβ-galactosidase/galactokinase gene-fusion protein prepared by Bulow and coworkers (Bulow et al. , 1985. Bio/Technology 3 :821-823 : Bulow, 1987. Eur. J. Biochem. 163:443-448; Ljungcrantz et al. , 1989. Biochemistry 28:8786-8792) to study the channeling phenomenon.
Chemical Crosslinking Methods
A large array of bifunctional protein-modification reagents, commonly referred to as "crosslinking agents" or "crosslinkers" , are available commercially, and their use in producing both intermolecular and intramolecular crosslinks in proteins has been extensively documented in the scientific and technical literature (Wong et al. 1993. Biocatalvst Design for Stability and Specificity, Vol. 516, American Chemical Society: Washington, DC, pp. 266-282; Gupta 1993. Biocatalvst Design for Stability and Specificity" , Vol. 516, American Chemical Society: Washington, DC, pp. 307-324) . The molecules of these reagents consist of a central linker portion of varying length, connecting two reactive chemical functionalities, each capable of combining with one or more of the types of functional groups commonly found on the surfaces of proteins (typically amine, carboxyl, sulfhydryl or guanidino groups, but including any group with active hydrogens in the case of nitrene-generating functional groups on the crosslinker) . The two reactive groups of a given reagent may be identical (as in a homobifunctional reagent) or different (as in heterobifunctional reagents) . The heterobifunctional reagents are further divided into one group of reagents in which both functional groups are intrinsically "armed" and active from the moment the molecules are placed into solution, and another group of reagents in which at least one of the end-groups requires very specific activation (such as, for example, exposure to light of a certain wavelength and intensity) before it becomes capable of reacting with protein surface groups. These two groups of heterobifunctional reagents might be termed "simultaneous" and "sequential" crosslinkers, respectively.
A major advantage of the "sequential" reagents with respect to the present invention is that they allow for the reaction of one end of the reagent with one of the two proteins that are to be connected, thus producing a uniformly modified and potentially activatable derivative of the protein. Excess free reagent, which might otherwise produce undesirable further reactions, can then be removed before the second protein is introduced and the activatable crosslinker groups now borne by the first protein are activated. If the first (crosslinker-bearing) protein can be immobilized during this step, rather than being free in solution, the probability of converting the protein starting material to undesirable side-products
(such as protein homodimers rather than the desired heterodimers) can be greatly reduced. The fact that the cellobiohydrolase can be immobilized on crystalline cellulose through its cellulose-binding domain, and later eluted in active form under conditions of low ionic strength and/or mild alkalinity (Gilkes et al., 1988. J. Biol. Chem. 263:10401-10407) makes the cellobiohydrolase/jβ-glucosidase system an especially attractive candidate for production of active protein heterodimers by such a strategy. Because microcrystalline cellulose is not only an absorbent but also a substrate for cellobiohydrolase, this approach also should provide for substantial protection of the cellobiohydrolase active site during crosslinking; i.e., it should favor reaction of the crosslinkers and attachment of the jβ-glucosidase in regions of the cellobiohydrolase molecule other than the immediate vicinity of the active site (where such modification might either chemically modify essential active-site functionalities or result in steric blockage of the active site) or the cellulose-binding face of the CBD (where modification would be equally disruptive of the intended function of the heteroconjugate) .
The above-described strategy, involving reversible immobilization of the cellobiohydrolase during the crosslinking procedure, is only one possible approach. A conceptually much simpler approach is simply to add a crosslinking reagent to a mixture of /β-glucosidase and cellobiohydrolase, with both enzymes free in solution, and then to use standard protein purification techniques to isolate the desired active heterodimers from the mixture of products to be expected. While simpler in execution than the approach that involves immobilization of one of the proteins, this free- solution approach is expected to result in lower yields of the desired product. Whatever crosslinking strategy is employed, there will be a need for separation and characterization of the components of a product mixture. The fact that oligomers of the proteins (which are of differing, but comparable sizes) will be substantially larger than the monomer of either will make size-exclusion chromatography a useful technique for preparative separations. Affinity chromatography on cellulose will be an effective means of differentiating those oligomeric species that possess a free and functional CBD from those oligomeric species that do not. Even among active, cellulose-binding dimers, there can be substantial heterogeneity traceable to the utilization of different groups on the protein surfaces for formation of crosslinks, with the resultant production of heterodimers having different rotational orientations of the two components. While some of these "orientational isomers" may be functionally equivalent in the saccharification, it may well be of advantage, in the developmental process, to resolve them and characterize them separately. In this respect, ion-exchange chromatography can be expected to be useful, in that different orientations of the two protein components can be expected to expose different ionic groups on outer surfaces of the conjugate as a whole. The hydrolysis of the chromogenic substrate p- nitrophenyl-jβ-D-glucopyranoside (or of the fluorogenic substrate 4-methylumbelliferyl-jβ-D-glucoside) provides a convenient assay for the presence of jβ-glucosidase activity in either chromatographic column fractions or in native polyacrylamide gel electrophoresis bands. There is not an equally convenient technique for the separate assay of cellobiohydrolase activity in oligomeric species, but monoclonal antibodies against the native, monomeric starting species provide a powerful technique for tracking the incorporation of the individual monomers into oligomers, whether the respective activities can be conveniently measured or not. The final proof that the desired active cellobiohydrolase/jβ-glucosidase dimer has been created and purified will of course be the direct production of glucose from crystalline cellulose by a purified oligomeric protein species, used in conjunction with an endoglucanase as the only other protein catalyst.
Gene Fusion Method The genetic fusion of two enzyme activities into a single polypeptide product differs from chemical methods in that only co-linear fusions of strings of amino acids are produced. Branched chains are not produced by normal genetic machinery. Chemical fusions commonly result from the formation of one or more covalent linkages, each connecting an amino acid located in one polypeptide chain, and an amino acid located in the other polypeptide chain. Linkages involving only the end residues of the two chains (which would produce co-linear polypeptide chains) are possible, but are statistically unlikely to dominate in the product mixture.
Gene fusion permits the controlled synthesis of novel, bifunctional heterodimers of cellobiohydrolase and /β-glucosidase. It requires the availability of clones for each of the desired proteins. Coding sequences can be obtained from cDNA or genomic clones, or by polymerase chain reaction (PCR) from genomic DNA, cloned DNA, or messenger RNA populations. If conveniently located restriction sites are available, the two coding sequences can be spliced together using standard molecular biology methods. In vitro mutagenesis might be employed to create such restriction sites within either or both coding sequences. PCR technology allows the production of any desired DNA sequence from a template molecule. Whatever strategy is adopted, it is of paramount importance to ensure that the coding regions of both polypeptides are in the same translational reading frame. This is essential for formation of the desired product. The novel coding sequence must also be coupled with DNA sequences which will promote its expression in vivo in an appropriate host organism
(e.g., probably a bacterial species, such as, Escherichia coli , Bacillus subtilis, or Streptomyces lividans) . Plasmid vectors are available which permit high level, inducible expression of foreign genes in each of these host organisms.
The gene fusion approach to formation of protein heterodimers avoids some problems associated with chemical crosslinking methods. Firstly, because of the inherent specificity of gene fusions, only heterodimers will be produced. Furthermore, each construction will yield a unique molecular species of heterodimer. While many possible constructions could be built, the previous statements will be true for each. Thus, each of a set of different heterodimers can be analyzed in isolation from all of the others. The degree of precision possible with the gene fusion approach to formation of heteroconjugates permits control over the exact position of the covalent linkage. Armed with some knowledge about the location of the essential regions of each of the proteins, fusions affecting the function of catalytic sites and the cellulose binding domain can be avoided. Enzyme Starting Materials
A large array of enzymes jβ-glucosidase activities have been identified, in both fungal and bacterial sources. The cellobiohydrolases I and II from T. reesei are somewhat unique in kinetic and biophysical properties and would not easily be replaced by other enzymes. The construction of the conjugates considered by this invention should employ one of these enzymes as the non-jβ-glucosidase component. Those enzyme selected for application to this invention should be available in large quantities and amenable to efficient purification schemes. The 3-glucosidase from the fungus Aspergillus niger and the cellobiohydrolase I from T. reesei are ideal enzymes for application to this invention.
EXAMPLE 1
Glutaraldehyde crosslinking has been used herein to form active homodimers of A. niger /β-glucosidase. This "first step" towards the ultimate goal of producing active, heterodimers of jβ-glucosidase and cellobiohydrolase shows that at least one proposed enzyme component of the system, the 3-glucosidase, is amenable to crosslinking methods (i.e., not easily denatured or otherwise inactivated during the exposure to crosslinking agent) . These crosslinking experiments were conducted by incubating jβ-glucosidase (0.2 mg/mL) with 0.35 M glutaraldehyde at 22°C for 30 minutes. After crosslinking, excess glutaraldehyde was removed by diafiltration and the modified enzyme was treated at pH 9.0 with 2.87 mM potassium borohydride to reduce and stabilize the Schiff-base crosslinks formed by the glutaraldehyde treatment. Analysis of the products by high-performance size-exclusion chromatography showed the product mixture to be high in a species very nearly twice the molecular weight of native, monomolecular A. niger jβ-glucosidase (240,000 Daltons) , and having kinetic parameters (Vmx and K essentially indistinguishable from those of the native enzyme.
The foregoing is considered as illustrative only on the principles of the invention. Further, because numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and operation shown and described, and accordingly all suitable modifications and equivalents may be resorted to falling within the scope of the invention as defined by the claims which follow.

Claims

Claims The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows: 1. A process for hydrolysis of cellulose to produce glucose in the presence of /3-glucosidase enzyme and cellobiohydrolase enzyme, wherein said enzymes are covalently linked together.
2. A process in accordance with claim 1, wherein said enzymes are linked by means of chemical crosslinking or genetic fusion of two coding sequences.
3. An improved process for producing ethanol from hydrolyzed cellulose, the improvement of which comprises hydrolyzing said cellulose in the presence of a first enzyme comprising /3-glucosidase and a second enzyme comprising cellobiohydrolase enzyme, wherein said first and second enzymes are covalently linked together.
4. A heteroconjugate comprising jβ-glucosidase and cellobiohydrolase connected to each other by means of a crosslinking agent or as regions of a single polypeptide chain produced by gene fusion.
5. A process in accordance with claim 1, wherein said jβ-glucosidase enzyme is from Aspergillus niger, and said cellobiohydrolase enzyme is from Trichodenna reesei .
6. A process in accordance with claim 3, wherein said jβ-glucosidase enzyme is from Aspergillus niger, and said cellobiohydrolase enzyme is from Trichodenna reesei .
7. A process in accordance with claim 4, wherein said jβ-glucosidase enzyme is from Aspergillus niger, and said cellobiohydrolase enzyme is from Trichodenna reesei .
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WO1998016633A1 (en) * 1996-10-11 1998-04-23 Novo Nordisk A/S Alpha-amylase fused to cellulose binding domain, for starch degradation
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US6818803B1 (en) 1997-06-26 2004-11-16 Wisconsin Alumni Research Foundation Transgenic plants as an alternative source of lignocellulosic-degrading enzymes
WO1999057250A1 (en) * 1998-05-01 1999-11-11 The Procter & Gamble Company Laundry detergent and/or fabric care compositions comprising a modified enzyme
WO1999057252A1 (en) * 1998-05-01 1999-11-11 The Procter & Gamble Company Laundry detergent and/or fabric care compositions comprising a modified enzyme
WO1999057157A1 (en) * 1998-05-01 1999-11-11 The Procter & Gamble Company Laundry detergent and/or fabric care compositions comprising a modified antimicrobial protein
US6468955B1 (en) 1998-05-01 2002-10-22 The Proctor & Gamble Company Laundry detergent and/or fabric care compositions comprising a modified enzyme
US6465410B1 (en) 1999-04-30 2002-10-15 The Procter & Gamble Laundry detergent and/or fabric care composition comprising a modified antimicrobial protein
KR20010007901A (en) * 1999-10-20 2001-02-05 서주원 β-Glucosidase obtained from oats and process for preparation of genistein or daidzein using it
WO2005093050A2 (en) * 2004-03-25 2005-10-06 Genencor International, Inc. Cellulase fusion protein and heterologous cellulase fusion construct encoding the same
WO2005093073A1 (en) * 2004-03-25 2005-10-06 Genencor International, Inc. Exo-endo cellulase fusion protein
WO2005093050A3 (en) * 2004-03-25 2006-08-24 Genencor Int Cellulase fusion protein and heterologous cellulase fusion construct encoding the same
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EP2029761A1 (en) * 2006-06-22 2009-03-04 Iogen Energy Corporation Enzyme compositions and methods for the improved enzymatic hydrolysis of cellulose
EP2029762A1 (en) * 2006-06-22 2009-03-04 Iogen Energy Corporation Enzyme compositions for the improved enzymatic hydrolysis of cellulose and methods of using same
EP2029761A4 (en) * 2006-06-22 2010-04-28 Iogen Energy Corp Enzyme compositions and methods for the improved enzymatic hydrolysis of cellulose
EP2029762A4 (en) * 2006-06-22 2010-04-28 Iogen Energy Corp Enzyme compositions for the improved enzymatic hydrolysis of cellulose and methods of using same
US8202709B2 (en) 2006-06-22 2012-06-19 Iogen Energy Corporation Enzyme compositions and methods for the improved enzymatic hydrolysis of cellulose
US8318461B2 (en) 2006-06-22 2012-11-27 Iogen Energy Corporation Enzyme compositions for the improved enzymatic hydrolysis of cellulose and methods of using same

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