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US 2004/0005674 A1 Duck Et Al US 2004.0005674A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0005674 A1 Duck et al. (43) Pub. Date: Jan. 8, 2004 (54) METHODS FOR ENZYMATIC HYDROLYSIS Related U.S. Application Data OF LIGNOCELLULOSE (60) Provisional application No. 60/376,527, filed on Apr. 30, 2002. Provisional application No. 60/432,750, (75) Inventors: Nicholas B. Duck, Apex, NC (US); filed on Dec. 12, 2002. Brian Carr, Raleigh, NC (US); Michael G. Koziel, Raleigh, NC (US); Publication Classification Nadine Carozzi, Raleigh, NC (US); (51) Int. Cl. ................................................. C12P 19/02 Brian Vande Berg, Durham, NC (US) (52) U.S. Cl. .............................................................. 435/105 Correspondence Address: (57) ABSTRACT ALSTON & BIRD LLP BANK OF AMERICA PLAZA Compositions and methods for biomass conversion are 101 SOUTH TRYON STREET, SUITE 4000 provided. Compositions comprise novel enzyme mixtures that can be used directly on lignocellulose Substrate. Meth CHARLOTTE, NC 28280-4000 (US) ods involve converting lignocellulosic biomass to free Sug (73) Assignee: Athenix Corporation arS and Small oligosaccharides with enzymes that break down lignocellulose. Novel combinations of enzymes are provided that provide a Synergistic release of Sugars from (21) Appl. No.: 10/426,111 plant biomass. Also provided are methods to identify enzymes, Strains producing enzymes, or genes that encode enzymes capable of degrading lignocellulosic material to (22) Filed: Apr. 29, 2003 generate SugarS. US 2004/0005674 A1 Jan. 8, 2004 METHODS FOR ENZYMATIC HYDROLYSIS OF potential energy in the form of Sugars, both five carbon and LIGNOCELLULOSE Six carbon Sugars that could be utilized for numerous industrial and agricultural processes. However, the enor CROSS REFERENCE TO RELATED mous energy potential of these carbohydrates is currently APPLICATION under-utilized because the Sugars are locked in complex 0001) This application claims the benefit of U.S. Provi polymers, and hence are not readily accessible for fermen sional Application Serial No. 60/376,527, filed Apr. 30, tation. Methods that generate SugarS from plant biomass 2002, and U.S. Provisional Application Serial No. 60/432, would provide plentiful, economically-competitive feed 750, filed Dec. 12, 2002, the contents of which are herein Stocks for fermentation into chemicals, plastics, and fuels. incorporated by reference in their entirety. 0008 Current processes to generate soluble Sugars from lignocellulose are complex. A key Step in the process is FIELD OF THE INVENTION referred to as pretreatment. The aim of pretreatment is to 0002 Methods to produce free Sugars and oligosaccha increase the accessibility of cellulose to cellulose-degrading rides from plant material are provided. enzymes, Such as the cellulase mixture derived from fer mentation of the fungus Trichoderma reesei. Current pre BACKGROUND OF THE INVENTION treatment processes involve Steeping lignocellulosic mate 0.003 Carbohydrates constitute the most abundant rial Such as corn Stover in Strong acids or bases under high organic compounds on earth. However, much of this carbo temperatures and preSSures. Such chemical pretreatments hydrate is Sequestered in complex polymers including Starch degrade hemicellulose and/or lignin components of ligno (the principle storage carbohydrate in Seeds and grain), and cellulose to expose cellulose, but also create unwanted a collection of carbohydrates and lignin known as lignocel by-products Such as acetic acid, furfural, hydroxymethyl lulose. The main carbohydrate components of lignocellulose furfural and gypsum. These products must be removed in are cellulose, hemicellulose, and glucans. These complex additional processes to allow Subsequent degradation of polymers are often referred to collectively as lignocellulose. cellulose with enzymes or by a co-fermentation process 0004 Starch is a highly branched polysaccharide of known as Simultaneous Saccharification and fermentation alpha-linked glucose units, attached by alpha-1,4 linkages to (SSF). form linear chains, and by alpha-1,6 bonds to form branches 0009. The conditions currently used for chemical pre of linear chains. Cellulose, in contrast, is a linear polysac treatments require expensive reaction vessels, and are charide composed of glucose residues linked by beta-1,4 energy intensive. Chemical pretreatment occurring at high bonds. The linear nature of the cellulose fibers, as well as the temperatures and extreme pH conditions (for example 160 Stoichiometry of the beta-linked glucose (relative to alpha) C. and 1.1% Sulfuric acid at 12 atm. pressure) are not generates Structures more prone to interStrand hydrogen compatible with known cellulose-degrading enzymes. Fur bonding than the highly branched alpha-linked Structures of ther, these reactions produce compounds that must be Starch. Thus, cellulose polymers are generally leSS Soluble, removed before fermentation can proceed. As a result, and form more tightly bound fibers than the fibers found in chemical pretreatment processes currently occur in Separate Starch. reaction vessels from cellulose degradation, and must occur 0005 Hemicellulose is a complex polymer, and its com prior to cellulose degradation. position often varies widely from organism to organism, and 0010 Thus, methods that are more compatible with the from one tissue type to another. In general, a main compo cellulose degradation process, do not require high tempera nent of hemicellulose is beta-1,4-linked Xylose, a five carbon tures and pressures, do not generate toxic waste products, Sugar. However, this Xylose is often branched as beta-1,3 and require less energy, are desirable. linkages, and can be Substituted with linkages to arabinose, galactose, mannose, glucuronic acid, or by esterification to 0011 For these reasons, efficient methods are needed for acetic acid. Hemicellulose can also contain glucan, which is biomass conversion. a general term for beta-linked six carbon Sugars. SUMMARY OF INVENTION 0006 The composition, nature of substitution, and degree of branching of hemicellulose is very different in dicot plants 0012 Methods for generating free Sugars and oligosac as compared to monocot plants. In dicots, hemicellulose is charides from lignocellulosic biomass are provided. These comprised mainly of Xyloglucans that are 1,4-beta-linked methods involve converting lignocellulosic biomass to free glucose chains with 1,6-beta-linked Xylosyl Side chains. In Sugars and Small oligosaccharides with enzymes that break monocots, including most grain crops, the principle com down lignocellulose. Enzymes used in the conversion pro ponents of hemicellulose are heteroxylans. These are pri ceSS can degrade any component of lignocellulose and marily comprised of 1,4-beta-linked Xylose backbone poly include but are not limited to: cellulases, Xylanases, ligni mers with 1,3-beta linkages to arabinose, galactose and nases, amylases, proteases, lipidases and glucuronidases. mannose as well as Xylose modified by ester-linked acetic The enzymes of the invention can be provided by a variety acids. Also present are branched beta glucans comprised of of Sources. That is, the enzymes may be bought from a 1,3- and 1,4-beta-linked glucosyl chains. In monocots, cel commercial Source. Alternatively, the enzymes can be pro lulose, heteroxylans and beta glucans are present in roughly duced recombinantly, Such as by expression either in micro equal amounts, each comprising about 15-25% of the dry organisms, fungi, i.e., yeast, or plants. matter of cell walls. 0013 Novel combinations of enzymes are provided. The 0007. The sequestration of such large amounts of carbo combinations provide a Synergistic release of Sugars from hydrates in plant biomass provides a plentiful Source of plant biomass. The Synergism between enzyme classes US 2004/0005674 A1 Jan. 8, 2004 requires less enzyme of each class and facilitates a more be useful in this invention. An auxiliary enzyme mix may be complete release of Sugars from plant biomass, allowing composed of enzymes from (1) commercial Suppliers; (2) more efficient conversion of biomass to Simple SugarS. cloned genes expressing enzymes; (3) complex broth (Such Efficient biomass conversion will reduce the costs of Sugars as that resulting from growth of a microbial Strain in media, useful to generate products including Specialty chemicals, wherein the Strains Secrete proteins and enzymes into the chemical feedstocks, plastics, Solvents and fuels by chemical media; (4) cell lysates of Strains grown as in (3); and, (5) conversion or fermentation. plant material expressing enzymes capable of degrading 0.014. Also provided are methods to identify enzymes, lignocellulose. Strains producing enzymes, or genes that encode enzymes 0020. It is recognized that any combination of auxiliary capable of degrading lignocellulosic material to generate enzymes may be utilized. The enzymes may be used alone Sugars. These methods involve assays based on degradation or in mixtures including, but not limited to, at least a of lignocellulosic biomass and quantitation of the released cellulase; at least a Xylanase; at least a ligninase; at least an Sugar. Additionally, methods that utilize Such assays to amylase; at least a protease; at least a lipidase; at least a Screen microbes, enzymes, or genes and quantify the ability glucuronidase; at least a cellulase and a Xylanase; at least a of the enzyme to degrade lignocellulose are provided. These cellulase and aligninase; at least
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