US 2004O231060A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0231060 A1 Burdette et al. (43) Pub. Date: Nov. 25, 2004 (54) METHODS TO ENHANCE THE ACTIVITY Related U.S. Application Data OF LIGNOCELLULOSE-DEGRADING ENZYMES (60) Provisional application No. 60/452,631, filed on Mar. 7, 2003. Provisional application No. 60/498,098, filed (75) Inventors: Jill Burdette, Morrisville, NC (US); on Aug. 27, 2003. Provisional application No. 60/502, Brian Vande Berg, Durham, NC (US); 727, filed on Sep. 12, 2003. Provisional application Brian Carr, Raleigh, NC (US); No. 60/538,334, filed on Jan. 22, 2004. Nicholas B. Duck, Apex, NC (US); Michael G. Koziel, Raleigh, NC (US); Publication Classification Nadine Carozzi, Raleigh, NC (US); Paresma R. Patel, Durham, NC (US) (51) Int. Cl. .................................................. D06M 10/00 (52) U.S. Cl. ............................................................. 8/115.51 Correspondence Address: (57) ABSTRACT ALSTON & BIRD LLP BANK OF AMERICA PLAZA Methods for hydrolyzing lignocellulose are provided, com 101 SOUTH TRYON STREET, SUITE 4000 prising contacting the lignocellulose with at least one chemi CHARLOTTE, NC 28280-4000 (US) cal treatment. Methods for pretreating a lignocellulosic material comprising contacting the material with at least one (73) Assignee: Athenix Corporation, Durham, NC chemical are also provided. Methods for liberating a sub stance Such as an enzyme, a pharmaceutical, or a nutraceu (21) Appl. No.: 10/795,102 tical from plant material are also provided. These methods are more efficient, more economical, and leSS toxic than (22) Filed: Mar. 5, 2004 current methods. Glucose Patent Application Publication Nov. 25, 2004 Sheet 1 of 4 US 2004/0231060 A1 FIG. 1 Patent Application Publication Nov. 25, 2004 Sheet 2 of 4 US 2004/0231060 A1 Sugars, H2O2 Sugars, H2O2 + Cellulase + Xylanase OmM 10mM 20mM 40mM 60mM FIG. 2 Patent Application Publication Nov. 25, 2004 Sheet 3 of 4 US 2004/0231060 A1 100 0.6 O) s C 0.5 V 80 o () E 60 s ?: 0.3 SP 40 (f) 0.2 ge C 20 g 0.1 .2g SS O O O 4 8 12 16 20 24 Time (hr) -A-% H2O2 remaining -- Soluble Sugars FIG. 3 Patent Application Publication Nov. 25, 2004 Sheet 4 of 4 US 2004/0231060 A1 1.6 . 6 1.4 C Wed 1.2 1 0 Stower Sugars 5 0.8 Glucose + Xylose s S 0.6 t is 0.4 0.2 O O 0.2 0.4 0.6 0.8 Growth Media Sugars (%) FIG. 4 US 2004/0231060 A1 Nov. 25, 2004 METHODS TO ENHANCE THE ACTIVITY OF removed in additional processes to allow Subsequent deg LIGNOCELLULOSE-DEGRADING ENZYMES radation of cellulose with enzymes or by a co-fermentation process known as Simultaneous Saccharification and fermen CROSS-REFERENCE TO RELATED tation (SSF). APPLICATIONS 0007. The harsh conditions needed for chemical pretreat 0001. The present application claims the benefit of U.S. ments require expensive reaction vessels, and are energy Provisional Application Serial No. 60/452,631, filed Mar. 7, intensive. Since the chemical treatment occurs at tempera 2003, U.S. Provisional Application No. 60/498,098, filed ture and pH conditions (for example 160° C. and 0.2% Aug. 27, 2003, U.S. Provisional Application No. 60/502, Sulfuric acid at 12 atm. pressure) incompatible with known 727, filed Sep. 12, 2003, and U.S. Provisional Application cellulosic enzymes, and produces compounds that must be No. 60/538,334, filed Jan. 22, 2004, the contents of which removed before fermentation, this process must occur in are herein incorporated by reference in their entirety. Separate reaction vessels from cellulose degradation, and must occur prior to cellulose degradation. Thus, novel FIELD OF THE INVENTION methods that are more compatible with the cellulose degra dation process, that do not generate toxic waste products, 0002 Methods to enhance the production of free Sugars and that require less energy would be desirable. Further, and oligosaccharides from plant material are provided. enzymatic processes that occur in conditions Similar to those used for cellulose degradation would allow development of BACKGROUND OF THE INVENTION co-treatment processes wherein the breakdown of hemicel lulose and cellulose occur in the same reaction vessel, or are 0.003 Plant biomass is comprised of Sugars and repre not separated in the manner in which current pre-treatment Sents the greatest Source of renewable hydrocarbon on earth. processes must be separated from cellulose breakdown and However, this enormous resource is under-utilized because the Sugars are locked in complex polymers. These complex Subsequent processes. In addition, processes that liberate polymers are often referred to collectively as lignocellulose. Sugars from lignocellulose without generating toxic prod SugarS generated from degradation of plant biomass could ucts may provide additional benefits due to the increased provide plentiful, economically competitive feedstocks for accessibility of nutrients present in lignocellulosic material fermentation into chemicals, plastics, and fuels, including Such as proteins, amino acids, lipids, and the like. ethanol as a Substitute for petroleum. 0008 For these reasons, efficient methods are needed for 0004 Commercial ethanol production in the U.S. is cur conversion of lignocellulose to Sugars and fermentation rently carried out in dry mill facilities, converting corn grain feedstockS. to ethanol. However corn grain is expensive, and has other SUMMARY OF INVENTION high value uses, Such as use in livestock feeds, and high 0009 Methods are provided for hydrolyzing lignocellu fructose corn syrups (Wyman, ed. (1999) Handbook On lose with increased efficiency without the need for a harsh Bioethanol. Production, and Utilization. Taylor & Francis, pretreatment. These methods involve a chemical treatment Washington, D.C., p.1). Alternate feedstocks for ethanol of the lignocellulose at mild or moderate conditions to production that allow production at a lower cost, and on a generate a treated lignocellulose, and contacting this treated larger commercial Scale, are desirable. lignocellulose with at least one enzyme capable of hydro 0005 Lignocellulosics such as corn stover, which is lyzing a component of lignocellulose. The chemical treat cheap, abundant, and has no competing markets, would be ment involves contacting lignocellulose with at least one preferred over grain for the production of ethanol. The chemical that acts in combination with enzyme treatment to limiting factor is the complex composition of the Sugar liberate Sugars. polymers. Starch in corn grain is a highly branched, water Soluble polymer that is amenable to enzyme digestion. In 0010 Methods are also provided for pretreating a ligno contrast, the carbohydrates comprising lignocellulosic mate cellulosic material comprising contacting the material with rials. Such as corn Stover are more difficult to digest. These at least one chemical under mild or moderate conditions to carbohydrates are principally found as complex polymers generate a treated lignocellulose. In Some embodiments, the including cellulose, hemicellulose and glucans, which form treated lignocellulose may be further treated with at least the Structural components of plant cell walls and Woody one enzyme capable of hydrolyzing lignocellulose. tissues. Starch and cellulose are both polymers of glucose. 0011 Methods for liberating substances from lignocellu losic material are also encompassed. These methods com 0006 Current processes to release the Sugars in lignocel prise a chemical treatment of the lignocellulosic material lulose involve many Steps. A key Step in the process is a under mild or moderate conditions. In Some embodiments, at harsh pretreatment. The aim of the current industry pretreat least one enzyme capable of hydrolyzing lignocellulose may ment is to increase the accessibility of cellulose to cellulose hydrolyzing enzymes, Such as the cellulase mixture derived be added Subsequent to the chemical treatment. Enzymes, from fermentation of the fungus Trichoderma reesei. Cur pharmaceuticals, and nutraceuticals may be released by rent pretreatment processes involve partial hydrolysis of treating lignocellulosic material by the methods of the lignocellulosic material, Such as corn Stover, in Strong acids invention. In Some embodiments, the lignocellulosic mate or bases under high temperatures and preSSures. Such rial has been engineered to contain the Substance to be chemical pretreatments degrade hemicellulose and/or lignin released. components of lignocellulose to expose cellulose, but also 0012 Chemicals for use in the above methods include create unwanted by-products Such as acetic acid, furfural, Oxidizing agents, denaturants, detergents, organic Solvents, and hydroxymethyl furfural. These products must be bases, or any combination thereof. US 2004/0231060 A1 Nov. 25, 2004 0013 Methods for hydrolyzing lignocellulose compris 0022 Methods for pretreating a lignocellulosic material ing contacting the lignocellulose with an oxidizing agent to comprising contacting the material under mild or moderate generate a treated lignocellulose, and contacting the treated conditions with at least one chemical are also provided. The lignocellulose with at least one enzyme capable of hydro treated lignocellulosic material may be further Subjected to lyzing lignocellulose are also provided. treatment with at least one enzyme capable of hydrolyzing lignocellulose. 0.014 Further provided are methods for hydrolyzing lignocellulose, comprising contacting the lignocellulose 0023. Further provided are methods for liberating a sub with a base