USOO8426158B2 (12) United States Patent (10) Patent No.: US 8.426,158 B2 Xu et al. (45) Date of Patent: Apr. 23, 2013 (54) METHODS FOR INCREASINGENZYMATIC FOREIGN PATENT DOCUMENTS HYDROLYSIS OF CELLULOSIC MATERAL WO WO 2005, O74656 8, 2005 IN THE PRESENCE OF A PEROXIDASE WO WO 2008, 134259 11, 2008 WO WO 2010/O12579 2, 2010 (75) Inventors: Feng Xu, Davis, CA (US); Jason Quinlan, Emeryville, CA (US) OTHER PUBLICATIONS Ramachandra et al "Characterization of an Extracellular Lignin (73) Assignee: Novozymes, Inc., Davis, CA (US) Peroxidase of the Lignocellulolytic Actinomycete Streptomyces virdosporus' Dec. 1988 Applied and Environmental Microbiology (*) Notice: Subject to any disclaimer, the term of this vol. 54 No. 12 pp. 3057-3063.* patent is extended or adjusted under 35 Nicholis et al., Enzymology and structure of catalases, 2001, Advances in Inorganic Chemistry. 51: 51-106. U.S.C. 154(b) by 568 days. Masaki et al., Differential role of catalase and glutathione peroxidase in cultured human fibroblasts under exposure of HO, or ultraviolet B (21) Appl. No.: 12/638,920 light, 1998, Archives of Dermatological Research 290: 113-118. Kedderis et al. Characterization of the N-Demethylation Reactions (22) Filed: Dec. 15, 2009 Catalyzed by Horseradish Peroxidase, 1983, J. Biol. Chem. 258: 8129-8138. (65) Prior Publication Data Jonsson et al., Detoxification of wood hydrolysates with laccase and peroxidase from the white-rot fungus Trametes versicolor, 1998, US 2010/O159509 A1 Jun. 24, 2010 Appl Microbiol Biotechnol, 49: 691-697. Lobarzewski et al., Lignocellulose biotransformation with immobi lized cellulose, D-glucose oxidase and fungal peroxidases, 1985, Related U.S. Application Data Enzymes Microb. Technol. 7: 564-566. Steffen et al., Differential degradation of oak (Quercus petraea) leaf (60) Provisional application No. 61/139,373, filed on Dec. litter by litter-decomposing basidiomycetes, 2007. Research in 19, 2008. Microbiology, 158: 447-455. Kedderis et al., Characterization of the N-Demethylation Reactions (51) Int. Cl. Catalyzed by Horseradish Peroxidase, 1983, J. of Biological Chem CI2P I/O (2006.01) istry, 258: 8129-8138. CI2P 13/04 (2006.01) Masaki et al., Differential role of catalase and glutathione peroxidase CI2P 7/00 (2006.01) in cultured human fibroblasts under exposure of H2O2 or ultraviolet CI2P 7/26 (2006.01) B light, 1998, Arch Dermatol Res, 290: 113-118. CI2P 7/02 (2006.01) (52) U.S. Cl. * cited by examiner USPC ............. 435/41; 435/106; 435/132; 435/148; 435/155 Primary Examiner — Herbert J Lilling (58) Field of Classification Search .................... 435/41, (74) Attorney, Agent, or Firm — Robert L. Starnes 435/106, 132, 148, 155 See application file for complete search history. (57) ABSTRACT The present invention relates to methods for increasing (56) References Cited hydrolysis of a cellulosic material, comprising: hydrolyzing the cellulosic material with an enzyme composition in the U.S. PATENT DOCUMENTS presence of a polypeptide having peroxidase activity. 8,148,495 B2 * 4/2012 Harris et al. .................. 530/350 2007/OO77630 A1 4/2007 Harris et al. 12 Claims, 3 Drawing Sheets U.S. Patent Apr. 23, 2013 Sheet 2 of 3 US 8,426,158 B2 45 ... 3. 3 . (HRP) (J/mL) Fig. 2 U.S. Patent Apr. 23, 2013 Sheet 3 of 3 US 8,426,158 B2 ?º, ?,, S N Fig. 3 US 8,426,158 B2 1. 2 METHODS FOR INCREASINGENZYMATIC The present invention also relates to methods for producing HYDROLYSIS OF CELLULOSIC MATERAL a fermentation product, comprising: IN THE PRESENCE OF A PEROXIDASE (a) Saccharifying a cellulosic material with an enzyme composition in the presence of a polypeptide having peroxi CROSS-REFERENCE TO RELATED dase activity; APPLICATION (b) fermenting the saccharified cellulosic material with one or more (several) fermenting microorganisms to produce the This application claims the benefit of U.S. Provisional fermentation product; and Application No. 61/139,373, filed Dec. 19, 2008, which (c) recovering the fermentation product from the fermen application is incorporated herein by reference. 10 tation. The present invention further relates to methods of fer REFERENCE TO ASEQUENCE LISTING menting a cellulosic material, comprising: fermenting the cellulosic material with one or more (several) fermenting This application contains a Sequence Listing filed elec microorganisms, wherein the cellulosic material is hydro tronically by EFS, which is incorporated herein by reference. 15 lyzed with an enzyme composition in the presence of a polypeptide having peroxidase activity. BACKGROUND OF THE INVENTION BRIEF DESCRIPTION OF THE FIGURES 1. Field of the Invention The present invention relates to methods for increasing FIG. 1 shows horseradish peroxidase mitigation of cellu hydrolysis of cellulosic material with an enzyme composi lase-inhibition by cellobiose dehydrogenase. Fractional cel tion. lulose conversion is plotted for various reaction conditions. 2. Description of the Related Art Solid bars: 1 day of hydrolysis; hatched bars: 3 days of Cellulose is a polymer of the simple Sugar glucose linked 25 hydrolysis. by beta-1,4-bonds. Many microorganisms produce enzymes FIG. 2 shows horseradish peroxidase enhancement of PCS that hydrolyze beta-linked glucans. These enzymes include hydrolysis. Circles: 1 day of hydrolysis; squares: 3 days of endoglucanases, cellobiohydrolases, and beta-glucosidases. hydrolysis. Endoglucanases digest the cellulose polymer at random loca FIG. 3 shows the effect of various peroxidases on PCS tions, opening it to attack by cellobiohydrolases. Cellobiohy 30 hydrolysis. Solid bars: 1 day of hydrolysis; hatched bars: 3 drolases sequentially release molecules of cellobiose from days of hydrolysis. Numbers indicate the volumes of the the ends of the cellulose polymer. Cellobiose is a water stock peroxidases added, as indicated in the text. Mn-perox: soluble beta-1,4-linked dimer of glucose. Beta-glucosidases manganese peroxidase; lignin-perox: lignin-peroxidase; and hydrolyze cellobiose to glucose. cellulase: Trichoderma reesei cellulase composition. It is well known in the art that oxidation of biomolecules 35 Such as DNA, lipids, or protein is a significant issue in bio DEFINITIONS logical systems. Consequently, treatment with a peroxidase may improve the performance of cellulose-hydrolyzing Peroxide-generating system: The term "peroxide-generat enzyme systems. ing system” is defined herein as either a peroxide generating Different peroxide-decomposing enzymes often have dif 40 enzyme as defined below, or as a chemical reaction leading to ferent specificities and potencies. For example, catalase is production of peroxide. Common examples of chemical very efficient only at high levels of hydrogen peroxide (0.1 M methods of peroxide generation include, but are not limited or above) because of its high Michaelis constant, K, on this to. UV-irradiation of Rose Bengal; the Reidl-Pfleiderer pro substance (K, ranges from 0.1 to 1 M: see Nicholls et al., cess of autooxidation of 2-ethyl-9,10-dihydroxyanthracene-- 2001, Advances in Inorg. Chem. 51:52-106; and Masaki et 45 O to 2-ethylanthraquinone--H2O, reaction of singlet state al., 1998, Archives of Dermatological Research 290: 113 molecular oxygen 'O, with ascorbate; the oxidation of 118). At low peroxide levels, a peroxidase can be significantly organic alcohols by molecular oxygen in the presence of more efficient (than catalase) to decompose the peroxide, various metal and metal complex catalysts; and the oxidation because of the enzyme’s high affinity (sub-mM ranges) for of unsaturated lipid by oxygen (after a radical initiation) to the peroxide. For example, horseradish peroxidase, an arche 50 form lipid peroxide. typical peroxidase, has a K, of 0.02 mM on hydrogen peroX Peroxide-generating enzyme: The term “peroxide-gener ide or ethyl hydroperoxide (Kedderis and Hollenberg, 1983, ating enzyme” is defined herein as an donor:oxygen oxi J. Biol. Chem. 258: 8129-8138), and glutathione peroxidase doreductase (E.C. number 1.1.3.x) that catalyzes the reaction has a K of O.025-0.06 mM (Masaki et al., 1998, supra). Since reduced substrate (2e)+O->oxidized substrate+HO, such many biomass conversion techniques are prone to generate 55 as glucose oxidase that catalyzes the reaction glucose-- low level peroxide, peroxidase may be more effective than O->gluconolactone--HO, and a donor: Superoxide oxi catalase to remove the peroxide to improve cellulose hydroly doreductase (E.C. 1.15.1.X). Such as Superoxide dismutase S1S. that catalyzes the reaction 20+2H->O+HO. Other The present invention provides methods for increasing examples of peroxide-generating enzymes are provided hydrolysis of cellulosic materials with enzyme compositions. 60 herein. Alternatively oxidoreductases with side activities, wherein molecular oxygen can be used as electron acceptor SUMMARY OF THE INVENTION by the enzyme, are also included within the term hydrogen peroxide-generating enzyme. In addition to hydrogen peroX The present invention relates to methods for degrading or ide, other peroxides may also be generated by these enzymes. converting a cellulosic material, comprising: treating the cel 65 Peroxidase activity: The term “peroxidase activity” is lulosic material with an enzyme composition in the presence defined herein as an enzyme activity that converts a peroxide, of a polypeptide having peroxidase activity. e.g., hydrogen peroxide, to a less oxidative species, e.g., US 8,426,158 B2 3 4 water. It is understood herein that a polypeptide having per substrate. The assay was established