USOO8962288B2 (12) United States Patent (10) Patent No.: US 8,962.288 B2 Quinlan et al. (45) Date of Patent: Feb. 24, 2015 (54) METHODS FOR PRODUCING A (56) References Cited FERMENTATION PRODUCT FROM CELLULOSC MATERAL IN THE U.S. PATENT DOCUMENTS PRESENCE OF A PEROXDASE 8,148,495 B2 * 4/2012 Harris et al. .................. 530/350 8,426,158 B2 * 4/2013 Xu et al. ......................... 435/41 (71) Applicant: Novozymes, Inc., Davis, CA (US) 2007, OO7763.0 A1* 4, 2007 Harris et al. .................. 435/105 (72) Inventors: Jason Quigley CA (US); Feng FOREIGN PATENT DOCUMENTS Xu, Davis, CA (US WO 2005074656 A2 8, 2005 (73) Assignee: Novozymes, Inc., Davis, CA (US) WO 2008134259 A1 11 2008 WO 2010O12579 A1 2, 2010 (*) Notice: Subject to any disclaimer, the term of this OTHER PUBLICATIONS patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days. Jonsson et al., 1998, Appl Microbiol Bioiechnol 49,691-697. Kedderis et al., 1983, J Biol Chem 258(13), 8129-8138. (21) Appl. No.: 13/861,838 Lobarzewski et al., 1985, Enzyme Microb Technol 7, 564-56. Masaki et al., 1998, Arch Dermatol Res 290, 113-118. 1-1. Nicholls et al., 2000, Adv Inorg Chem PS006-02, 51-106. (22) Filed: Apr. 12, 2013 Ramachandraetal, 1988, App Environ Microbiol 54(12), 3057-3063. O O Steffen et al., 2007, Res Microbiol 158, 447-455. (65) Prior Publication Data Asgarietal. 1998, Canadian Journal of Chemistry 76, 1606-1615. Goldstein et al. 1993, FreeRadical Biology & Medicine 15,435-445. US 2013/0203127 A1 Aug. 8, 2013 Sippola 2006, Industrial Chemistry Publication Series, 21, 1-59. * cited by examiner Related U.S. Application Data y (62) Division of application No. 12/638,920, filed on Dec. Primary Examiner — Ralph Gitomer 15, 2009, now Pat. No. 8,426,158. (74) Attorney, Agent, or Firm — Kelly Reynolds (60) Provisional application No. 61/139,373, filed on Dec. (57) ABSTRACT 19, 2008. A method for production of a fermentation product from 51) Int. C cellulosic material where a cellulosic material is saccharified (51) 2/06 2006.O1 with an enzyme composition in the presence of a peroxide ( .01) generating system and a polypeptide having peroxidase activ (52) U.S. Cl. ity, the resultant saccharified cellulosic material is fermented USPC - - - - - - - - - - - grgrrr. 435/161; 435/165 and a fermentation product is recovered. Inclusion of the (58) Field of Classification Search polypeptide having peroxidase activity increases saccharifi USPC .................................................. 435/1 61, 165 cation of the cellulosic material. IPC ................. C12N 1/22; C12P 7/10; Y02E 50/16 See application file for complete search history. 16 Claims, 3 Drawing Sheets U.S. Patent Feb. 24, 2015 Sheet 2 of 3 US 8,962.288 B2 4. 35 3 O. Fig. 2 U.S. Patent Feb. 24, 2015 Sheet 3 of 3 US 8,962.288 B2 Hæ,, N . Fig. 3 US 8,962,288 B2 1. 2 METHODS FOR PRODUCING A lulosic material with an enzyme composition in the presence FERMENTATION PRODUCT FROM of a polypeptide having peroxidase activity. CELLULOSIC MATERAL IN THE The present invention also relates to methods for producing PRESENCE OF A PEROXIDASE a fermentation product, comprising: (a) Saccharifying a cellulosic material with an enzyme CROSS-REFERENCE TO RELATED composition in the presence of a polypeptide having peroxi APPLICATIONS dase activity; (b) fermenting the saccharified cellulosic material with one This application is a divisional application of U.S. appli or more (several) fermenting microorganisms to produce the cation Ser. No. 12/638,920, filed Dec. 15, 2009, now U.S. Pat. 10 fermentation product; and No. 8,426,158, which claims the benefit of U.S. Provisional (c) recovering the fermentation product from the fermen Application No. 61/139,373, filed Dec. 19, 2008, which tation. applications are fully incorporated herein by reference. The present invention further relates to methods of fer menting a cellulosic material, comprising: fermenting the REFERENCE TO ASEQUENCE LISTING 15 cellulosic material with one or more (several) fermenting microorganisms, wherein the cellulosic material is hydro This application contains a Sequence Listing filed elec lyzed with an enzyme composition in the presence of a tronically by EFS, which is incorporated herein by reference. polypeptide having peroxidase activity. BACKGROUND OF THE INVENTION BRIEF DESCRIPTION OF THE FIGURES 1. Field of the Invention FIG. 1 shows horseradish peroxidase mitigation of cellu The present invention relates to methods for increasing lase-inhibition by cellobiose dehydrogenase. Fractional cel hydrolysis of cellulosic material with an enzyme composi lulose conversion is plotted for various reaction conditions. tion. 25 Solid bars: 1 day of hydrolysis; hatched bars: 3 days of 2. Description of the Related Art hydrolysis. Cellulose is a polymer of the simple Sugar glucose linked FIG. 2 shows horseradish peroxidase enhancement of PCS by beta-1,4-bonds. Many microorganisms produce enzymes hydrolysis. Circles: 1 day of hydrolysis; squares: 3 days of that hydrolyze beta-linked glucans. These enzymes include hydrolysis. endoglucanases, cellobiohydrolases, and beta-glucosidases. 30 FIG. 3 shows the effect of various peroxidases on PCS Endoglucanases digest the cellulose polymer at random loca hydrolysis. Solid bars: 1 day of hydrolysis; hatched bars: 3 tions, opening it to attack by cellobiohydrolases. Cellobiohy days of hydrolysis. Numbers indicate the volumes of the drolases sequentially release molecules of cellobiose from stock peroxidases added, as indicated in the text. Mn-perox: the ends of the cellulose polymer. Cellobiose is a water manganese peroxidase; lignin-perox: lignin-peroxidase; and soluble beta-1,4-linked dimer of glucose. Beta-glucosidases 35 cellulase: Trichoderma reesei cellulase composition. hydrolyze cellobiose to glucose. It is well known in the art that oxidation of biomolecules DEFINITIONS Such as DNA, lipids, or protein is a significant issue in bio logical systems. Consequently, treatment with a peroxidase Peroxide-generating system: The term "peroxide-generat may improve the performance of cellulose-hydrolyzing 40 ing system” is defined herein as either a peroxide generating enzyme systems. enzyme as defined below, or as a chemical reaction leading to Different peroxide-decomposing enzymes often have dif production of peroxide. Common examples of chemical ferent specificities and potencies. For example, catalase is methods of peroxide generation include, but are not limited very efficient only at high levels of hydrogen peroxide (0.1 M to. UV-irradiation of Rose Bengal; the Reidl-Pfleiderer pro or above) because of its high Michaelis constant, K, on this 45 cess of autooxidation of 2-ethyl-9,10-dihydroxyanthracene-- substance (K, ranges from 0.1 to 1 M: see Nicholls et al., O to 2-ethylanthraquinone--H2O, reaction of singlet state 2001, Advances in Inorg. Chem. 51:52-106; and Masaki et molecular oxygen 'O, with ascorbate; the oxidation of al., 1998, Archives of Dermatological Research 290: 113 organic alcohols by molecular oxygen in the presence of 118). At low peroxide levels, a peroxidase can be significantly various metal and metal complex catalysts; and the oxidation more efficient (than catalase) to decompose the peroxide, 50 of unsaturated lipid by oxygen (after a radical initiation) to because of the enzyme’s high affinity (sub-mM ranges) for form lipid peroxide. the peroxide. For example, horseradish peroxidase, an arche Peroxide-generating enzyme: The term “peroxide-gener typical peroxidase, has a K, of 0.02 mM on hydrogen peroX ating enzyme” is defined herein as an donor:oxygen oxi ide or ethyl hydroperoxide (Kedderis and Hollenberg, 1983, doreductase (E.C. number 1.1.3.x) that catalyzes the reaction J. Biol. Chem. 258: 8129-8138), and glutathione peroxidase 55 reduced substrate (2e)+O->oxidized substrate+HO, such has a K, of 0.025-0.06 mM (Masaki et al., 1998, Supra). Since as glucose oxidase that catalyzes the reaction glucose-- many biomass conversion techniques are prone to generate O >gluconolactone--H2O, and a donor: Superoxide oxi low level peroxide, peroxidase may be more effective than doreductase (E.C. 1.15.1.X). Such as Superoxide dismutase catalase to remove the peroxide to improve cellulose hydroly that catalyzes the reaction 20+2H->O+HO. Other S1S. 60 examples of peroxide-generating enzymes are provided The present invention provides methods for increasing herein. Alternatively oxidoreductases with side activities, hydrolysis of cellulosic materials with enzyme compositions. wherein molecular oxygen can be used as electron acceptor by the enzyme, are also included within the term hydrogen SUMMARY OF THE INVENTION peroxide-generating enzyme. In addition to hydrogen peroX 65 ide, other peroxides may also be generated by these enzymes. The present invention relates to methods for degrading or Peroxidase activity: The term “peroxidase activity” is converting a cellulosic material, comprising: treating the cel defined herein as an enzyme activity that converts a peroxide, US 8,962,288 B2 3 4 e.g., hydrogen peroxide, to a less oxidative species, e.g., the filter paper assay using Whatman No 1 filter paper as the water. It is understood herein that a polypeptide having per substrate. The assay was established by the International oxidase activity encompasses a peroxide-decomposing Union of Pure and Applied Chemistry (IUPAC) (Ghose, enzyme (defined below). 1987, Measurement of cellulase activities, Pure Appl. Chem. Peroxide-decomposing enzyme: The term “peroxide-de 59: 257-68). composing enzyme’ is defined herein as an donor:peroxide For purposes of the present invention, cellulolytic activity oxidoreductase (E.C. number 1.11.1.x) that catalyzes the is determined by measuring the increase in hydrolysis of a reaction reduced substrate (2e)+ROOR'->oxidized sub cellulosic material by cellulolytic enzyme(s) under the fol strate+ROH--ROH; such as horseradish peroxidase that cata lowing conditions: 1-20 mg of cellulolytic protein/g of cellu lyzes the reaction phenol--HO equinone--H2O, and cata 10 lose in PCS for 3-7 days at 50-65° C.