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(12) Patent Application Publication (10) Pub. No.: US 2015/0211037 A1 Graham Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2015/0211037 A1 Graham Et Al US 2015021 1 037A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0211037 A1 Graham et al. (43) Pub. Date: Jul. 30, 2015 (54) POLYPEPTIDES FOR USE IN THE Related U.S. Application Data DECONSTRUCTION OF CELLULOSE (60) Provisional application No. 61/369,588, filed on Jul. 30, 2010. (75) Inventors: Joel Edward Graham, Baltimore, MD Publication Classification (US); Melinda E. Clark, San Francisco, CA (US); Frank Thomson Robb, (51) Int. Cl. Gaithersburg, MD (US); Douglas S. CI2P 19/14 (2006.01) Clark, Orinda, CA (US); Harvey W. CI3K L/02 (2006.01) Blanch, San Francisco, CA (US) CI2N 9/42 (2006.01) CI2P 19/02 (2006.01) (73) Assignees: University of Maryland, Baltimore, (52) U.S. Cl. Baltimore, MD (US); The Regents of CPC ................. CI2P 19/14 (2013.01); C12P 19/02 the University of California, Oakland, (2013.01); CI3K I/02 (2013.01); C12N 9/2437 CA (US) (2013.01) (21) Appl. No.: 13/813,154 (57) ABSTRACT Hydrolysis and degradation of cellulose-containing biomass (22) PCT Filed: Jul. 14, 2011 by use of a polypeptide having cellulase activity is provided. (86). PCT No.: PCT/US11A4074 Also provided are polypeptides having cellulase activity, Such as archaeal cellulases, polynucleotides encoding the polypep S371 (c)(1), tides, and compositions containing the polypeptides, and (2), (4) Date: Mar. 20, 2014 methods of use thereof. Patent Application Publication Jul. 30, 2015 Sheet 1 of 28 US 2015/0211037 A1 S SS. Patent Application Publication Jul. 30, 2015 Sheet 2 of 28 US 2015/0211037 A1 30 DAYS 50 DAYS 65 DAYS 65 DAYS S. 200 66 31 215 6.5 Patent Application Publication Jul. 30, 2015 Sheet 3 of 28 US 2015/0211037 A1 M N B W1 175 58 46 FIG. 4 Patent Application Publication Jul. 30, 2015 Sheet 4 of 28 US 2015/0211037 A1 CLOSTRIDIUM THERMOCELLUM CTL-6 (FJ599513) CANDDATUSKORARCHAEUM CRYPTOFILUM OPF8 (CPOOO968) METHANOPYRUS KANDLER AW19 (M59932) SULFOLOBUS TOKODAIISTRAIN 7 (AB022438) 1OO SULFOLOBUS SHIBATAE (M32504) 100 SULFOLOBUS SOLFATARICUS P2 (D26490) 100 ACIDILOBUS SACCHAROVORANS STRAIN 345-15 (AY350586) 99 SULFOPHOBOCOCCUS ZILLIG|| K1 (X98064) DESULFURCCOCCUS KAMCHATKENSIS 100 95 STRAIN 1221N (EU167539) DESULFURC)COCCUS FERMENTANS STRAINZ-1312 (AY264344) 71 THERMOSPHAERA AGGREGANS 99 STAPHYLOTHERMUSRSX-1138.895 MARINUS F1 (X99560) 100 - STAPHYLOTHERMUSHELLENCUS DSM 12710 (AJO12645) 100 GNISPHAERA-LIKE ORGANISM IGNISPHAERA SP. TOK 10A.S1 (DQ060322) 100 IGNISPHAERAAGGREGANSDSM 17230 96 (DP060321) IGNISPHAERA SP. TOK 37.31 (DQ000323) AEROPYNUMPERNIXK1 (D83259) HYPERTHERMUSBUTYICUS DSM 5456 (X99553) 100- PYRODICTIUM OCCULTUMPL-19 (M21087) 100 THERMOFILUM-LIKE ORGANISM THERMOFILISCESE STR. SRI-370 (AF266606) 76 1OO THERMOFILUMPENDENS HWV 3 (X14835) 100 THERMOFILUMPENDENS HRK 5 (CP000505) WULCANSAETA DISTRIBUTADSM 14429 (AB063630) 100 THERMOPROTEUS TENAXKRA1 (M35966) 991 100 PYROBACULUM CALIDIFONTIS JCM 11548 (AB078332) PYROBACULUMNEUTROPHILUM (X81886) 100 PYROBACULUM-LIKE ORGANISM 88 PYROBACULUMISLAND CUM DSM 4184 (LO7511) FIG. 5 Patent Application Publication US 2015/0211037 A1 Patent Application Publication Jul. 30, 2015 Sheet 6 of 28 US 2015/0211037 A1 Patent Application Publication Jul. 30, 2015 Sheet 7 of 28 US 2015/0211037 A1 Patent Application Publication Jul. 30, 2015 Sheet 8 of 28 US 2015/0211037 A1 - GC•ZT0* GILLLº?LdiLo?dia?ILº?LÕLÂN?ATVZVŽAVIXI Patent Application Publication Jul. 30, 2015 Sheet 9 of 28 US 2015/0211037 A1 |ad?NN-––EVAGTVÆDVIH,HÆVÆÐIHMVH Patent Application Publication Jul. 30, 2015 Sheet 11 of 28 US 2015/0211037 A1 1808SE||VQ8SE| Patent Application Publication Jul. 30, 2015 Sheet 15 of 28 US 2015/0211037 A1 Patent Application Publication Patent Application Publication Jul. 30, 2015 Sheet 17 of 28 US 2015/0211037 A1 CIENIVILSCIENOSONOO‘9%9||-%8EC||WITANOV‘OWNO%9ZOINV/>H9OWNWZ Patent Application Publication Jul. 30, 2015 Sheet 18 of 28 US 2015/0211037 A1 dp m1 m2 O 1 2 3 4 5 6 7 FIG. 12 Patent Application Publication Jul. 30, 2015 Sheet 19 of 28 US 2015/0211037 A1 AMMONIUMSULFATE FRACTIONATION OF EB5326244 S 2O 40 60 90 1.O O.4 O.2 1.O. O.4 O.2 10 O.4 O.2 10 O.4 O.2 1.0 FIG. 13 FLOW THROUGH FRACTION WASH (10CV) FIG. 14 Patent Application Publication Jul. 30, 2015 Sheet 20 of 28 US 2015/0211037 A1 S.S 25KD O.8 O.7 O.6 O.5 O.4 O.3 O.2 O. 1 O 7O 75 80 85 90 95 1 OO 105 11 O 115 TEMPERATURE (°C) FIG. 16 Patent Application Publication Jul. 30, 2015 Sheet 21 of 28 US 2015/0211037 A1 2.0 4.5 9 4.0 2 as 3.5 O2S 2 3.0 i 2.5 cd Oo O CN w co o O O r r r v- y 2 3 2.0 v- v- v- v- v- v- v 5 TEMPERATURE (C) < 1.5 CCD 1.0 sn 0.5 O O -0.5 6O 65 7O 75 8O 85 90 95 1 OO 105 11 O 115 120 TEMPERATURE (C) DNS ASSAY: 1/2 FP WHATMAN #1 + EEI-44 SUPER (6.7.2010) IN 50mMSODIUMACETATE, pH 5.0 18 16 S 14 St 12 310 n5 8 6 O 4 2 O 85 90 95 100 TEMPERATURE (C) FIG. 18 Patent Application Publication Jul. 30, 2015 Sheet 22 of 28 US 2015/0211037 A1 THERMOSTABILITY OF EB5326244 0.1 T1/2 =34 min ACTIVITYC O.OO1 -- 1 OOC O.OO1 -- 105C O.OOO1 O.OOOO1 HOURS PRETREATMENT FIG. 19 Ln REMAINING ACTIVITY O 1 2 3 4 5 TIME (HOURS) FIG. 20 Patent Application Publication Jul. 30, 2015 Sheet 23 of 28 US 2015/0211037 A1 Ln REMAINING ACTIVITY O 10 20 30 40 50 60 70 80 90 100 110 TIME (min) FIG 21 OM 2M 4M SATURATING OM 1M 2M SATURATING Patent Application Publication Jul. 30, 2015 Sheet 24 of 28 US 2015/0211037 A1 CMCASE ACTIVITY OF EB5326244 IN HIGH SALT INHEPPS pH 6.8AND 90C 1200 1 OOO MICROGRAMS –0- CMC(90C) 600 -x- NaCl(2.5M) PRODUCT -H KCI(3.0M) 400 2OO O 1 15 2 HOURS FIG. 23 2.4 2.2 2.0 1.8 1.6 1.4 FOLD INCREASE 12 1.O O.8 0.6 O.4 O2 BUFFER TWEEN TRITON NP-40 CHAPS ONLY 2O X-100 SUB FIG. 24 Patent Application Publication Jul. 30, 2015 Sheet 25 of 28 US 2015/0211037 A1 1100 1000 900 800 700 PRODUCT 600 (M) 500 400 300 200 100 O O 0.2 0.4 0.6 0.8 1.0 12 14 1.6 1.8 2.0 2.2 TIME (hrs) FIG. 25 PRODUCT (MICROGRAMS) 64 66 68 70 72 74 76 78 80 TEMPERATURE (C) FIG. 26 Patent Application Publication Jul. 30, 2015 Sheet 26 of 28 US 2015/0211037 A1 TEMPERATURE MAXIMUM VSIONIC LIQUID (DMIM) DMP 110 105 100 a. 95 TEMPERATURE 90 -- T-MAX (C) 85 80 75 70 O 10 20 30 40 50 60 % DMIM) DMP FIG. 27 10 9 8 7 6 5 -- BUFFER 4 -H 40% 3 - A - 50% 2 1 O 75 77 79 81 83 85 87 89.91 93 95 97 99 101 FIG. 28 Patent Application Publication Jul. 30, 2015 Sheet 27 of 28 US 2015/0211037 A1 THERMOSTABILITY OF RECOMBINANT EB-5326244AT 1 OOC min O 10 2O 40 O 10 2O 40 230 150 8O 50 PHOSPHATE BUFFER PHOSPHATE BUFFER ONLY +SDS pH PROFILE OF EBI244 O 5 5 g g o os pH FIG. 30 Patent Application Publication Jul. 30, 2015 Sheet 28 of 28 US 2015/0211037 A1 18 a 1.6 E S 14 S Y 1.2 8 1.0 - 9 0.8 2 3 0.6 s 0.4 b< 0.2 O E. 2 3 4 5 6 7 8 9 10 11 US 2015/021 1 037 A1 Jul. 30, 2015 POLYPEPTIDES FOR USE IN THE binant thermostable endoglucanase of Aquifex aeolicus pro DECONSTRUCTION OF CELLULOSE duced in E. coli showed maximal activity at 80°C. and pH 7.0 with a half-life of 2 hat 100° C. (Kim J S. Lee YY. Torget, R CROSS-REFERENCE TO RELATED W (2001). Cellulose hydrolysis under extremely low sulfuric APPLICATION acid and high-temperature conditions. Appl. Biochem. Bio 0001. This application claims the benefit of U.S. Provi technol. 91-93.331-340)). The endoglucanases produced by sional Application No. 61/369,388, filed Jul. 30, 2010, which Anaerocellum thermophilum and Caldicellulosiruptor sac is hereby incorporated by reference, in its entirety. charolyticus are multidomain enzymes composed of two catalytic domains, linked to carbohydrate binding domains by SUBMISSION OF SEQUENCE LISTING AS proline-threonine-rich regions (Zverlov V. Mahr S, Riedel K. ASCII TEXT FILE Bronnenmeier K (1998a), “Properties and gene structure of a bifunctional cellulolytic enzyme (CelA) from the extreme 0002 The content of the following submission on ASCII thermophile Anaerocellum thermophilum with separate text file is incorporated herein by reference in its entirety: a glycosyl hydrolase family 9 and 48 catalytic domains. computer readable form (CRF) of the Sequence Listing (file Microbiology 144 (Pt 2): 457-465; Te'o V S, Saul DJ, name: 677792.000940SEQLIST.txt, date recorded: Jun. 30, Bergquist P L (1995), “celA, another gene coding for a mul 2011, size: 206 KB). tidomain cellulase from the extreme thermophile Caldocel lum saccharolyticum,' Appl Microbiol Biotechnol 43: 291 FIELD 296; Saul et al. 1990. The recombinant endoglucanase of 0003. The present disclosure relates to hydrolysis of cel Rhodothermus marinus has a pH optimum of 6.0–7.0 and a lulose-containing polysaccharides and degradation of biom temperature optimum at 100° C. (Halldórsdóttir S, Thórólfs ass using polypeptides having cellulase activity, including dóttir ET, Spilliaert R. Johansson M, Thorbjarnardóttir SH, hyperthermophilic polypeptides.
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