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WO 2017/202946 Al 30 November 2017 (30.11.2017) W !P O PCT

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WO 2017/202946 Al 30 November 2017 (30.11.2017) W !P O PCT (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2017/202946 Al 30 November 2017 (30.11.2017) W !P O PCT (51) International Patent Classification: Published: C12N 9/5 (2006.01) — with international search report (Art. 21(3)) (21) International Application Number: — before the expiration of the time limit for amending the PCT/EP2017/062598 claims and to be republished in the event of receipt of amendments (Rule 48.2(h)) (22) International Filing Date: — with sequence listing part of description (Rule 5.2(a)) 24 May 2017 (24.05.2017) (25) Filing Language: English (26) Publication Langi English (30) Priority Data: 16170964.7 24 May 2016 (24.05.2016) EP (71) Applicant: NOVOZYMES A/S [DK/DK]; Krogshoejvej 36, 2880 Bagsvaerd (DK). (72) Inventors: CARSTENSEN, Lone; Krogshoejvej 36, 2880 Bagsvaerd (DK). SPODSBERG, Nikolaj; Krogshoejvej 36, 2880 Bagsvaerd (DK). GJERMANSEN, Morten; Krogshoejvej 36, 2880 Bagsvaerd (DK). SALOMON, Jes- per; Krogshoejvej 36, 2880 Bagsvaerd (DK). KROGH, Kristian, B,R,M,; Krogshoejvej 36, 2880 Bagsvaerd (DK). (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY,TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). (54) Title: COMPOSITIONS COMPRISING POLYPEPTIDES HAVING GALACTANASE ACTIVITY AND POLYPEPTIDES o HAVING BETA-GALACTOSIDASE ACTIVITY (57) Abstract: The present invention relates to compositions comprising polypeptides having galactanase activity and polypeptides having beta-galactosidase activity for use in e.g. animal feed. The present invention further relates to polypeptides having beta-galac- o tosidase activity, polypeptides having galactanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypep tides. COMPOSITIONS COMPRISING POLYPEPTIDES HAVING GALACTANASE ACTIVITY AND POLYPEPTIDES HAVING BETA-GALACTOSIDASE ACTIVITY Reference to a Sequence Listing This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference. Background of the Invention Field of the Invention The present invention relates to compositions comprising polypeptides having galactanase activity and polypeptides having beta-galactosidase activity for use in e.g. animal feed. The present invention further relates to polypeptides having beta-galactosidase activity, polypeptides having galactanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides. Description of the Related Art Soybean is a species of legume native to East Asia and is the second biggest feed crop globally and the biggest protein source applied in animal feed. Soybean can be manufactured (defatted) to produce soybean meal (SBM), and SBM is a significant and cheap source of high quality protein for animal feeds. Other common types of legume are chickpea, lupin, lentil, peanut, beans or peas which can also be processed and used as animal feed. Legumes, such as soybean, contain significant amounts of galactan polysaccharides which need to be degraded by enzymes to release the sugars, and thus energy, therein. However, there are few solutions which efficiently degrade galactan polysaccharides and thus energy which is in the legume cannot be properly utilised by an animal. Up to 70% of a farmers expenses is from the cost of animal feed. Shipkowski et aiin Applied and Environmental Microbiology, 2006, 72(12), 7730 discloses the beta-galactosidase LacA and the galactanase GalA and the use of these two enzymes in degrading galactan. Tabachnikov et al in Febs Journal, 2013, 280(3), 950 discloses the beta- galactosidase GanB and the galactanase GanA and the use of these two enzymes in degrading galactan. However, as disclosed herein these prior art solutions are not very effective at releasing galactose from legumes, such as soybean. Thus the object of this invention is to provide a solution which efficiently releases sugars from galactan polysaccharides and thereby improves the nutritional value of the legumes to reduce the feed costs by reformulation of the diet or to provide more energy to the animal resulting in improved animal growth. Summary of the Invention The present invention relates to compositions comprising one or more GH53 polypeptides having galactanase activity and one or more GH42 polypeptides having beta-galactosidase activity. The present application further relates to granules comprising the polypeptide(s) of the invention; animal feed additives comprising the polypeptide(s) of the invention; liquid formulations comprising the polypeptide(s) of the invention; animal feed and pelleted animal feed comprising the polypeptide(s) of the invention; methods of releasing galactose from plant based material; methods of improving one or more performance parameters of an animal and use of the composition of the invention in animal feed, in animal feed additives, in the preparation of a composition for use in animal feed, for improving the nutritional value of an animal feed, for increasing digestibility of the animal feed, for improving one or more performance parameters in an animal and/or for releasing galactose from plant based material. The invention also relates to isolated polypeptide having beta-galactosidase or galactanase activity, polynucleotides encoding the polypeptides of the present invention; nucleic acid constructs; expression vectors; recombinant host cells comprising the polynucleotides and methods of producing the polypeptides. Overview of Sequence Listing SEQ ID NO: 1 is the gene sequence of the GH53 galactanase as isolated from Cohnella sp-60555. SEQ ID NO: 2 is the amino acid sequence as deduced from SEQ ID NO: 1. SEQ ID NO: 3 is the amino acid sequence of the mature GH53 galactanase from Cohnella sp-60555. SEQ ID NO: 4 is the amino acid sequence of SEQ ID NO: 3 with His-tag. SEQ ID NO: 5 is the gene sequence of the GH53 galactanase as isolated from Cohnella xylanilytica. SEQ ID NO: 6 is the amino acid sequence as deduced from SEQ ID NO: 5 . SEQ ID NO: 7 is the amino acid sequence of the mature GH53 galactanase from Cohnella xylanilytica. SEQ ID NO: 8 is the amino acid sequence of SEQ ID NO: 7 with His-tag. SEQ ID NO: 9 is the gene sequence of the GH53 galactanase as isolated from Paenibacillus tundrae. SEQ ID NO: 10 is the amino acid sequence as deduced from SEQ ID NO: 9. SEQ D NO: is the amino acid sequence of the mature GH53 galactanase from Paenibacillus tundrae. SEQ ID NO: 12 is the amino acid sequence of SEQ ID NO: 11 with His-tag. SEQ ID NO: 13 is the gene sequence of the GH53 galactanase as isolated from Paenibacillus barcinonensis. SEQ ID NO: 14 is the amino acid sequence as deduced from SEQ ID NO: 13. SEQ ID NO: 15 is the amino acid sequence of the mature GH53 galactanase from Paenibacillus barcinonensis. SEQ ID NO: 16 is the amino acid sequence of SEQ ID NO: 15 with His-tag. SEQ ID NO: 17 is the gene sequence of the GH53 galactanase as isolated from Paenibacillus sp-62603. SEQ ID NO: 18 is the amino acid sequence as deduced from SEQ ID NO: 17. SEQ ID NO: 19 is the amino acid sequence of the mature GH53 galactanase from Paenibacillus sp-62603. SEQ ID NO: 20 is the amino acid sequence of SEQ ID NO: 19 with His-tag. SEQ D NO: 2 1 is the gene sequence of the GH53 galactanase as isolated from Paenibacillus xylanilyticus. SEQ ID NO: 22 is the amino acid sequence as deduced from SEQ ID NO: 2 1. SEQ ID NO: 23 is the amino acid sequence of the mature GH53 galactanase from Paenibacillus xylanilyticus. SEQ ID NO: 24 is the amino acid sequence of SEQ ID NO: 23 with His-tag. SEQ ID NO: 25 is the gene sequence of the GH53 galactanase as isolated from Paenibacillus sp-1 8 179. SEQ ID NO: 26 is the amino acid sequence as deduced from SEQ ID NO: 25. SEQ ID NO: 27 is the amino acid sequence of the mature GH53 galactanase from Paenibacillus sp-1 8 179. SEQ ID NO: 28 is the amino acid sequence of SEQ ID NO: 27 with His-tag. SEQ ID NO: 29 is the gene sequence of the GH53 galactanase as isolated from Paenibacillus peoriae. (D448RG) SEQ ID NO: 30 is the amino acid sequence as deduced from SEQ ID NO: 29. SEQ ID NO: 3 1 is the amino acid sequence of the mature GH53 galactanase from Paenibacillus peoriae.
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