Glycosyl hydrolase enzymes and uses thereof

• US 10,273,465 B2
• Filed: 07/27/2015
• Issued: 04/30/2019
• Est. Priority Date: 09/23/2009
• Status: Active Grant

• Current Assignee: Danisco USA Incorporated (International Flavors & Fragrances Incorporated)
• Original Assignee: Danisco USA Incorporated (International Flavors & Fragrances Incorporated)
• Inventors: Bower, Benjamin, Hsi, Megan Yee, Kaper, Thijs, Kelemen, Bradley R., Lantz, Suzanne E., Larenas, Edmund A., Mitchinson, Colin, Kim, Steven, Hitz, William D., Emptage, Mark, Wing, Keith Dumont
• Primary Examiner(s): Monshipouri, Maryam
• Application Number:
  US14/810,434
• Publication Number:
  US 20160177279A1
• Time in Patent Office: 1,373 Days
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/0065 acting on hydrogen peroxide as acceptor (1.11)
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/20 Triglyceride splitting, e.g. by means of lipase
• CPC Class Codes: C12N 9/2402 hydrolysing O- and S- glycosyl compounds (3.2.1)
• CPC Class Codes: C12N 9/2437 Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
• CPC Class Codes: C12N 9/2445 Beta-glucosidase (3.2.1.21)
• CPC Class Codes: C12N 9/248 Xylanases
• CPC Class Codes: C12N 9/2485 Xylan endo-1,3-beta-xylosidase (3.2.1.32), i.e. endo-1,3-beta-xylanase
• CPC Class Codes: C12P 19/00 Preparation of compounds containing saccharide radicals ketoaldonic acids C12P7/58
• CPC Class Codes: C12P 19/02 Monosaccharides 2-ketogulonic acid C12P7/60
• CPC Class Codes: C12P 19/14 produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase , e.g. by cellulase, hemicellulase
• CPC Class Codes: C12Y 302/01 Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
• CPC Class Codes: C12Y 302/01004 Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• CPC Class Codes: C12Y 302/01008 Endo-1,4-beta-xylanase (3.2.1.8)
• CPC Class Codes: C12Y 302/01021 Beta-glucosidase (3.2.1.21)
• CPC Class Codes: C12Y 302/01037 Xylan 1,4-beta-xylosidase (3.2.1.37)
• CPC Class Codes: C12Y 302/01055 Alpha-N-arabinofuranosidase (3.2.1.55)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel
• CPC Class Codes: Y02E 50/30 Fuel from waste, e.g. synthetic alcohol or diesel

Abstract

The present disclosure is generally directed to glycosyl hydrolase enzymes, compositions comprising such enzymes, and methods of using the enzymes and compositions, for example for the saccharification of cellulosic and hemicellulosic materials into sugars.

Claims

1. A non-naturally occurring composition comprising:

(a) a first polypeptide having f3-xylosidase activity, wherein the first polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO;

28 or the amino acid sequence corresponding to residues (i) 20-341, (ii) 21-350, (iii) 107-341, or (iv) 107-350 of SEQ ID NO;

28; and

(b) a second polypeptide having xylanase activity;

wherein the non-naturally occurring composition comprises one or more polypeptides produced from a host cell, wherein the host cell is engineered to express the first polypeptide having β

-xylosidase activity and wherein the first polypeptide is heterologous to the host cell.

2. The composition of claim 1, wherein the second polypeptide is a Trichoderma xylanase or an Aspergillus xylanase.

3. The composition of claim 2, wherein the Trichoderma xylanase is a Trichoderma reesei Xyn3 polypeptide or a Trichoderma reesei Xyn2 polypeptide, or the Aspergillus xylanase is an Aspergillus fumigatus AfuXyn2 polypeptide or an Aspergillus fumigatus AfuXyn5 polypeptide, wherein:

said Trichoderma reesei Xyn3 polypeptide has at least 90% sequence identity to SEQ ID NO;

42, or to residues 17-347 of SEQ ID NO;

42, or is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO;

41;

said Trichoderma reesei Xyn2 polypeptide has at least 90% sequence identity to SEQ ID NO;

43, or to residues 33-222 of SEQ ID NO;

43;

said Aspergillus fumigatus AfuXyn2 polypeptide has at least 90% sequence identity to SEQ ID NO;

24, or to residues 19-228 of SEQ ID NO;

24, or is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO;

23;

said Aspergillus fumigatus AfuXyn5 polypeptide has at least 90% sequence identity to SEQ ID NO;

26, or to residues 20-313 of SEQ ID NO;

26, or is encoded by a nucleic acid having at least 90% sequence identity to SEQ ID NO;

25.

4. The composition of claim 1, further comprising one or more accessory proteins selected from the group consisting of mannanases, galactanases, arabinases, ligninases, amylases, glucuronidases, proteases, esterases, lipases, xyloglucanases, glycoside hydrolase Family 61 polypeptides, CIP1, CIP2, swollenin, expansins, CIP1-like proteins, CIP2-like proteins, cellobiose dehydrogenases, and manganese peroxidases.

5. The composition of claim 1, further comprising a biomass.

6. The composition of claim 5, wherein the biomass is pretreated biomass, optionally wherein the biomass is ammonia-pretreated.

7. The composition of claim 6, wherein the combined weight of polypeptides having xylanase activity is 0.5 g to 40 g per 1 kg of hemicellulose in the biomass, or the combined weight of polypeptides having f3-xylosidase activity is 0.5 g to 50 g per 1 kg of hemicellulose in the biomass.

8. The composition of claim 7, wherein the biomass comprises one or more of corncob, corn stover, corn fiber, switchgrass, sorghum, paper, pulp, sugarcane bagasse, or Miscanthus.

9. A fermentation broth comprising the composition of claim 1.

10. The fermentation broth of claim 9, which is the fermentation broth of a filamentous fungus, wherein the filamentous fungus is a Trichoderma, Humicola, Fusarium, Aspergillus, Myceliophthora, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora, Endothia, Mucor, Cochliobolus, Pyricularia, Phanerochaete, or Chrysosporium.

11. A saccharification process comprising treating a material comprising hemicellulose with the composition of claim 1.

12. The process of claim 11, wherein the material comprising hemicellulose is one or more of corncob, corn stover, corn fiber, switchgrass, sorghum, paper, pulp, sugarcane bagasse, or Miscanthus;

optionally wherein the process yields at least 60% xylose from hemicellulose xylan of the material comprising hemicellulose.