Method for reducing viscosity in saccharification process

• US 10,190,108 B2
• Filed: 08/06/2015
• Issued: 01/29/2019
• Est. Priority Date: 03/17/2011
• Status: Active Grant

• Current Assignee: Danisco USA Incorporated (International Flavors & Fragrances Incorporated)
• Original Assignee: Danisco USA Incorporated (International Flavors & Fragrances Incorporated)
• Inventors: Mitchinson, Colin, Li, Mian, Kelemen, Bradley R., Lantz, Suzanne E., Wing, Keith D., Hitz, William D.
• Primary Examiner(s): Desai, Anand U
• Application Number:
  US14/819,989
• Publication Number:
  US 20170096651A1
• Time in Patent Office: 1,272 Days
• Field of Search: None
• 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: 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 302/01004 Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase

Abstract

The present invention relates to compositions that can be used in hydrolyzing biomass such as compositions comprising a polypeptide having glycosyl hydrolase family 61/endoglucanase activity, methods for hydrolyzing biomass material, and methods for reducing viscosity of biomass mixture using a composition comprising a polypeptide having glycosyl hydrolase family 61/endoglucanase activity.

Claims

1. A biomass saccharification mixture comprising:

a. a pretreated biomass material; and

b. a non-naturally occurring enzyme composition comprising a glycosyl hydrolase family 61 (“

GH61”

) polypeptide having GH61/endoglucanase activity, wherein the GH61 polypeptide

i) has at least 65% in sequence identity to residues 22-344 of SEQ ID NO;

27;

or

ii) is encoded by a polynucleotide sequence or a complement thereof that has at least 65% sequence identity to SEQ ID NO;

30;

or

iii) is encoded by a polynucleotide sequence that hybridizes under high stringency conditions to a sequence that is complementary to SEQ ID NO;

30;

wherein the enzyme composition is a whole cellulase comprising at least one polypeptide having cellobiohydrolase activity, at least one polypeptide having endoglucanase activity that is different from the GH61 polypeptide, and at least one polypeptide having beta-glucosidase activity and wherein the whole cellulase is derived from a host cell containing a heterologous expression cassette comprising a nucleic acid encoding the GH61 polypeptide, wherein the GH61 polypeptide is present in the whole cellulase in an amount of at least 6 wt % and no more than 50 wt % based on the total weight of protein in the whole cellulase and wherein said biomass saccharification mixture has a lower viscosity than a biomass saccharification mixture without the GH61 polypeptide and/or is capable of increasing the level of saccharification in the mixture as compared to the level of saccharification in a mixture having no or a lower level of GH-61 polypeptide.

2. The biomass saccharification mixture of claim 1, wherein the level of saccharification is measured by the yield of fermentable sugar after the mixture is incubated for a period of time sufficient to cause saccharification of the biomass.

3. The biomass saccharification mixture of claim 1 wherein the GH61 polypeptide having GH61/endoglucanase activity is derived from a filamentous fungus.

4. The biomass saccharification mixture of claim 3, wherein the filamentous fungus is one selected from the group:

Trichoderma, Humicola, Fusarium, Aspergillus, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora, Endothia, Mucor, Cochliobolus, Pyricularia, Chrysosporium, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium lucknowense, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Neurospora intermedia, Penicillium purpurogenum, Penicillium canescens, Penicillium solitum, Penicillium funiculosum Phanerochaete chrysosporium, Phlebia radiate, Pleurotus eryngii, Talaromyces flavus, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride, Geosmithia emersonii, or G. stearothermophilus.

5. The biomass saccharification mixture of claim 1, wherein the at least one polypeptide having cellobiohydrolase activity is T. reesei CBH1, Af7A (SEQ ID NO:

150), Af7B (SEQ ID NO;

151), Cg7A (SEQ ID NO;

152), Cg7B (SEQ ID NO;

153), Tt7A (SEQ ID NO;

154), Tt7B (SEQ ID NO;

155), T. reesei CBH2, Tt6A (SEQ ID NO;

156), St6A (SEQ ID NO;

157), St6B (SEQ ID NO;

158), ora variant thereof having at least 90% sequence identity thereto.

6. The biomass saccharification mixture of claim 1, wherein the at least one polypeptide having beta-glucosidase activity is:

a. Fv3C (SEQ ID NO;

100), Pa3D (SEQ ID NO;

94), Fv3G (SEQ ID NO;

96), Fv3D (SEQ ID NO;

98), Tr3A (SEQ ID NO;

102), Tr3B (SEQ ID NO;

104), Te3A (SEQ ID NO;

106), An3A (SEQ ID NO;

108), Fo3A (SEQ ID NO;

110), Gz3A (SEQ ID NO;

112), Nh3A (SEQ ID NO;

114), Vd3A (SEQ ID NO;

116), Pa3G (SEQ ID NO;

118), Tn3B (SEQ ID NO;

119), or a variant thereof having at least 90% sequence identity thereto;

or

b. a polypeptide encoded by a polynucleotide (1) having at least 90% sequence identity to SEQ ID NO;

99, 93, 95, 97, 101, 103, 105, 107, 109, 111, 113, 115, or 117;

or (2) that hybridizes under high stringency conditions to a sequence that is complementary to SEQ ID NO;

99, 93, 95, 97, 101, 103, 105, 107, 109, 111, 113, 115, or 117.

7. The biomass saccharification mixture of claim 1, wherein the enzyme composition further comprises:

(1) a polypeptide having xylanase activity, (2) a polypeptide having beta-xylosidase activity; and

(3) a polypeptide having L-alpha-arabinofuranosidase activity.

8. The biomass saccharification mixture of claim 7, wherein the polypeptide having xylanase activity is:

a. T. reesei Xyn3 (SEQ ID NO;

76), T. reesei Xyn2 (SEQ ID NO;

77), AfuXyn2 (SEQ ID NO;

58), AfuXyn5 (SEQ ID NO;

60), or a variant thereof having at least 90% sequence identity thereto;

or

b. a polypeptide encoded by a polynucleotide (1) having at least 90% sequence identity to SEQ ID NO;

75, 57, or 59;

or (2) that hybridizes under high stringency conditions to a sequence that is complementary to SEQ ID NO;

75, 57, or 59.

9. The biomass saccharification mixture of claim 7, wherein the at least one polypeptide having beta-xylosidase activity is:

a. Fv3A (SEQ ID NO;

36), Fv43A (SEQ ID NO;

44), Pf43A (SEQ ID NO;

38), Fv43D (SEQ ID NO;

62), Fv39A (SEQ ID NO;

42), Fv43E (SEQ ID NO;

40), Fo43A (SEQ ID NO;

52), Fv43B (SEQ ID NO;

46), Pa51A (SEQ ID NO;

48), Gz43A (SEQ ID NO;

50), T. reesei Bxl1 (SEQ ID NO;

78), or a variant thereof having at least 90% sequence identity thereto;

or

b. a polypeptide encoded by a polynucleotide (1) having at least 90% sequence identity to SEQ ID NO;

35, 43, 37, 61, 41, 39, 51, 45, 47, 49, or 159;

or (2) that hybridizes under high stringency conditions to a sequence that is complementary to SEQ ID NO;

35, 43, 37, 61, 41, 39, 51, 45, 47, 49, or 159.

10. The biomass saccharification mixture of claim 7, wherein the at least one polypeptide having L-alpha-arabinofuranosidase activity is:

a. Af43A (SEQ ID NO;

54), Fv43B (SEQ ID NO;

46), Pf51A (SEQ ID NO;

56), Pa51A (SEQ ID NO;

48), Fv51A (SEQ ID NO;

66), or a variant thereof having at least 90% sequence identity thereto;

or

b. a polypeptide encoded by a polynucleotide (1) having at least 90% sequence identity to SEQ ID NO;

53, 45, 55, 47, or 65;

or (2) that hybridizes under high stringency conditions to a sequence that is complementary to SEQ ID NO;

53, 45, 55, 47, or 65.

11. The biomass saccharification mixture of claim 7, wherein the GH61 polypeptide is present in the whole cellulase in an amount of at least 8 wt % and no more than 35 wt % based on the total weight of protein in the whole cellulase.

12. The biomass saccharification mixture of claim 1, wherein the pretreated biomass material is subjected to pretreatment with an acid or a base, and wherein the pretreated biomass is adjusted to pH of about 4.0 to 6.5 before mixing with the enzyme composition.

13. The biomass saccharification mixture of claim 1, wherein the biomass material is present in the mixture in an amount of about 5 wt. % to about 60 wt. %, about 10 wt. % to about 50 wt. %, about 15 wt. % to about 40 wt. %, about 15 wt. % to about 30 wt. %, or about 20 wt. % to about 30 wt. %, referring to the amount of biomass material in its solid state relative to the total weight of the mixture.

14. A method of hydrolyzing a biomass material comprising incubating the biomass saccharification mixture of claim 1, under conditions suitable for hydrolyzing the biomass materials in the biomass saccharification mixture and for a sufficient period of time.

15. The method of claim 14, wherein the conditions suitable for hydrolyzing the biomass materials in the biomass saccharification mixture comprises:

(1) a pH of about 3.5 to about 7.0;

(2) for a duration of about 2 hours or longer; and

/or (3) a temperature of about 20°

C. to about 75°

C.;

or wherein the sufficient period of time comprises a time period of about 8 hours to about 72 hours.

16. The method of claim 14, wherein at any given time above 2 hours, the amount of fermentable sugars is produced by the biomass saccharifcation mixture is increased by at least about 5% as compared to the amount of fermentable sugars produced by a control biomass saccharification mixture comprising the same amount and type of biomass material, and the same composition of enzyme components but in the absence of the GH-61 polypeptide having GH61/endoglucanase activity.

17. The method of claim 14, wherein the viscosity of the biomass saccharification mixture is reduced by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 35%, as compared to the viscosity of the control biomass saccharification mixture comprising the same amount and type of biomass material, and the same composition of enzyme components but in the absence of the GH61 polypeptide having GH61/endoglucanase activity.

18. A method of using the composition of claim 1 to convert a biomass material into fermentable sugars in a merchant enzyme supply model or an on-site bio-refinery model.

19. The biomass saccharification mixture of claim 1, wherein the polypeptide having endoglucanase activity is comprises T. reesei EG1 or T. reesei EG2.