WO2010009515A1 - Procédé d'hydrolyse enzymatique - Google Patents

Procédé d'hydrolyse enzymatique Download PDF

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WO2010009515A1
WO2010009515A1 PCT/AU2009/000950 AU2009000950W WO2010009515A1 WO 2010009515 A1 WO2010009515 A1 WO 2010009515A1 AU 2009000950 W AU2009000950 W AU 2009000950W WO 2010009515 A1 WO2010009515 A1 WO 2010009515A1
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orf
polysaccharide
thermophilic
hydrolytic enzymes
enzymes
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WO2010009515A8 (fr
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Valentino Setoa Junior Te'o
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Applimex Systems Pty Ltd
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Priority claimed from AU2008903814A external-priority patent/AU2008903814A0/en
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Publication of WO2010009515A1 publication Critical patent/WO2010009515A1/fr
Publication of WO2010009515A8 publication Critical patent/WO2010009515A8/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/12—Disaccharides
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
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    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01072—Acetylxylan esterase (3.1.1.72)
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
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    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01021—Beta-glucosidase (3.2.1.21)
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01037—Xylan 1,4-beta-xylosidase (3.2.1.37)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/0105—Alpha-N-acetylglucosaminidase (3.2.1.50)
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    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01055—Alpha-N-arabinofuranosidase (3.2.1.55)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/0108—Fructan beta-fructosidase (3.2.1.80)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01091—Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01113—Mannosyl-oligosaccharide 1,2-alpha-mannosidase (3.2.1.113), i.e. alpha-1,2-mannosidase
    • C—CHEMISTRY; METALLURGY
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01151—Xyloglucan-specific endo-beta-1,4-glucanase (3.2.1.151), i.e. endoxyloglucanase
    • C—CHEMISTRY; METALLURGY
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01155—Xyloglucan-specific exo-beta-1,4-glucanase (3.2.1.155), i.e. exoxyloglucanase
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the invention relates to methods for the enzymatic hydrolysis of materials. More specifically, the invention relates to a two stage enzyme hydrolysis method for the hydrolysis of polysaccharides. The invention further relates to sugar fragments produced by the enzymatic hydrolysis methods.
  • the optimal operational temperature for most fungal enzymes is normally moderate (around 40°C-50°C).
  • polysaccharide components such as xylan can precipitate back onto the cellulose and/or hemicellulose as the alkali concentration decreases with temperature, as has previously been observed during kraft pulping (Yllner et al., "A study of the removal of constituents of pine wood in the sulphate process using a continuous liquor flow method” (1957), Svensk Papperstid, 60: 795-802; Meller, "The retake of xylan during alkaline pulping. A critical appraisal of the literature” (1965), Holzforschung, 19: 118-124).
  • lignin was found to precipitate back onto the surface of lignocellulose biomass following pretreatment of corn stover under acidic and neutral pH, and was shown to have a negative impact on cellulose conversion by fungal enzymes (Selig et al., "Deposition of lignin droplets produced during dilute acid pretreatment of maize stems retards enzymatic hydrolysis of cellulose, " Biotechnol. Prog. 23: 1333-1339, 2007).
  • the enzymatic hydrolysis of pre-treated raw material and subsequent fermentation of released mono-sugars can be performed separately or simultaneously. These two processes are commonly referred to as SHF (separate hydrolysis fermentation) or SSF (simultaneous saccharification fermentation).
  • SSF has generally been regarded as more cost-effective over SHF in ethanol production.
  • the enzymatic hydrolysis rate during SHF is strongly affected by end- product inhibition (e.g. by cellobiose and glucose) and requires the addition of adequate amounts of ⁇ -glucosidase, reducing cost-efficiency.
  • end- product inhibition e.g. by cellobiose and glucose
  • ⁇ -glucosidase reducing cost-efficiency.
  • SSF seeks to avoid this problem by means of a continuous removal of glucose by the fermenting organism, the conditions during the combined SSF process are not optimal as the temperature optimum for the hydrolyzing enzymes ( ⁇ 50°C) is different to the optimal growth temperature of the fermenting organism (e.g. ⁇ 35°C).
  • ⁇ 50°C the temperature optimum for the hydrolyzing enzymes
  • it is difficult to optimize a continuous SSF process because of solid residues resulting from the hydrolysis which prevents recovery and therefore recycling of the fermenting organism.
  • Described herein is a two-stage enzyme hydrolysis method for the production of fermentable sugars.
  • the method involves using a combination of mesophilic and thermophilic hydrolytic enzymes to hydrolyse polysaccharides into smaller fermentable units.
  • the method is demonstrated herein to provide higher yields of fermentable sugars compared to the use of mesophilic or thermophilic hydrolytic enzymes alone, and is more cost-effective in comparison to many existing enzyme hydrolysis systems.
  • the invention provides a method for hydrolysing a polysaccharide, the method comprising the steps of: (i) contacting a polysaccharide with an enzyme mixture comprising mesophilic and thermophilic hydrolytic enzymes,
  • step (ii) incubating the polysaccharide and enzyme mixture of step (i) at a temperature suitable for activity of the mesophilic hydrolytic enzymes; and (iii) incubating the polysaccharide and enzyme mixture of step (ii) at a temperature suitable for activity of the thermophilic hydrolytic enzymes, wherein said polysaccharide is cleaved by said hydrolytic enzymes to produce at least one fragment of said polysaccharide.
  • the invention provides a method for hydrolysing a polysaccharide, the method comprising the steps of:
  • step (ii) incubating the polysaccharide and mesophilic hydrolytic enzymes of step (i) at a temperature suitable for activity of the mesophilic hydrolytic enzymes,
  • thermophilic hydrolytic enzymes contacting the polysaccharide of step (ii) with thermophilic hydrolytic enzymes
  • the invention provides a method for hydrolysing a polysaccharide, the method comprising the steps of:
  • the invention provides a method for hydrolysing a polysaccharide, the method comprising the steps of:
  • thermophilic hydrolytic enzymes
  • thermophilic hydrolytic enzymes incubating the reaction mixture at a temperature suitable for activity of the thermophilic hydrolytic enzymes, wherein the polysaccharide is hydrolysed by said mesophilic and thermophilic hydrolytic enzymes to produce two or more polysaccharide fragments.
  • the temperature suitable for activity of the mesophilic hydrolytic enzymes is above about 1O 0 C and below 60°C. In another embodiment of the above aspects, the temperature suitable for activity of the mesophilic hydrolytic enzymes is between about 30°C and below 60 0 C.
  • the temperature suitable for activity of the mesophilic hydrolytic enzymes is between about 40°C and below 60 0 C.
  • the temperature suitable for activity of the mesophilic hydrolytic enzymes is between about 45°C and about 55°C.
  • the temperature suitable for activity of the mesophilic hydrolytic enzymes is about 50 0 C.
  • the temperature suitable for activity of the thermophilic hydrolytic enzymes is 60 0 C, or above 60 0 C. In one embodiment of the above aspects, the temperature suitable for activity of the thermophilic hydrolytic enzymes is above 60 0 C.
  • the temperature suitable for activity of the thermophilic hydrolytic enzymes is above about 65°C.
  • the temperature suitable for activity of the thermophilic hydrolytic enzymes is about 70 0 C, or above about 70 0 C.
  • the temperature suitable for activity of the thermophilic hydrolytic enzymes is above about 80 0 C.
  • the temperature suitable for activity of the thermophilic hydrolytic enzymes is above about 90 0 C.
  • the polysaccharide is selected from the group consisting of cellulose, hemicellulose and mixtures thereof.
  • the polysaccharide is a component of lignocellulosic biomass.
  • the lignocellulosic biomass may be pre-treated with a supercritical solvent, acid hydrolysis or base hydrolysis.
  • the mesophilic or thermophilic hydrolytic enzyme is a hydrolase.
  • the mesophilic or thermophilic hydrolase is a glycosylase.
  • the glycosylase may be selected from the group consisting of xylanases, glucosidases, cellulases, xylosidases, mannanases, mannosidases, arabinosidases, glycoside hydrolases, dextranases, exoglucanases, and endoglucanases.
  • thermophilic hydrolytic enzymes are derived from at least one bacterial strain.
  • the bacterial strain may be selected from the group consisting of Acetogenium kuvui, Acetomicrobium faecalis, Acidothermus cellulolyticus, Anaerocellum thermophilum, Chloroflexus auranticus, Desulfotomaculum nigrificans, Desulfovibrio thermophilus, Dictyoglomus thermophilum, Dictyoglomus thermophilum strain Rt46B.l, Bacillus acidocaldarius, Bacillus stearothermophilus, Bacillus caldolyticus, Bacillus caldotenax, Bacillus caldovelox, Bacillus thermoglucosides, Bacillus thermoglucosidasius, Bacillus thermocatenulatus, Bacillus schlegelii, Bacillus ⁇ avothermus, Bacillus tusciae, Bacillus sp.
  • KSM-S237 Caldicellulosiruptor saccharolyticus (formerly known as Caldocellum saccharolyticum), Caldicellulosiruptor strain Rt69B.l, Caldicellulosiruptor strain Tok7B.l, Clostridium stercorarium, Clostridium thermocellum, Clostridium thermosulfurogenes, Clostridium thermohydrosulfuricum, Clostridium autotrophicum, Clostridium fervidus, Clostridium. thermosaccharolyticum, Caldohacterium hydrogenophilum, Fervidobacterium nodosum, F.
  • Rhodothermus marinus Saccharococcus thermophilus, Streptomyces sp., Synechococcus lividus, Thermoleophilum album, Thermoleophilum minutum, Thermoanaerobium brockii, Thermospiro africanus, Thermoanaerobacter ethanolicus, Thermoanaerobacterium lactoethylicum, Thermodesulfobacterium commune, Thermobacteroides acetoethylicus, Thermobacteroides leptospartum,
  • thermophilic hydrolytic enzyme is selected from the group consisting of ⁇ -glucosidase (BgIA) from Caldicellulosiruptor saccharolyticus TP8.3.3.1, Xylanase A (XynA) from Dictyoglomus thermophilum, bifunctional Cellulase B (CeIB) from Caldicellulosiruptor saccharolyticus, ⁇ -xylosidase (XynB) from Caldicellulosiruptor saccharolyticus, mannanase (ManA) from Dictyoglomus thermophilum, mannanase from Caldicellulosiruptor Rt8B.4, mannosidase
  • BgIA ⁇ -glucosidase
  • XynA Xylanase A
  • CeIB bifunctional Cellulase B
  • XynB ⁇ -xylosidase
  • ManA from Dic
  • thermophilic hydrolytic enzyme is selected from the group consisting of ⁇ -glucosidase (BgIA) from Cs. saccharolyticus
  • TP8.3.3.1 Cellulase/Cellobiohydrolase (CeIA) from Cs. saccharolyticus, Arabinofuranosidase (XynF) from Cs. saccharolyticus, Xylanases (xynE and xynl) from Cs. saccharolyticus.
  • CeIA Cellulase/Cellobiohydrolase
  • XynF Arabinofuranosidase
  • xynE and xynl Xylanases
  • CelECterm and CelB5 from Caldicellulosiruptor Tok7B.l and Mannanase (ManA) from Caldicellulosiruptor Tok7B.l and Mannanase (ManA) from Caldicellulosiruptor Tok7B.l and Mannanase (ManA) from Caldicellulosiruptor Tok7B.l and Mannanase (ManA) from Caldicellulosiruptor Tok7B.l and Mannanase (ManA) from Caldicellulosiruptor Tok7B.l and Mannanase (ManA) from Caldicellulosiruptor Tok7B.l and Mannanase (ManA) from Caldicellulosiruptor Tok7B.l and Mannanase (ManA) from Caldicellulosiruptor Tok7B.l and Mannanase (ManA) from
  • thermophilic hydrolytic enzymes are produced from a synthetic polynucleotide sequence.
  • the mesophilic hydrolytic enzymes are derived from fungi.
  • the fungi may be selected from the group consisting of Trichoderma, Aspergillus, Humicola, Chrysosporium, Doratomyces, Fusarium, Gliocladium,
  • the mesophilic hydrolytic enzymes are endogenously produced mesophilic enzymes derived from at least one fungal strain.
  • the fungal strain may be selected from the group consisting of Trichoderma, Aspergillus,
  • the mesophilic hydrolytic enzymes are endogenously produced mesophilic enzymes derived from the fungal strain Trichoderma reesei.
  • the endogenous enzymes may be selected from the group consisting of:
  • the mesophilic hydrolytic enzymes are endogenously produced mesophilic enzymes derived from at least one recombinant fungal strain capable of producing thermophilic hydrolytic enzymes.
  • the thermophilic hydrolytic enzymes may comprise thermophilic xylanase B (XynB).
  • the thermophilic xylanase B may be derived from Dictyoglomus thermophilum.
  • the thermophilic xylanase B may be derived from Dictyoglomus thermophilum Rt46B.l s
  • the fragment is selected from the group consisting of oligosaccharides, disaccharides, monosaccharides and mixtures thereof.
  • the disaccharides may be selected from the group consisting of sucrose, lactose, maltose, trehalose, cellobiose, laminaribiose, xylobiose, gentiobiose, isomaltose, mannobiose, kojibiose, rutinose, nigerose, and melibiose.
  • the monosaccharides may be selected fromo the group consisting of trioses, tetroses, pentoses, hexoses, heptoses, octoses and nonoses.
  • the method comprises an additional step of contacting the polysaccharide with a cryophilic hydrolytic enzyme and incubating the polysaccharide and cryophilic hydrolytic enzyme at a temperature suitable for the activity of the cryophilic hydrolytic enzyme.
  • the invention provides a method for producing a fermented sugar product, the method comprising: hydrolysing a polysaccharide using the method according to any one of the first, second, third or fourth aspects to produce at least two polysaccharide fragments, and fermenting the polysaccharide fragments to produce the fermented sugar product.0
  • the step of fermenting may be performed utilising a microorganism selected from the group consisting of fungi, bacteria, and combinations thereof.
  • the fungi may be selected from the group consisting of Basidiomycetes,
  • Trichocladium Trichocladium, Geotrichum, Aspergillus, Penicillium, Fusarium, Saccharomyces,
  • the bacteria may be selected from the group consistingS of Zymomonas, Leuconostoc, Lactobacillus, Oenococcus, Leuconostoc and
  • the fermented sugar product may be an alcohol or an organic acid.
  • the alcohol may be selected from the group consisting of xylitol, mannitol, arabinol, butanol and ethanol.
  • the invention provides a fragment produced in accordance with the method of the first, second, third or fourth aspects.
  • the invention provides a fermented sugar product produced in accordance with method of the fifth aspect.
  • the invention provides a composition for the enzymatic hydrolysis of a polysaccharide, said composition comprising at least one mesophilic hydrolytic enzyme, and at least one thermophilic hydrolytic enzyme.
  • the invention provides use of a composition for the enzymatic hydrolysis of a polysaccharide, said composition comprising at least one mesophilic hydrolytic enzyme and at least one thermophilic hydrolytic enzyme.
  • the mesophilic hydrolytic enzyme is used to hydrolyze said polysaccharide at a temperature of above about 10°C and below 60°C
  • the mesophilic hydrolytic enzyme is used to hydrolyze said polysaccharide at a temperature of between about 30°C and below 60°C.
  • the mesophilic hydrolytic enzyme is used to hydrolyze said polysaccharide at a temperature of between about 40 0 C and below 60°C.
  • the temperature suitable for activity of the mesophilic hydrolytic enzymes is between about 45°C and about 55°C. In one embodiment of the ninth aspect, the mesophilic hydrolytic enzyme is used to hydrolyze said polysaccharide at a temperature of about 5O 0 C.
  • thermophilic hydrolytic enzyme is used to hydrolyze said polysaccharide at a temperature of 60°C, or above 60 0 C.
  • thermophilic hydrolytic enzyme is used to hydrolyze the polysaccharide at a temperature of about 70°C, or above about 70°C.
  • thermophilic hydrolytic enzyme is used to hydrolyze the polysaccharide at a temperature of above about 80 0 C.
  • thermophilic hydrolytic enzyme is used to hydrolyze the polysaccharide at a temperature of above about 90 0 C.
  • the hydrolytic enzyme is a glycosylase selected from the group consisting of xylanases, glucosidases, cellulases, xylosidases, mannanases, glycoside hydrolases, dextranases, cellobiohydrolases and endoglucanases.
  • the polysaccharide is selected from the group consisting of cellulose, hemicellulose and mixtures thereof.
  • Figure 1 is a graph showing the yield of reducing sugars produced from enzyme hydrolysis of hemicellulose liquor from supercritical ethanol-treated Pinus radiata sawdust substrate, as detected by a dinitrosalicyclic acid (DNS) colour-based assay.
  • Sample numbers (1-4) are indicated on the x-axis
  • absorbance values (measured at ⁇ 540 ) are indicated on the y-axis.
  • Figure 2 is a graph showing the yield of reducing sugars produced from enzyme hydrolysis of supercritical ethanol-treated Pinus radiata sawdust substrate, as detected by a dinitrosalicyclic acid (DNS) colour-based assay.
  • Sample numbers (1-4) are indicated on the y-axis, absorbance values (measured at ⁇ 54 o) are indicated on the z-axis, temperatures utilised in the assay are indicated on the x-axis. Dark columns: absorbance at ⁇ 540 after 16 hour incubation at 50°C; light columns: absorbance at ⁇ 54 o after a further 16 hour incubation at 70°C.
  • Figure 3 is a graph showing the yield of reducing sugars produced from enzyme hydrolysis of supercritical ethanol-treated Pinus radiata sawdust substrate, as detected by a dinitrosalicyclic acid (DNS) colour-based assay.
  • Sample numbers (1-5) are indicated on the x-axis, absorbance values (measured at ⁇ s 4 o) are indicated on the y-axis.
  • Figure 4a is a graph showing the yield of reducing sugars produced from enzyme hydrolysis of supercritical ethanol-treated Pinus radiata sawdust substrate, as detected by a dinitrosalicyclic acid (DNS) colour-based assay.
  • DNS dinitrosalicyclic acid
  • Sample numbers (1-8) are indicated on the x-axis, absorbance values (measured at ⁇ 54 o) are indicated on the y-axis. Different shaded columns represent absorbance at ⁇ 54 o measured at different timepoints in the assay (3, 6, 22, 25, 30 and 46.5 hours) as indicated on the legend key.
  • Figure 4b is a graph showing the yield of reducing sugars produced from enzyme hydrolysis of ethanol-supercritical-treated Pinus radiata sawdust substrate, as detected by a dinitrosalicyclic acid (DNS) colour-based assay. Time (in hours) is indicated on the x- axis, absorbance values (measured at X ⁇ AO ) are indicated on the y-axis. The various combinations/concentrations of mesophilic and/or thermophilic hydrolytic enzymes utilised on different samples are indicated on the legend key. SubCont: substrate control.
  • Figure 5 is a high performance liquid chromatography (HPLC) trace overlay comparison of higher molecular weight oligosaccharides released in the enzyme-substrate samples at 50°C (black line) compared to 70°C (blue line).
  • Figure 6 is a graph showing the yield of glucose, xylose and arabinose produced from enzyme hydrolysis of ethanol-supercritical-treated Pinus radiata kraft pulp substrate, as detected by HPLC. Sample numbers (1-4) are indicated on the x-axis. GIu, glucose; XyI, Xylose; Ara; Arabinose. Sugar concentration (g/L) is indicated on the y- axis.
  • a plant cell also includes a plurality of plant cells.
  • a polynucleotide “comprising” a sequence encoding a protein may consist exclusively of that sequence or may include one or more additional sequences.
  • polysaccharide encompasses any molecule comprising two or more monosaccharide units.
  • mesophilic hydrolytic enzyme and “mesophilic enzyme” encompasses any hydrolytic enzyme having optimal enzymatic activity at a temperature above about 10 0 C and below 6O 0 C. Accordingly, a temperature "suitable for the activity" of a mesophilic hydrolytic enzyme will be a temperature above about 10 0 C and below 60 0 C.
  • thermophilic hydrolytic enzyme and “thermophilic enzyme” encompasses any hydrolytic enzyme having optimal enzymatic activity at a temperature of 60 0 C, and any hydrolytic enzyme having optimal enzymatic activity at a temperature higher than 60 0 C. Accordingly, a temperature "suitable for the activity" of a thermophilic hydrolytic enzyme will be a temperature of 60°C, or higher than 60°C.
  • the yield of fermentable sugars from complex polysaccharides can be increased using a two-stage enzyme hydrolysis method.
  • the method involves using a combination of mesophilic and thermophilic hydrolytic enzymes to fragment polysaccharides into smaller fermentable sugars.
  • the method is suitable for use on any material comprising polysaccharides and can be utilised, for example, in existing bioethanol production methods.
  • the enzymatic hydrolysis is performed in a two-stage method whereby, in the first stage, the polysaccharide substrate is hydrolysed at a reaction temperature suitable for the activity of mesophilic hydrolytic enzymes.
  • the reaction temperature is raised to favour the activity of thermophilic hydrolytic enzymes.
  • the increase in temperature not only favours thermophilic enzyme activities at elevated temperatures, but is also believed to assist in "opening up" the polysaccharide substrate (lower substrate viscosity) allowing further penetration by the enzymes. Accordingly, the elevated reaction temperature facilitates increased hydrolysis of the polysaccharide substrate by making parts of the polysaccharide substrate accessible for hydrolysis that were not available to the enzymes at the lower reaction temperature.
  • the two-stage enzyme hydrolysis method described herein is demonstrated to provide higher yields of fermentable sugars compared to the use of mesophilic or thermophilic hydrolytic enzymes alone.
  • the method also provides the advantage of being more cost-effective in comparison to a number of existing enzyme hydrolysis methods used in biofuel production.
  • polysaccharides The two-stage hydrolysis method described herein is suitable for hydrolysing any polysaccharide.
  • the polysaccharide may be a homopolysaccharide, a heteropolysaccharide, a branched, cross-linked or linear polysaccharide, a large complex polysaccharide such as a carbohydrate, a non-starch polysaccharide such as cellulose or hemicellulose, or a short polysaccharide chain such as an oligosaccharide or disaccharide.
  • the polysaccharide will have at least two monosaccharide units, such that a fragment comprising at least one monosaccharide unit may be derived from hydrolysing the polysaccharide.
  • Non-limiting specific examples of polysaccharides suitable for hydrolysis using the two-stage hydrolysis method described herein include complex carbohydrates such as starch, raff ⁇ nose, stachyoses, maltotriose, maltotetraose, glycogen, amylose, amylopectin, polydextrose, dextran, pectin and maltodextrin, non-starch polysaccharides such as cellulose, hemicellulose (including xylan, mannans and galactans), pectins, glucans, gums, mucilages, inulin, and chitin, oligosaccharides including mannan-oligosaccharides, fructo-oligosaccharides and galacto-oligosaccharides, and disaccharides including sucrose lactose, maltose, trehalose, cellobiose, laminaribiose, xylobiose, gentiobiose, isomal
  • the polysaccharide is cellulose, hemicellulose, or a mixture thereof.
  • the cellulose, hemicellulose or mixture thereof may be in a pure or substantially pure form, or be associated with one or more additional components (e.g. lignin).
  • Polysaccharides for use in the methods described herein may be derived from any source, for example plants, animals, microorganisms, processed materials, foodstuffs or synthetic sources. Polysaccharides or materials comprising polysaccharides may be pre-treated prior to performing the two-stage enzyme hydrolysis process (e.g. degradation, separation, purification etc.) although it will be understood that pre-treatment is not a requirement.
  • the polysaccharide is derived from biomass, and in particular, lignocellulosic biomass.
  • Lignocellulosic biomass for use in accordance with the methods described herein may be derived from any source, examples of which include, but are not limited to, woody plant matter, fibrous plant matter, and products and byproducts comprising lignocellulosic matter.
  • woody plant matter examples include pine (e.g. Pinus radiata), birch, eucalyptus, beech, spruce, fir, cedar, poplar and aspen.
  • suitable fibrous plant matter examples include grass, grass clippings, flax, corn cobs, corn stover, reed (e.g. Arundo donax), bamboo, bagasse, hemp, sisal, jute, cannibas, hemp, straw, wheat straw, abaca, cotton plant, kenaf, rice hulls, and coconut hair.
  • the grass is switch grass (Panicum vergatum).
  • Suitable products and byproducts comprising lignocellulosic matter include wood-related materials (for example, sawmill and paper mill discards, saw dust, particle board and leaves) and industrial products such as pulp (e.g. kraft pulp), paper, papermaking sludge, textiles and cloths, dextran, and rayon.
  • Lignocellulosic biomass when used as a source of polysaccharides for the methods described herein may first be subjected to one or more pre-treatment steps, although it will be understood that pre-treatment is not a requirement.
  • Pre-treatment of the lignocellulosic biomass may be performed to degrade the material and/or to separate the material into one or more its basic components (e.g. cellulose, hemicellulose and lignin).
  • pre-treatment may be used to improve the capacity of polysaccharides (e.g. hemicellulose and/or cellulose) within the biomass material to be digested by hydrolytic enzymes.
  • Lignocellulosic biomass may be pre-treated using mechanical methods to disrupt its structure.
  • Mechanical pre-treatment methods may include, for example, pressure, grinding, agitation, shredding, milling, compression/expansion, or other types of mechanical action.
  • Mechanical pre-treatment of lignocellulosic matter may be performed using a mechanical device, for example, an extruder.
  • Steam explosion pre-treatment methods may be used to disrupt the structure of lignocellulosic biomass.
  • the biomass may be exposed to high pressure steam in a contained environment before the resulting product is explosively discharged to an atmospheric pressure.
  • Pre-treatment with steam explosion may also involve agitation of the biomass.
  • lignocellulosic biomass may be pre-treated using chemical methods.
  • Chemical methods for the pre-treatment of lignocellulosic biomass are generally known in the art.
  • lignocellulosic biomass may be pre-treated with a solvent to solubilise cellulosic and/or hemicellulosic matter within the biomass.
  • the solvated cellulosic and/or hemicellulosic polysaccharides may then be subjected to enzymatic hydrolysis in accordance with the methods described herein.
  • Chemical pre-treatment of lignocellulosic matter may be performed using a supercritical solvent. The supercritical treatment may be used, for example, to remove lignin from other components of the biomass.
  • Such methods generally involve the application to the biomass of a supercritical solvent heated above its critical temperature and pressurized above its critical pressure, thereby facilitating its degradation.
  • suitable solvents for the supercritical treatment of lignocellulosic biomass include water, and alcohols (e.g ethanol).
  • Methods involving the supercritical treatment of lignocellulosic biomass are known in the art and are described, for example, in United States Patent No. 4644060.
  • chemical pre-treatment of lignocellulosic matter may be performed using an acid/base hydrolysis treatment.
  • Such treatments generally involve the application of an acidic or alkaline aqueous solution (e.g.
  • Patent No. 4880473 US Patent No. 5410034, United States Patent No. 5424417 and
  • Pre-treatment of lignocellulosic matter may also be performed by hydrolysis in aqueous solution of neutral or substantially neutral pH (i.e. about pH 7.0), normally in conjunction with heat.
  • neutral or substantially neutral pH i.e. about pH 7.0
  • lignocellulosic biomass is fractionated into one or more components comprising purified or substantially purified hemicellulose and/or cellulose.
  • lignin is removed from lignocellulosic matter prior to enzymatic hydrolysis in accordance with the methods described herein.
  • this is not essential and the enzymatic hydrolysis method may be utilised on lignocellulosic biomass, pre-treated lignocellulosic biomass and/or fractionated components thereof in which lignin has not been removed.
  • the two-stage hydrolysis method involves contacting one or more polysaccharides or a material comprising one or more polysaccharides with mesophilic and thermophilic hydrolytic enzymes.
  • a mesophilic hydrolytic enzyme encompasses any hydrolytic enzyme having optimal enzymatic activity at a temperature above about 10°C and below 60 0 C. Accordingly, a temperature "suitable for the activity" of a mesophilic hydrolytic enzyme as contemplated herein will be a temperature above about 10 0 C and below 60 0 C.
  • a thermophilic hydrolytic enzyme as referred to herein encompasses any hydrolytic enzyme having optimal enzymatic activity at a temperature of 60°C, and any hydrolytic enzyme having optimal enzymatic activity at a temperature higher than 60°C.
  • a temperature "suitable for the activity" of a thermophilic hydrolytic enzyme as contemplated herein will be a temperature of 60 0 C, or higher than 60 0 C.
  • the optimal enzymatic activity of a hydrolytic enzyme may be determined by measuring the product of the reaction between the enzyme and its substrate.
  • the optimal temperature at which a given mesophilic or thermophilic hydrolytic enzyme functions can be determined by exposing the enzyme to an appropriate substrate under suitable conditions and measuring the release of hydrolysis products over a range of different reaction temperatures. Suitable conditions for determining the activity of the enzyme at a given temperature can be readily determined by a person of ordinary skill in the field without inventive effort.
  • reaction mixture may be adjusted to modify factors such as pH and isotonicity, and reagents such as buffers and/or enzyme cofactors may be added to the reaction mixture to augment the activity of the hydrolytic enzyme. Additionally or alternatively, the amount of substrate (e.g. polysaccharide starting material) and/or concentration of hydrolytic enzymes may be adjusted.
  • factors such as pH and isotonicity
  • reagents such as buffers and/or enzyme cofactors
  • concentration of hydrolytic enzymes may be adjusted.
  • Measurement of the release of hydrolysis products for determining optimal enzymatic activity can be performed using techniques generally known in the art.
  • methods which may be used to measure reaction products include, but are not limited to, measurement of the transfer or incorporation of a radioactive or other labelled atom or group, spectrophotometric or colorimetric measurement of the concentration of the product, measurement of fluorescence from a fluorescent product, measurement of light output from a luminescent or chemiluminescent reaction, immunoassays, other immunochemical procedures, and other competitive binding assays.
  • Enzyme activity can also be measured using any of the procedures mentioned above to detect the product of secondary reaction(s) that rely on the product of the reaction of interest as a substrate or a cofactor.
  • Hydrolysis products may be measured as reducing sugars assayed by the dinitrosalicyclic acid (DNS) method (see, for example, the assays described in Bernfeld P., "Amylases a and ⁇ " In: Methods in Enzymology, vol 1. Colowick, Kaplan (Eds) (1955), Academic, New York, pl49-158; Miller GL, "Use of aminosalicylic acid reagent or determination of reducing sugar” Analytical Chemistry, (1959), 31, 426-428; Aibba et al. "Applied Environmental Microbiology", (1983) Vol. 46, p. 1059-1065). Additionally or alternatively, hydrolysis products may be analyzed using ion- exchange chromatography.
  • DMS dinitrosalicyclic acid
  • hydrolysis products may be analyzed using anion exchange high-performance liquid chromatography (HPLC) as described, for example, in Tenkanen and Siika-aho "An alpha-glucuronidase of Schizophyllum commune acting on polymeric xylan " J. Biotechnol., (2000), 78:149-61; or Clarke et al, "The compositional analysis of bacterial extracellular polysaccharides by high- performance anion-exchange chromatography", (1991), Anal. Biochem.
  • HPLC anion exchange high-performance liquid chromatography
  • Mesophilic and thermophilic hydrolytic enzymes used in the two-stage enzyme hydrolysis method may be applied to the polysaccharide separately or in combination.
  • the polysaccharide is contacted with mesophilic hydrolytic enzymes, or a combination of mesophilic and thermophilic hydrolytic enzymes, thereby forming an enzyme/substrate mixture.
  • thermophilic hydrolytic enzymes or a combination of mesophilic and thermophilic hydrolytic enzymes may be achieved by mixing the polysaccharide with said thermophilic and/or mesophilic hydrolytic enzymes.
  • one or more microorganisms capable of producing said thermophilic and/or mesophilic hydrolytic enzymes may be mixed with the polysaccharide. Thermophilic and/or mesophilic hydrolytic enzymes produced by the microorganisms may then contact the polysaccharide.
  • the mixture comprising the polysaccharide and hydrolytic enzymes is incubated at a reaction temperature suitable for the activity of mesophilic hydrolytic enzymes.
  • the reaction temperature utilised during the first phase of the two-stage enzyme hydrolysis method is above about 10°C and less than 60 0 C.
  • the reaction temperature utilised during the first phase of the hydrolysis method is between about 15°C and 6O 0 C, between about 2O 0 C and 60°C, between about 25°C and 60 0 C, between about 30 0 C and 60 0 C, between about 35°C and 6O 0 C, between about 40 0 C and 60 0 C, between about 45°C and about 55°C, between about 45°C and 60 0 C, between about 50 0 C and 60 0 C, between about 55°C and 60 0 C, between about 10 0 C and about 55°C, between about 10 0 C and about 50 0 C, between about 10 0 C and about 45°C, between about 10 0 C and about 4O 0 C, between about 10 0 C and about 35°C, between about 10°C and about 30°C, between about 10°C and about 25°C, between about 10°C and about 20°C, and between about 10°C and about 15°C.
  • the reaction temperature utilised during the first stage of the two-stage enzyme hydrolysis method is about 50°C.
  • the mixture comprising the polysaccharide and hydrolytic enzymes is incubated (at any of the temperatures referred to above) for a time period sufficient for the hydrolysis of the polysaccharide to occur.
  • the mixture may be incubated for a period of between about 1 hour and about 30 hours.
  • the mixture is incubated for a period of between about 5 hours and 25 hours, more preferably between about 10 hours and about 20 hours, and still more preferably between about 15 hours and about 20 hours.
  • the polysaccharide is subjected to hydrolysis at a reaction temperature suitable for the activity of thermophilic hydrolytic enzymes.
  • the polysaccharide may be contacted with thermophilic hydrolytic enzymes prior to commencing and/or during the first stage of the two-stage enzyme hydrolysis method. Additionally or alternatively, the polysaccharide may be contacted with thermophilic hydrolytic enzymes upon completion of the first stage of the enzyme hydrolysis method.
  • thermophilic hydrolytic enzymes may be achieved by mixing the polysaccharide with thermophilic hydrolytic enzymes. Additionally or alternatively, one or more microorganisms capable of producing thermophilic hydrolytic enzymes may be mixed with the polysaccharide. Thermophilic hydrolytic enzymes produced by the microorganisms may then contact the polysaccharide.
  • the reaction temperature utilised during the second stage of the hydrolysis method is 60°C, or above 60°C.
  • the reaction temperature utilised during the second stage of the two-stage enzyme hydrolysis method is above 60°C, above about 63 °C, above about 65°C, above about 68°C, about 70°C, above about 73°C, above about 75°C, above about 78°C, above about 80°C, above about 83°C, above about 85°C, above about 88 0 C, above about 9O 0 C, above about 95°C, above about 100 0 C, above about 110 0 C, and above about 120 0 C.
  • the reaction temperature utilised during the second stage of the hydrolysis method is about 60°C.
  • the reaction temperature utilised during the second stage of the hydrolysis method is about 70°C.
  • the mixture comprising the polysaccharide and hydrolytic enzymes is incubated (at any of the temperatures referred to above) for a time period sufficient for further hydrolysis of the polysaccharide to occur.
  • the mixture may be incubated for a period of between about 1 hour and about 40 hours.
  • the mixture is incubated for a period of between about 1 hour and about 20 hours, between about 5 hours and about 15 hours, more preferably between about 5 hours and about 12 hours, and still more preferably between about 7 hours and 10 hours.
  • the two-stage hydrolysis method may optionally be combined with a third stage comprising the step of contacting the polysaccharide with a cryophilic hydrolytic enzyme and incubating the polysaccharide and enzyme at a temperature suitable for the activity of the cryophilic hydrolytic enzyme.
  • the optional third additional stage may be performed prior to the first stage of the two-stage enzyme hydrolysis method, between the first and second stages of the two-stage enzyme hydrolysis method, and/or after the second stage of the two-stage enzyme hydrolysis method.
  • cryophilic hydrolytic enzymes will have optimal (or substantial) activity at a temperature of below about 10 0 C. Accordingly, it will be understood a temperature "suitable for the activity" of a cryophilic hydrolytic enzyme as contemplated herein will be a temperature of below about 10°C. It will also be understood that "cryophilic" hydrolytic enzymes as contemplated herein encompass “pyschrophilic” hydrolytic enzymes. It will be understood that contacting the polysaccharide with cryophilic hydrolytic enzymes may be achieved by mixing the polysaccharide with cryophilic hydrolytic enzymes. Additionally or alternatively, one or more microorganisms capable of producing cryophilic hydrolytic enzymes may be mixed with the polysaccharide. Cryophilic hydrolytic enzymes produced by the microorganisms may then contact the polysaccharide.
  • the reaction temperature utilised during the optional third stage is below about 10°C, below about 7 0 C, or below about 5°C.
  • the mixture comprising the polysaccharide and cryophilic hydrolytic enzymes in the optional third stage is incubated (at any of the temperatures referred to above) for a time period sufficient for hydrolysis of the polysaccharide to occur.
  • the mixture may be incubated for a period of between about 1 hour and about 40 hours.
  • the mixture is incubated for a period of between about 1 hour and about 20 hours, between about 5 hours and about 15 hours, more preferably between about 5 hours and about 12 hours, and still more preferably between about 7 hours and 10 hours.
  • Cryophilic hydrolytic enzymes may be optionally combined with thermophilic and/or mesophilic hydrolytic enzymes and utilised in the first stage of the hydrolysis method.
  • optimal reaction conditions for each stage of the two-stage enzyme hydrolysis method can be determined readily by a person of ordinary skill in the field without inventive effort. Optimal conditions will ultimately depend on factors including the type of polysaccharide under treatment and the specific hydrolytic enzymes utilised. For example, factors such as the pH of the reaction mixture, isotonicity, the amount of polysaccharide starting material, concentration of mesophilic and thermophilic hydrolytic enzymes, and time of incubation may be routinely varied in order to determine optimal conditions. Enzyme hydrolysis conditions (e.g. pH) may be optimized by the addition of buffering agents, for example, sodium acetate, dilute HCl and/or dilute KOH.
  • buffering agents for example, sodium acetate, dilute HCl and/or dilute KOH.
  • suitable dosages of hydrolytic enzymes and optimal conditions for enzymatic treatment will be influenced by the level of activity of the hydrolytic enzymes utilized, and the structure and purity of the polysaccharide under treatment.
  • Suitable buffers and/or enzyme cofactors may be included in the hydrolysis reactions to augment the activity of the hydrolytic enzymes and increase the efficiency of the hydrolysis reactions in general.
  • the two-stage enzyme hydrolysis method preferably employs enzymes in dissolved state, it is equally possible to use enzymes immobilized on a solid support.
  • optimal reaction conditions may be determined by performing the two-stage enzyme hydrolysis method with varying reaction parameters such as those described above and measuring the release of hydrolysis products (i.e. fragments of the polysaccharide).
  • the release of hydrolysis products may be analysed using techniques generally known in the art.
  • hydrolysis products may be measured as reducing sugars assayed by the dinitrosalicyclic acid (DNS) method (see, for example, Bernfeld P. "Amylases a and ⁇ . " In: Methods in Enzymology, vol 1.
  • NDS 3,5-dinitrosalicylic acid
  • 3- amino,5-nitrosalicylic acid under alkaline conditions
  • a yellow colouration of the solution which can be measured spectrophotomerically at ⁇ 540 .
  • known amounts of sugars e.g. glucose and xylose
  • sugars e.g. glucose and xylose
  • hydrolysis products may be analysed using ion- exchange chromatography.
  • hydrolysis products may be analysed using anion exchange high-performance liquid chromatography (HPLC) as described, for example, in Tenkanen and Siika-aho "An alpha-glucuronidase of Schizophyllum commune acting on polymeric xylan " J.
  • HPLC anion exchange high-performance liquid chromatography
  • hydrolysis enzymes suitable for use in the methods described herein are those classified under EC 3 (hydrolases) of the enzyme nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB)
  • hydrolytic enzymes utilized in the methods described herein are those classified under class EC 3.2 (glycosylases) of the NC-IUBMB enzyme nomenclature.
  • hydrolytic enzymes suitable for use in the methods described herein are those classified under subclass 3.2.1 (Glycosidases, i.e. enzymes hydrolyzing O- and S-glycosyl compounds) of the NC-IUBMB nomenclature.
  • the enzymes that may be utilised are those classified under subclass EC 3.2.2 (Hydrolysing N-Glycosyl Compounds) of the NC-IUBMB nomenclature.
  • hydrolytic enzymes that may be utilized are those classified under subclass EC 3.2.3 (Hydrolysing 5-Glycosyl Compounds) of the NC-IUBMB nomenclature.
  • glycoside hydrolases and carbohydrases suitable for use in the methods described herein and commercial sources of those enzymes are described in US Patent Publication No. 20060073193.
  • Preferred examples include cellulases, carbohydrases, glycoside hydrolases, endoxylanases, exoxylanases, ⁇ -glucosidases, ⁇ - xylosidases, mannanases, galactanases, dextranases, endoglucanases, exoglucanases, mannosidases, arabinosidases, and alpha-galactosidase.
  • thermophilic hydrolytic enzymes include, but are not limited to, ⁇ -glucosidase enzyme (BgIA) from Caldicellulosiruptor saccharolyticus TP8.3.3.1, Xylanase A (XynA) from Dictyoglomus thermophilum (e.g strain Rt46B.l), xylanase (XynB) from Dictyoglomus thermophilum (e.g strain Rt46B.l), Cellulase/Cellobiohydrolase (CeIA) from Caldicellulosiruptor saccharolyticus, bifunctional Cellulase B (CeIB) from Caldicellulosiruptor saccharolyticus, Arabinofuranosidase (XynF) from Cs.
  • BgIA ⁇ -glucosidase enzyme
  • XynA Xylanase A
  • XynB xylanase
  • Saccharolyticus Saccharolyticus , ⁇ -xylosidase (XynB) from Caldicellulosiruptor saccharolyticus, mannanase from Caldicellulosiruptor Rt8B.4, Mannanase (ManA) from Dictyoglomus thermophilum, Mannosidase (Man2) from Thermotoga neapolitana, endoxylanase (XynA) from Thermoanaerobacterium saccharolyticum, xylanase (XynX) from Clostridium thermocellum, ⁇ -glycanases from Caldicellulosiruptor saccharolyticus, Xylosidase (XynD) from Cs.
  • Xylanases xynE and xynl
  • Saccharolyticus Saccharolyticus , ⁇ -xylosidase (XynB) from Caldicellulos
  • the polysaccharide is subjected to hydrolysis using a reaction temperature suitable for the activity of mesophilic hydrolytic enzymes.
  • mesophilic hydrolytic enzymes may be derived from any source.
  • mesophilic hydrolytic enzymes may be derived from mesophilic microorgansisms such as mesophilic fungi, mesophilic bacteria, and mesophilic yeasts.
  • a "mesophilic" microorganism as contemplated herein is any microorganism that exists optimally at a temperature of above about 10°C and less than 60°C.
  • the mesophilic microorganism may express endogenous mesophilic hydrolytic enzymes and/or be genetically modified to express mesophilic hydrolytic enzymes.
  • mesophilic hydrolytic enzymes used in the two stage enzyme hydrolysis method are derived from a microorganism which endogenously expresses mesophilic hydrolytic enzymes.
  • the microorganism is a fungus.
  • the mesophilic hydrolytic enzymes are endogenous mesophilic enzymes derived from Trichoderma reesei.
  • the endogenous enzymes may be one or more of:
  • Non-limiting examples of suitable mesophilic fungi from which hydrolytic enzymes may be derived include, but are not limited to, Trichoderma ⁇ e.g. T. reesei, T. viride, T. koningii, T. harzianum), Aspergillus ⁇ e.g. A. awamori, A. niger and A. oryzae),
  • Emericella Emericella, Humicola ⁇ e.g. H. insolens and H. g ⁇ sea), Chrysosporium ⁇ e.g. C. lucknowense), Doratomyces ⁇ e.g. D. stemonitis), Fusarium, Gliocladium, Geomyces,
  • Candida Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, and Yarrowia.
  • Non-limiting examples of suitable mesophilic bacteria from which suitable hydrolytic enzymes may be derived include, but are not limited to, Streptomyces, Micromonospora, and Clostridium.
  • the polysaccharide is subjected to hydrolysis using a reaction temperature suitable for the activity of thermophilic hydrolytic enzymes.
  • thermophilic hydrolytic enzymes may be derived from any source.
  • thermophilic hydrolytic enzymes may be derived from thermophilic microorganisms including, but not limited to, thermophilic fungi, thermophilic bacteria, and Archaea. It will be understood that a "thermophilic" microorganism as contemplated herein is any microorganism that exists optimally at a temperature of above 60°C.
  • the thermophilic microorganism may express endogenous thermophilic hydrolytic enzymes and/or be genetically modified to express thermophilic hydrolytic enzymes.
  • thermophilic hydrolytic enzymes are derived from thermophilic bacteria.
  • thermophilic fungi from which suitable hydrolytic enzymes may be derived include, but are not limited to, Acremonium ⁇ e.g. A. thermophilum), Talaromyces ⁇ e.g. T. emersoni ⁇ ), Cladosporium, Melanocarpus ⁇ e.g. M. albomyces), Rhizomucor ⁇ e.g. R. pusillus), Sporotrichum, Thermonospora ⁇ e.g. T. curvata), Thermoascus (e.g. T. aurantiacus, T. lanuginosa), Thermomyces ⁇ e.g.
  • Thermomyces lanuginosa Chaetomium (e.g. C. thermophilum, C. thermophila), Myceliophthera (e.g. M. thermophila), Thielavia (e.g. T. terrestris), Corynascus (e.g. C. thermophilus), Aureobasidium, Candida and Hansenula.
  • Chaetomium e.g. C. thermophilum, C. thermophila
  • Myceliophthera e.g. M. thermophila
  • Thielavia e.g. T. terrestris
  • Corynascus e.g. C. thermophilus
  • Aureobasidium Candida and Hansenula.
  • thermophilic bacteria from which suitable hydrolytic enzymes may be derived include, but are not limited to, Acetogenium kuvui, Acetomicrobium faecalis, Acidothermus cellulolyticus, Anaerocellum thermophilum, Chloroflexus auranticus, Desulfotomaculum nigrificans, Desulfovibrio thermophilus, Dictyoglomus thermophilum, Dictyoglomus thermophilum strain Rt46B.l, Bacillus acidocaldarius, Bacillus stearothermophilus, Bacillus caldolyticus, Bacillus caldotenax, Bacillus caldovelox, Bacillus thermoglucosides, Bacillus thermoglucosidasius, Bacillus thermocatenulatus, Bacillus schlegelii, Bacillus flavothermus, Bacillus tusciae, Bacillus sp.
  • KSM-S237 Caldicellulosiruptor saccharolyticus (formerly known as Caldocellum saccharolyticum), Caldicellulosiruptor strain Rt69B.l, Caldicellulosiruptor strain Tok7B.l, Clostridium stercorarium, Clostridium thermocellum, Clostridium thermosulfurogenes, Clostridium thermohydrosulfuricum, Clostridium autotrophicum, Clostridium fervidus, Clostridium. thermosaccharolyticum, Caldobacterium hydrogenophilum, Fervidobacterium nodosum, F.
  • thermophilic hydrolytic enzyme is selected from the group consisting of ⁇ -glucosidase (BgIA) from Caldicellulosiruptor saccharolyticus TP8.3.3.1, Xylanase A (XynA) from Dictyoglomus thermophilum, bifunctional Cellulase B (CeIB) from Caldicellulosiruptor saccharolyticus, ⁇ -xylosidase (XynB) from Caldicellulosiruptor saccharolyticus, mannanase (ManA) from Dictyoglomus thermophilum, mannanase from Caldicellulosiruptor Rt8B.4, mannosidase 2 (Man2) from Thermotoga neapolitana, endoxylanase (XynA) from Thermoanaerobacterium saccharolyticum, xylanase (XynX
  • the two-stage hydrolysis method may optionally be combined with a third stage comprising the step of contacting the polysaccharide with a cryophilic hydrolytic enzyme.
  • suitable microorganisms from which cryophilic hydrolytic enzymes may be derived include cryophilic bacteria, archaea and/or fungi.
  • mesophilic hydrolytic enzymes used in the two-stage enzyme hydrolysis method are endogenous mesophilic hydrolytic enzymes produced by a strain of T. reesei genetically modified to express one or more thermophilic hydrolytic enzymes.
  • the genetically modified strain of T. reesei may be used as a source of both mesophilic and thermophilic hydrolytic enzymes for the two-stage enzyme hydrolysis method.
  • Mesophilic and/or thermophilic enzymes from other sources may be used to supplement hydrolytic enzymes produced by the genetically modified strain of T. reesei.
  • the genetically modified strain of T. reesei may express any thermophilic hydrolytic enzyme.
  • thermophilic hydrolytic enzymes that may be expressed by the genetically modified form of T. reesei include XynB, CeIE 1/2, CeIE- Cterm, CelB5, CeIA, ManA, XynE, XynF, XynD, Xynl and BgIA.
  • the genetically modified form of T. reesei expresses thermophilic XynB.
  • the XynB is derived from Dictyoglomus thermophilum Rt46B.1.
  • Mesophilic and thermophilic hydrolytic enzymes for use in the methods described herein may be obtained from microorganisms capable of producing such enzymes. Accordingly, suitable microorganisms that naturally produce hydrolytic enzymes, for example, any of the fungi or bacteria referred to above, may be cultured under suitable conditions for propagation and/or expression of the hydrolytic enzyme or enzymes of interest. Methods and conditions suitable for the culture of microorganisms are generally known in the art and are described in, for example, Current Protocols in Microbiology (Coico et al (Eds), John Wiley and Sons, Inc, 2007).
  • hydrolytic enzymes suitable for use in the methods described herein may be produced using recombinant DNA technology.
  • one or more genes encoding a hydrolytic enzyme may be used to transform a host cell such as a fungus, yeast or bacterium, and the hydrolytic enzyme/s then expressed by the host cell.
  • Methods for the production of recombinant organisms are generally known in the art and are described in, for example, Molecular Cloning: A Laboratory Manual, (Joseph Sambrook, David W Russell, 3 rd . Edition, Cold Spring Harbour Press 2001), Current Protocols in Molecular Biology (Ausubel F. M. et al.
  • one or more genes encoding hydrolytic enzymes may be cloned into a vector.
  • the vector may be a plasmid vector, a viral vector, a phosmid, a cosmid or any other vector construct suitable for the insertion of foreign sequences, introduction into cells and subsequent expression of the introduced sequences.
  • the vector is an expression vector comprising expression control and processing sequences such as a promoter, an enhancer, polyadenylation signals and/or transcription termination sequences.
  • the vector construct may also include a selectable marker, for example, an antibiotic-resistance gene such as ampicillin, chloramphenicol, tetracycline, hygromycin or bleomycin.
  • Genetic material for insertion into the vector construct may be generated, for example, by chemical synthesis techniques such as the phosphodiester and phosphotriester methods (see, for example, Narang et al. "Improved phosphotriester method for the synthesis of gene fragments " , (1979), Meth. Enzymol. 68:90; Brown, E. L. et al., "Chemical synthesis and cloning of a tyrosine tRNA gene ", (1979), Meth. Enzymol.
  • Genetic material for insertion into the vector construct may be amplified in number by performing the polymerase chain reaction (PCR) on DNA or cDNA sequences encoding hydrolytic enzymes, or RT-PCR on RNA sequences encoding hydrolytic enzymes. The resulting nucleic acids may then be inserted into the construct, for example, by restriction-ligation reactions or by the TA cloning method.
  • PCR polymerase chain reaction
  • Suitable methods for the introduction of vector constructs and other foreign nucleic acid material into host cells are generally known in the art, and are described, for example, in Current Protocols in Molecular Biology, (Ausubel et al. (Eds), New York: John Wiley & Sons, 2007) and Molecular Cloning: A Laboratory Manual, (Sambrook et al. 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001).
  • host cells may be transformed with vector constructs by the "heat shock” method.
  • the cells are chilled in the presence of divalent cations such as Ca 2+ , which causes cell wall permeability.
  • Cells are incubated on ice with the construct and briefly heat shocked (e.g. at 42 °C for 0.5-2 minutes) causing the vector construct to enter the cell.
  • host cells may be transformed with vector constructs by electroporation, a method involving briefly shocking the cells with an electric field causing the cells to briefly develop holes through which the vector construct may enter the cell. Natural membrane-repair mechanisms rapidly close these holes after the shock.
  • Transformation of vector constructs into filamentous fungi can be performed using the biolistic bombardment approach as described in "Biolistic transformation of Trichoderma reesei using the seven-barrels using the Bio-Rad seven barrels Hepta Adaptor system " (2002), Te'o et al., J. Microbiol. Methods, 51 :393-399. Briefly, microcarrier particles ⁇ e.g. gold and tungsten) may be coated with the vector construct, and introduced into fungal conidia using helium as a carrier gas under high pressure and vacuum. Vector constructs may also be introduced into fungal cells (e.g. protoplast cells) using methods described in "A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei” (1987), Penttila et al., Gene, 61:155-164.
  • the host cell may be cultured under conditions suitable to facilitate reproduction.
  • Methods for the culture of microorganisms such as bacteria and fungi are well known in the art and described in, for example, Current Protocols in Microbiology, (Coico, et al. (Eds), John Wiley & Sons, Inc., 2007).
  • the culture may be performed in medium containing a substrate that facilitates the identification of transformed strains, for example, an antibiotic such as ampicillin, chloramphenicol, kanamycin, tetracycline, hygromycin B or bleomycin.
  • Transformed host cells may be selected and propagated.
  • transformed host cells may be identified by expression of the marker or markers.
  • a drug resistance gene such as a chloramphenicol resistance gene
  • host cell transformants that grow in the selection medium containing chloramphenicol can be identified as transformants.
  • double transformants may be identified by the ability to grow in the selection media containing multiple selection determinants.
  • Hydrolytic enzymes may be purified from the cultured microorganism. Any suitable methods of screening or purification may be used, taking into account various factors such as size and the structural, enzymatic and functional features of the desired hydrolytic enzyme. Methods and assays suitable for purification of the hydrolytic enzymes from microorganism cultures are known in the art, and are described, for example, in Current Protocols in Protein Science, Coligan et al., (Eds) John Wiley and Sons, Inc. 2007. The screening and purification step may comprise, for example, chromatography methods, methods that can accelerate solvent extraction, or combinations thereof.
  • Chromatography methods may include, for example, reverse phase chromatography, normal phase chromatography, affinity chromatography, thin layer chromatography, counter current chromatography, ion exchange chromatography and reverse phase chromatography.
  • Other methods include precipitation with ammonium sulphate, PEG, antibodies and the like, or heat denaturation, followed by centrifugation, isoelectric focusing, gel electrophoresis, selective precipitation techniques, and combinations of those and other techniques.
  • Hydrolytic enzymes utilized in the two stage enzyme hydrolysis method may also be genetically engineered to contain various affinity tags or carrier proteins to aid purification.
  • Histidine and protein tags may be engineered into an expression vector comprising, for example, a streptavidin subunit to facilitate purification by its high non-covalent affinity to biotin, and histidine-tagged proteins can be purified using metal- chelate chromatography (MCAC) under either native and denaturing conditions.
  • MCAC metal- chelate chromatography
  • the purification of secondary metabolites may also be "scaled-up" for large-scale production purposes.
  • hydrolytic enzymes for use in the two stage enzyme hydrolysis method are expressed and isolated from recombinant Trichoderma reesei host cells.
  • the enzymes are thermophilic hydrolytic enzymes.
  • the thermophilic enzyme may be thermophilic Xylanase B (XynB).
  • the thermophilic xylanase B may be derived from a strain of Dictyoglomus thermophilum.
  • the strain of Dictyoglomus thermophilum may be Dictyoglomus thermophilum Rt46B.1.
  • thermophilic hydrolytic enzymes are produced in accordance with methods described in PCT patent publication number WO 2009/076709 entitled “Multiple promoter platform for gene expression", the entire contents of which are incorporated herein by reference.
  • hydrolytic enzymes for use in the methods described herein are produced synthetically (e.g. using a commercial facility).
  • the invention also provides compositions for the enzymatic hydrolysis of a polysaccharide.
  • the compositions comprise at least one mesophilic hydrolytic enzyme and at least one thermophilic hydrolytic enzyme.
  • the hydrolytic enzymes may be any enzyme capable of hydrolyzing a polysaccharide. Suitable hydrolytic enzymes include, for example, those classified under EC 3 (hydrolases) of the enzyme nomenclature of the
  • compositions of the invention comprise hydrolytic enzymes classified under class EC 3.2 (glycosylases) of the NC-IUBMB enzyme nomenclature.
  • compositions of the invention comprise hydrolytic enzymes classified under subclass 3.2.1 (Glycosidases, i.e. enzymes hydrolyzing O- and S-glycosyl compounds) of the NC-IUBMB nomenclature.
  • the compositions comprise hydrolytic enzymes classified under subclass EC 3.2.2 (Hydrolysing iV-Glycosyl Compounds) of the NC-IUBMB nomenclature.
  • the compositions comprise hydrolytic enzymes classified under subclass EC 3.2.3 (Hydrolysing S-Glycosyl Compounds) of the NC-IUBMB nomenclature.
  • mesophilic and thermophilic hydrolytic enzymes suitable for the compositions described herein are provided above in the section entitled "Hydrolytic enzymes”.
  • compositions of the invention may comprise hydrolytic enzymes derived from any source. Suitable sources of hydrolytic enzymes are described above in the section entitled
  • Hydrolytic enzymes For example, mesophilic and thermophilic hydrolytic enzymes may be derived from a suitable microorganism, or produced using recombinant DNA technology (see section entitled “Hydrolytic enzymes” above).
  • compositions comprise synthetically produced hydrolytic enzymes.
  • Thermophilic hydrolytic enzymes for inclusion in the compositions of the invention may be produced in accordance with methods described in Australian Provisional Patent application number 2007906984 entitled “Multiple promoter platform for gene expression", the entire contents of which are incorporated herein by reference.
  • compositions of the invention may be used for the enzymatic hydrolysis of a polysaccharide.
  • the polysaccharide is cellulose, hemicellulose or a mixture thereof.
  • use of the compositions involves contacting the composition with a polysaccharide.
  • the polysaccharide may then be hydrolyzed at a temperature suitable for the activity of a mesophilic hydrolytic enzyme, followed by hydrolysis at a temperature suitable for the activity of a thermophilic enzyme.
  • a temperature suitable for the activity of mesophilic hydrolytic enzymes may be above about 10°C and below 60°C.
  • the temperature for the activity of mesophilic hydrolytic enzymes is about 50°C.
  • a temperature suitable for the activity of thermophilic hydrolytic enzymes may be 60°C, or above 60 0 C.
  • the temperature for the activity of thermophilic enzymes is about 70 0 C.
  • the temperature suitable for the activity of thermophilic enzymes may be above about 70 0 C.
  • polysaccharide fragments In general, the hydrolytic enzymes will be capable of cleaving one or more bonds within the polysaccharide structure thereby facilitating the release of a fragment or fragments comprising one or more monosaccharide units.
  • bonds within the structure of a polysaccharide that may be cleaved by hydrolytic enzymes in accordance with the methods described herein are S-glycosidic bonds, N-glycosidic bonds, C-glycosidic bonds, O-glycosidic bonds, ⁇ -glycosidic bonds, ⁇ -glycosidic bonds, 1 ,2-glycosidic bonds, 1,3-glycosidic bonds, 1,4-glycosidic bonds and 1 ,6-glycosidic bonds, ether bonds, hydrogen bonds and/or ester bonds.
  • the two-stage hydrolysis method described herein can be used to derive a smaller fragment or smaller fragments from larger, more complex polysaccharides.
  • the fragments will comprise one or more monosaccharide units.
  • the fragment will be of a size suitable for fermentation, for example, by a microorganism such as a yeast, fungus or bacterium.
  • oligosaccharide fragments that may be produced by the methods described herein include, but are not limited to, oligosaccharides including mannan- oligosaccharides, fructo-oligosaccharides and galacto-oligosaccharides.
  • disaccharide fragments that may be produced by the methods described herein include, but are not limited to, sucrose, lactose, maltose, trehalose, cellobiose, laminaribiose, xylobiose, gentiobiose, isomaltose, mannobiose, kojibiose, rutinose, nigerose, and melibiose.
  • monosaccharide fragments that may be produced by the methods described herein include, but are not limited, to trioses including aldotrioses (e.g. glyceraldehyde) and ketotrioses (e.g. dihydroxyacetone), tetroses including aldotetroses
  • ketotetroses e.g. erythrulose
  • pentoses including aldopentoses (e.g. lyxose, ribose, arabinose, deoxyribose and xylose) and ketopentoses
  • hexoses including aldohexoses (e.g. glucose, mannose, altrose, idose, galactose, allose, talose and gulose) and ketohexoses (e.g. fructose, psicose, tagatose and sorbose), heptoses including keto-heptoses (e.g. sedoheptulose and mannoheptulose), octoses including octolose and 2-keto-3-deoxy-manno-octonate, and nonoses including sialose.
  • aldohexoses e.g. glucose, mannose, altrose, idose, galactose, allose, talose and gulose
  • ketohexoses e.g. fructose, psicose, tagatose and sorbose
  • keto-heptoses e.g. sedoheptulose and
  • the invention also relates to polysaccharide fragments produced in accordance with the two-stage enzyme hydrolysis method described herein.
  • Fermentation Fragments derived from the enzymatic hydrolysis of polysaccharides in accordance with the methods described herein may be fermented to produce one or more fermented sugar products.
  • a "fermented sugar product" encompasses any product obtainable by the fermentation of a polysaccharide fragment produced in accordance with the two-stage hydrolysis method described herein.
  • fermentation may be performed using any microorganism capable of converting fragments of polysaccharides produced in accordance with the methods described herein into one or more desired products.
  • the microorganism may be capable of converting polysaccharide fragments into alcohols (including ethanol), or organic acids (for example succinic acid and glutamic acid).
  • Suitable microorganisms for fermentation include but are not limited to yeasts, bacteria, fungi, and/or recombinant varieties of these organisms.
  • the microorganism is capable of fermenting polysaccharide fragments produced in accordance with the methods described herein into one or more alcohols, non-limiting examples of which include xylitol, mannitol, arabinol, butanol and ethanol.
  • 5-carbon saccharides (pentoses) derived from the hydrolysis of hemicellulose in accordance with the methods described herein are fermented to produce alcohols, examples of which include but are not limited to xylitol, mannitol, arbinol and ethanol.
  • fragments derived from the hydrolysis of cellulose in accordance with the methods described herein are fermented to produce alcohols, examples of which include but are not limited to ethanol and butanol.
  • Non-limiting examples of microorganisms capable of producing ethanol from polysaccharide fragments produced in accordance with the methods described herein include Zymomonas ⁇ e.g. Z. mobilis), Saccharomyces (e.g. S. cerevisiae), Candida (e.g.
  • Pichia e.g. P. stipitis
  • Microorganisms suitable for the fermentation of polysaccharide fragments produced in accordance with the methods described herein to mannitol include, for example, yeast, fungi and lactic acid bacteria. Suitable microorganisms will, in general, express enzymes necessary for mannitol production (e.g. mannitol dehydrogenase).
  • Examples of bacterial species that may be used for the fermentation of polysaccharide fragments to mannitol include Zymomonas, Leuconostoc (e.g. Leuconostoc mesenteroides), Lactobacillus (e.g. L. bevis, L. buchnei, L. communyitum, L.sanfranciscensis), Oenococcus (e.g. O. oeni), Leuconostoc (e.g. L. mesenteriode) and Mycobacterium (e.g. M. smegmatis).
  • Leuconostoc e.g. Leuconostoc mesenteroides
  • Lactobacillus e.g. L. bevis, L. buchnei, L. consumeryitum, L.sanfranciscensis
  • Oenococcus e.g. O. oeni
  • Leuconostoc e.g. L. me
  • fungi suitable for the fermentation of saccharides to produce mannitol include, but are not limited to, Candida (e.g. C. zeylannoide, C. lipolitica), Cryptococcus
  • Torulopsis e.g. T. mannitofaciens
  • Basidiomycetes Trichocladium, Geotrichum., Fusarium, Mucor (e.g. M. rouxii), Aspergillus (e.g. A. nidulans), and Penicillium (e.g. P. scabrosum).
  • Microorganisms suitable for the fermentation of polysaccharide fragments produced in accordance with the methods described herein to xylitol include, for example, fungi such as Saccharomyces, Candida (e.g. C. magnoliae, C. tropicalis, C. guilliermondi ⁇ ), Pichia , and Debaryomyces (e.g. D. hansenii).
  • fermentation of polysaccharide fragments produced in accordance with the methods described herein is performed using one or more recombinant microorganisms.
  • Methods for the production of recombinant microorganisms are generally known in the art and are described, for example, in Ausubel et al. (Eds) Current Protocols in Molecular Biology (2007) John Wiley & Sons, and Sambrook et al. Molecular Cloning: A Laboratory Manual, (2000) 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  • recombinant microorganisms suitable for use in the methods described herein will express one or more genes encoding enzymes necessary for the conversion of polysaccharide fragments into the desired product.
  • the recombinant microorganism may express one or more genes encoding one or more of the following enzymes: Hexokinase, Phosphogluco- mutase, Phosphomannose isomerase, Phosphohexose isomerase, Phosphofructokinase-1, Aldolase, Triose phosphate isomerase, Glyceraldehyde 3-phosphate dehydrogenase, Phosphoglycerate kinase, Phosphoglycerate mutase, Enolase, Pyruvate kinase, Pyruvate decarboxylase and Alcohol dehydrogenase.
  • Examples of preferred recombinant ethanologenic microorganisms are those which express alcohol dehydrogenase and pyruvate decarboxylase. Genes encoding alcohol dehydrogenase and pyruvate decarboxylase may be obtained, for example, from Zymomonas mobilis. Examples of recombinant microorganisms expressing one or both of these enzymes and methods for their generation are described, for example, United States Patent No. 5000000, United States Patent No. 5028539, United States Patent No. 424202, and United States Patent No. 5482846. Suitable recombinant microorganisms may be capable of converting both pentoses and hexoses to ethanol.
  • microorganisms may be cultured at a temperature of between about 30°C and about 40°C, and a pH of between about 5.0 and about 7.0. In may be advantageous to add cofactors for the enzymes and/or nutrients for the microorganisms to optimize the enzymatic fermentation.
  • cofactors such as NADPH and/or NAD may be added to the culture to assist the activity of fermentation enzymes (e.g. xylose reductase and xylitol dehydrogenase).
  • fermentation enzymes e.g. xylose reductase and xylitol dehydrogenase
  • Carbon, nitrogen and sulfur sources may also be included in the culture.
  • the invention also relates to fermented sugar products derived from polysaccharide fragments produced in accordance to the methods described herein. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, The invention will now be described with reference to specific examples, which should not be construed as in any way limiting.
  • thermophilic hydrolytic enzyme Xylanase B (xynB) from Dictyoglomus thermophilum strain Rt46B.l was synthesised synthetically using an overlap PCR strategy with its codon usage optimised to suit expression in T. reesei (described in Te'o et al., "Codon optimization of xylanase gene xynB from the thermophilic bacterium Dictyoglomus thermophilum for expression in the filamentous fungus Trichoderma reesei ", (2000), FEMS Microbiol. Lett. 190: 13-19).
  • thermophilic hydrolytic enzymes were used for hydrolysis experiments with their wild type codon usages intact: the ⁇ -glucosidase gene (bglA) (see GenBank accession number X12575) from Cs. saccharolyticus TP8.3.3.1 (see also Love et al., "Sequence structure and expression of a cloned beta-glucosidase gene from an extreme thermophile", (1988), MoI. Gen.
  • Mannanase gene (matiA) (see GenBank accession number AFO 13989) from Dictyoglomus thermophilum coding for a mannanase (see also Gibbs et al., "Sequencing and expression of a beta-mannanase gene from the extreme thermophile Dictyoglomus thermophilum s Rt46B.l, and characteristics of the recombinant enzyme", 1999, Curr. Microbiol. 39: 351- 357).
  • Example 2 Expression of genes encoding thermophilic hydrolytic enzymes in E. coli
  • Polynucleotide sequences encoding hydrolytic enzymes described in Example 1o were inserted into plasmids for amplification and/or expression in E. coli host cells, to test for production of functional proteins in E. coli.
  • thermophilic hydrolytic enzymes in E. coli were: pJLA602 (see Schauder et al., "Inducible expression vectors incorporating the Escherichia coli atpE translational initiation region.”, 1987, Gene, 52:s 279-283), pET (see Studier et al., "Use of T7 RNA polymerase to direct expression of cloned genes", 1990, Methods Enzymol.
  • the polynucleotide sequences encoding the different thermophilic0 hydrolytic enzymes were inserted into the appropriate E. coli expression plasmids after digestion of the insertion fragment and vector with the appropriate restriction enzymes, in a vector to insertion fragment ratio of 1:1, 1 :3 and/or 3:1, using the enzyme T4 DNA ligase supplied by Roche (www.roche-applied-science.com/, catalogue number 10481220001), in accordance with the manufacturer's instructions.
  • T4 DNA ligase supplied by Roche (www.roche-applied-science.com/, catalogue number 10481220001), in accordance with the manufacturer's instructions.
  • thermophilic hydrolytic enzyme xylanase B (XynB) from Dictyoglomus thermophilum strain Rt46B.l with codon usage optimised to suit expression in T. reesei (see Example 1 above) was inserted into T. reesei expression vectors for expression in T. reesei hosts.
  • the different plasmids used for gene expression in T. reesei were: pCBHlcorlin, pHEN54, pHEN54RQ and pHEN54xynlpro, pHEN54xyn2pro (wherein gene/s of interest are expressed under the control of a cbhl gene promoter), pCBHIIsigpro and pCBHIIcbmlin (wherein gene/s of interest are expressed under the control of a cbh2 gene promoter), pEGLIIsigpro and pEGLIIcbmlin (wherein gene/s of interest are expressed under the control of an egl2 gene promoter), pXYNIIsigpro (wherein gene/s of interest are expressed under the control of the xyn2 gene promoter) and pHEXl (wherein gene/s of interest are expressed under the control of a hexl gene promoter).
  • the filamentous fungus Trichoderma reesei strain Rut-C30 (publicly available from the American Type Culture Collection: ATCC# 56765) was used as an expression host for expression of the synthetic xynB gene using multiple expression vectors. Expression vectors were introduced into T.
  • Conidia from fungal transformants were cultivated in shake flasks in 50 mL minimal salt-based cultures (as described in Penttila et al., "A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei", 1987, Gene, 61 :155- 164) supplemented with Avicel cellulose (2% w/v), Soy hydrolysate (1.5% w/v) and Lactose (1% w/v), pH 6.5. Following a seven day cultivation at 28°C, supernatants were harvested by centrifugation and used for further analysis.
  • thermophilic hydrolytic enzymes in E. coli typically involved the inoculation of a 250-300 mL fresh LB culture (as described in Luria and Burrous, "Hybridization between Escherichia coli and Shigella " (1957), J. Bact. 74: 461-476) with 25-30 mL of an overnight culture of the appropriate E. co //-recombinant strain and incubation at 28°C (for pJLA602-recombinant based plasmids) or 37°C (for pET- recombinant and pUC18/19-recombinant based plasmids) until the OD ⁇ oo reached between 0.8-1.0.
  • thermophilic hydrolytic enzymes For induction of the thermophilic hydrolytic enzymes, E. coli strains carrying pJLA602-recombinant plasmids were induced at 42°C and grown for 3-4 hours, whilst E. coli strains carrying either pET or pUC18/19-recombinant plasmids, were induced at 37°C for a similar time-period but with the addition of isopropyl ⁇ -D-1- thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM.
  • IPTG isopropyl ⁇ -D-1- thiogalactopyranoside
  • TES buffer (10 mM Tris, 1 mM EDTA, 1 mM NaCl, pH 7.5) and lysed with two or three passages through a French press cylinder. The lysed cultures were heat-treated at 70°C to precipitate out most of the mesophilic E. coli proteins. Following centrifugation at -20,000 g for 30 minutes, the clear supernatants containing the thermophilic enzymes were removed and kept at 4 0 C or used immediately in enzyme-substrate hydrolysis experiments.
  • T. reesei For production of hydrolytic enzymes from T. reesei (Rut-C30) and T. reesei-Xy ⁇ B transformant(s), fifty millilitres of medium containing minimal salts (as described in Penttila et al., "A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei ", (1987), Gene, 61:155-164) supplemented with Avicel cellulose (2- 2.5% w/v), soy bean flour (1.5% w/v) and lactose (1% w/v), pH 4.5-6.5 were inoculated with 10 -10 conidia isolated from fungal non-transformants and transformants and grown on potato dextrose agar (PDA) plates.
  • PDA potato dextrose agar
  • culture supernatants containing secreted enzymes and hyphae were collected by centrifugation and the clear supernatant containing only the soluble and secreted enzymes was removed and used for further analysis.
  • Fermentation cultivations (IL - 10L) were performed using the same medium as for 50 mL shake cultures. Typical batch fermentations were run for 5-7 days, with agitation set at 400 rpm, aeration kept at -2.5 LPM, and the pH was maintained at between 5.0 and 6.5. Further control on d ⁇ 2 uptake was linked to a cascade setup with an agitation span of 400 - 700 rpm.
  • the culture supernatant from T. reesei Rut-C30, containing the full suite of cellulolytic and hemicellulolytic mesophilic enzymes was separated from the fungal mycelia,following centrifugation at -20,000 g for 30 minutes,and used for the hydrolysis experiments.
  • the culture supernatant from the T. reesei-XynB recombinant strain was heat- treated at 70°C to remove most of the fungal mesophilic enzymes and the resulting crude thermophilic XynB enzyme was used also in the hydrolysis experiments.
  • a total of 100 mg of SC-treated P. radiata sawdust substrate was used per assay.
  • the hemicellulose liquor was adjusted to pH -5.4 with NaOH, before use.
  • the Pinus radiata kraft pulp was used at 2.5% (w/v) concentration.
  • Each two-stage enzyme hydrolysis assay comprised a first stage in which the enzyme/substrate mixture incubated at a temperature in the range of 45-50 °C for a time period of between 15 and 20 hours and a second stage in which the enzyme/substrate mixture was incubated at a temperature in the range of 65-75°C for a time period of between, 5 and 20 hours.
  • the specific temperatures and times utilised in each individual assay are indicated in the relevant examples below.
  • Example 7 Enzyme activity assays and sugar analysis
  • Example 8 Two-stage enzyme hydrolysis assay using hemicellulose liquor from high temperature-high pressure-treated Pinus radiata sawdust as substrate
  • a two-stage enzyme hydrolysis assay was performed using hemicellulose liquor from high temperature-high pressure-treated Pinus radiata sawdust as a substrate.
  • Enzymes utilised in this assay included mesophilic enzymes (5% v/v) derived from T. reesei Rut-C30 and a thermophilic enzyme cocktail (5% v/v) made up of XynB, CelEl/2,
  • Hemicellulose liquor sample (900 ⁇ l) prepared as indicated in Example 5 above was added to four tubes.
  • Tube one endogenous mesophilic enzymes expressed by T. reesei Rut-C30 (5% v/v).
  • Tube two T. ree.se/-XynB transformant enzymes (5% v/v) (i.e. endogenous mesophilic enzymes derived from T. reesei Rut-C30 and the thermophilic XynB enzyme).
  • Tube 3 thermophilic enzymes cocktail (5% v/v) made up of XynB, CeIE 1/2, ManA and
  • Tube 4 combination (5% v/v total) of endogenous mesophilic enzymes expressed by T. reesei and thermophilic enzyme cocktail made up of the XynB, CeIE 1/2, ManA and BgIA thermophilic enzymes. Tubes 2 and 4 were subjected to a first incubation at 48°C for 17 hours followed by a second incubation at 70°C for a further 6 hours. Tube 1 was incubated at 48°C for 23 hours, while tube 3 was incubated at 70°C for 23 hours. All reactions were performed in duplicate. Following enzyme hydrolysis, the release of reducing sugars from the substrate was assayed using the DNS-based method as described in Example 7. Results are shown in
  • sample 1 (compared to T. reesei only, sample 1) may have arisen from increased xylobiose, xylotriose, longer xylo-oligomers and mannose sugars (in samples 2 and 4) released due to the action of the thermophilic XynB and ManA when the incubation temperature was increased up to 70°C.
  • Example 9 Two-stage enzyme hydrolysis assays using supercritical ethanol-treated P. radiata sawdust
  • T. reesei-XynB culture supernatant/enzyme sample containing up to 500 ⁇ g of total protein (produced from a batch fermentation run as described in the Examples 3 and 4 above) comprising thermophilic XynB (up to about 6,250 nkatals) and endogenous mesophilic enzymes derived from T. reesei Rut-C30 containing up to 500 ⁇ g of total protein (was added to each of two tubes containing lOOmg of substrate (tubes 2 and 3).
  • Tube 1 had lOOmg of substrate only (no enzyme added) and tube 4 had the same enzyme mixture applied to tubes 3 and 4 but water was added instead of substrate.
  • the OU 54 o value from the enzyme sample only control was subtracted from OD values obtained from samples 2 and 3 before plotting the data. All tubes were incubated with rotation at 50°C for 16 hours. Samples (250 ⁇ L) were then removed and stored at 4°C. The remainder of the enzyme-substrate samples were then further incubated for another 16 hours at 70°C. All tubes were then collected and the supernatants were removed and kept at 4 0 C for further analysis. The hydrolysates were analysed for release of reducing sugars, as described in
  • Example 7 Results are shown in Figure 2, where release of reducing sugars from the substrate was first established after incubation of the substrate and enzyme samples at 50 0 C. A low background level was detected from the substrate only control ( Figure 2, sample 1). A further increase of reducing sugars in the order of ⁇ 9-17 % was observed when the temperature was raised from 50 0 C to 70 0 C. While not being bound to a particular mode of action, it is postulated that the shift of temperature up to 7O 0 C substantially denatures the mesophilic fungal enzymes while the thermophilic enzymes of the enzyme-substrate mix are still functional and hydrolyse the substrate more effectively at the elevated temperature. The two-step incubation utilising a shift from 5O 0 C up to 7O 0 C demonstrated improved sugar release compared to the single hydrolysis incubation at 50 0 C.
  • Table 1 also shows a 13.92-fold increase in xylose hydrolysis product was achieved from incubation of enzyme-substrate at 50°C compared to the control. An 18.47-fold increase in xylose hydrolysis product was obtained compared to the control after two- stage incubation at 5O 0 C and 70°C, when compared to an enzyme sample only control at temperatures of 50°C and 70°C, respectively.
  • Table 1 Glucose and xylose products derived from two stage enzyme hydrolysis of supercritical ethanol-treated P. radiata sawdust.
  • the high increase in xylose from the enzyme-substrate sample (Table 1) produced during the two-stage enzyme hydrolysis process is thought to arise from the combined action of Trichoderma 's own xylanases at 50 0 C (which includes a xylosidase that can cleave xylobiose down to xylose monomers) and the endogenously produced thermophilic XynB at 70 °C.
  • the peak labelled 8 in Figure 5 (most probably disaccharides) has an area corresponding to 7.467 in 70°C (blue line) and 2.415 in 50°C (black line), representing a 3.09-fold increase during the shift from 50 0 C to 70°C.
  • Example 10 Two-stage enzyme hydrolysis assays using P. radiata kraft pulp Pinus radiata kraft pulp was used as a substrate for the two-stage enzyme hydrolysis process. Samples were prepared as follows:
  • Sample one endogenous mesophilic enzymes expressed by T. reesei Rut-C30 (5% v/v).
  • Sample two thermophilic enzyme cocktail made up of XynB, CelEl/2, CelE-Cterm, CeIA, ManA, XynE, XynF, XynD, Xynl and BgIA (5% v/v total).
  • Sample three endogenous mesophilic enzymes expressed by T.
  • thermophilic enzyme cocktail made up of XynB, CelEl/2, CelE-Cterm, CeIA, ManA, XynE, XynF, XynD, Xynl and BgIA (5% v/v).
  • Kraft pulp samples were used in the assay at 2.5% (w/v).
  • Sample three was subjected to a first incubation at 50 0 C for a total of 18 hours followed by a second incubation at 70 0 C for a further 18 hours. All reactions were performed in duplicate.
  • Results from the DNS-reducing sugar assays performed on the hydrolysates are shown in Figure 3.
  • the combination of T. reesez-thermophilic enzymes (sample 3) produced the highest amount of reducing sugars based on the DNS-colour based assay following the total hydrolysis time of 36 hours.
  • Sample one endogenous mesophilic enzymes expressed by T. reesei Rut-C30 (5% v/v).
  • Sample two thermophilic enzyme cocktail made up of XynB, CelEl/2, CeIE-
  • Sample four endogenous mesophilic enzymes expressed by T. reesei Rut-C30 (5% v/v) mixed with thermophilic enzyme cocktail made up of XynB, CelEl/2, CelE-Cterm, CeIA, ManA, XynE, XynF, XynD, Xynl and BgIA (5% v/v).
  • thermophilic enzyme cocktail made up of XynB, CeIE 1/2, CeIE- Cterm, CeIA, ManA, XynE, XynF, XynD, Xynl and BgIA (10% v/v).
  • Sample six endogenous mesophilic enzymes expressed by T. reesei Rut-C30 (10% v/v) mixed with thermophilic enzyme cocktail made up of XynB, CelEl/2, CelE-Cterm, CeIA, ManA, XynE, XynF, XynD, Xynl and BgIA (5% v/v).
  • Kraft pulp samples were used in the assay at 2.5% (w/v). Each of samples one, three and seven were incubated at 50°C for a total of 46.5 hours. Each of samples two, five and eight were incbated at 70 °C for a total of 46.5 hours. Each of samples four and six were subjected to a first incubation at 50 °C for 22 hours followed by a second incubation at 70°C for a further 24.5 hours.
  • the DNS-based assay (see Example 7) was performed on the hydrolysates and the results shown in Figures 4a and 4b.
  • thermophilic hydrolytic enzymes Figure 4a, samples 4 and 6
  • thermophilic hydrolytic enzymes Figure 4a, samples 2 and 5
  • thermophilic enzymes Figure 4a, sample 5 compared to sample 2
  • T. reesei and thermophilic enzymes at a combined total enzyme concentration of 5% (v/v) Figure 4a, sample 4
  • T. reesei enzymes only at a total enzyme concentration of 5% (v/v) Figure 4a, sample 3
  • the conditions may have been somewhat favourable towards T. reesei enzymes as only half the amount of Trichoderma enzymes was present in sample 4 (combination) compared to sample 3 ⁇ Trichoderma enzymes only).
  • HPLC analysis also was performed on hydrolysates from P. radiata Kraft pulp following enzyme hydrolysis.
  • up to 12.78 % increase in glucose levels was generated during the first 18 hours of hydrolysis from the combination of T. reesei (5 % v/v) and thermophilic enzymes (5 % v/v) ( Figure 6, sample ) when compared to T. reesei enzymes (5 % v/v) alone ( Figure 6, sample 1)
  • up to 22.6 % increase in glucose levels was generated using the T. reesei plus thermophilic enzyme combination ( Figure 6, sample 4) when compared to T. reesei enzymes alone ( Figure 6, sample 2).
  • the two-stage hydrolysis conditions were kept within limited but near optimal working conditions (temperature and pH) so as to favour most of the Trichoderma and thermophilic hydrolytic enzymes and thus minimse costs related to energy consumption. While the multiple and different enzymes used in these examples have different temperature and pH optima's, the hydrolysis conditions used in these examples favoured the majority of the hydrolytic enzymes from both T. reesei ( ⁇ 50°C) and the thermophilic enzymes (70°C). Previous results have indicated that fungal enzyme hydrolysis at 60 0 C to 70°C resulted in a rapid drop in enzyme activities over time.
  • thermophilic hydrolytic enzymes such as XynB have a temperature optimum of ⁇ 85°C (and others even higher) but most have half lives of at least 24-48 hours at 70°C and perform well at a pH range of between 5.0 to 6.0.
  • thermophilic enzymes used in these assays were produced in E. coli it would be considered advantageous to produce the selected thermophilic enzymes in T. reesei as was the case for the assay described in Example 8 (results shown in Figure 1) where a T. reesei-XynB transformant was used as a source of both mesophilic and thermophilic hydrolytic enzymes.
  • Culture supernatants containing the mixtures of both T. reesei mesophilic and thermophilic enzymes could be used for the hydrolysis of any renewable biomaterial, such as cellulose and hemi cellulose.

Abstract

L'invention concerne des procédés d'hydrolyse enzymatique de matériaux. Plus spécifiquement, l'invention concerne un procédé d'hydrolyse d'un polysaccharide, le procédé comprenant les étapes suivantes : (i) mise en contact du polysaccharide avec des enzymes hydrolytiques mésophiles et thermophiles afin de produire un mélange réactionnel, (ii) incubation du mélange réactionnel à une température appropriée à l'activité des enzymes hydrolytiques mésophiles, et (iii) incubation du mélange réactionnel à une température appropriée à l'activité des enzymes hydrolytiques thermophiles, le polysaccharide étant hydrolysé par lesdites enzymes hydrolytiques mésophiles et thermophiles pour produire deux fragments polysaccharidiques ou davantage. L'invention concerne en outre des fragments sucres produits par les procédés d'hydrolyse enzymatique.
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EP2585606A4 (fr) * 2010-06-26 2016-02-17 Virdia Ltd Mélanges de sucres, méthodes de production et d'utilisation associées
US9512495B2 (en) 2011-04-07 2016-12-06 Virdia, Inc. Lignocellulose conversion processes and products
CN107164436A (zh) * 2017-05-12 2017-09-15 南京林业大学 β‑葡萄糖苷酶在转化淫羊藿总黄酮制备宝霍苷I中的应用
CN107189994A (zh) * 2017-07-05 2017-09-22 中国科学院青岛生物能源与过程研究所 β‑木糖苷酶及其应用
KR101839440B1 (ko) 2015-06-24 2018-04-26 주식회사 에이더블유바이오 고온에서 단백질 분해 활성을 갖는 퍼비도박테리움 아이슬란디쿰 aw-1 또는 이의 조효소액
WO2020089703A1 (fr) * 2018-10-29 2020-05-07 Roberto Bassi Micro-algues transgéniques pour la production d'enzymes dégradant la paroi de cellules végétales ayant une activité cellulolytique thermostable
US10760138B2 (en) 2010-06-28 2020-09-01 Virdia, Inc. Methods and systems for processing a sucrose crop and sugar mixtures
US10759727B2 (en) 2016-02-19 2020-09-01 Intercontinental Great Brands Llc Processes to create multiple value streams from biomass sources
CN112159774A (zh) * 2020-09-22 2021-01-01 自然资源部第一海洋研究所 一株高效降解硒化卡拉胶的深海芽孢杆菌n1-1及其应用
US11078548B2 (en) 2015-01-07 2021-08-03 Virdia, Llc Method for producing xylitol by fermentation
US11091815B2 (en) 2015-05-27 2021-08-17 Virdia, Llc Integrated methods for treating lignocellulosic material
WO2024062148A1 (fr) * 2022-09-19 2024-03-28 Universidade De Vigo Processus de production de produits dérivés de l'hémicellulose
US11965220B2 (en) 2012-05-03 2024-04-23 Virdia, Llc Methods for treating lignocellulosic materials

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