ENZYME HYDROLYSIS METHOD

Technical Field

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.

Background There is currently considerable global interest in the production of biofuels from abundant and renewable bio-materials such as cellulose and hemicelluloses due to increasing fuel prices and the recognition of depleting global oil reserves. Countries such as Brazil and the USA currently lead the way in bioethanol production, using agricultural crops such as sugarcane and corn. However, converting food grade raw materials into sugars and fuel ethanol has had a negative impact on food costs for local communities and has induced debate at political and governmental levels concerning the food/energy tradeoff. As a result of these factors, there is increasing interest in the development of effective processes for extraction of fermentable sugars from alternative, renewable resources such as cellulose and hemicellulose available from abundant woody materials. Currently, most technologies utilise fungal enzyme mixtures in the production of fermentable sugars. However, while high-secreting fungal strains have been produced and their enzymes tested, current enzyme hydrolysis processes cannot be considered to be optimal due to inherent enzymatic deficiencies. One deficiency is that certain exo-type enzymes are too 'slow' in their performance. Another deficiency is that some enzymes are believed to attach too tightly to the substrate (due to the presence of substrate binding domains), thereby preventing recycling of the enzyme.

In addition, the optimal operational temperature for most fungal enzymes is normally moderate (around 40°C-50°C). At these temperatures 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). This back-precipitation may inhibit

further access and enzymatic activities of the enzymes. More recently, 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 β-glucosidase, reducing cost-efficiency. Although 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). Furthermore, 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.

There is a need for improved enzyme hydrolysis methods in fermentable sugar production that overcome the deficiencies of current techniques involving fungal hydrolysis enzymes at moderate reaction temperatures. There is also a need for enzyme hydrolysis methods which increase the cost-efficiency of fermentable sugar production.

Summary of the Invention

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.

In a first aspect 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,

(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.

In a second aspect the invention provides a method for hydrolysing a polysaccharide, the method comprising the steps of:

(i) contacting the polysaccharide with mesophilic hydrolytic enzymes,

(ii) incubating the polysaccharide and mesophilic hydrolytic enzymes of step (i) at a temperature suitable for activity of the mesophilic hydrolytic enzymes,

(iii) contacting the polysaccharide of step (ii) with thermophilic hydrolytic enzymes; and

(iv) incubating the polysaccharide and hydrolytic enzymes 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. In a third aspect the invention provides a method for hydrolysing a polysaccharide, the method comprising the steps of:

(i) contacting the polysaccharide with mesophilic and thermophilic hydrolytic enzymes to produce a reaction mixture,

(ii) incubating the reaction mixture at a temperature suitable for activity of the mesophilic hydrolytic enzymes, and

(iii) 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. In a fourth aspect the invention provides a method for hydrolysing a polysaccharide, the method comprising the steps of:

(i) contacting the polysaccharide with mesophilic hydrolytic enzymes to produce a reaction mixture,

(ii) incubating the reaction mixture at a temperature suitable for activity of the mesophilic hydrolytic enzymes,

(iii) contacting the reaction mixture with thermophilic hydrolytic enzymes, and

(iv) 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.

In one embodiment of the above aspects, 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.

In another embodiment of the above aspects, the temperature suitable for activity of the mesophilic hydrolytic enzymes is between about 40°C and below 60 ⁰ C.

In another embodiment of the above aspects, the temperature suitable for activity of the mesophilic hydrolytic enzymes is between about 45°C and about 55°C.

In another embodiment of the above aspects, the temperature suitable for activity of the mesophilic hydrolytic enzymes is about 50 ⁰ C.

In one embodiment of the above aspects, 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.

In one embodiment of the above aspects, the temperature suitable for activity of the thermophilic hydrolytic enzymes is above about 65°C.

In another embodiment of the above aspects, the temperature suitable for activity of the thermophilic hydrolytic enzymes is about 70 ⁰ C, or above about 70 ⁰ C.

In another embodiment of the above aspects, the temperature suitable for activity of the thermophilic hydrolytic enzymes is above about 80 ⁰ C.

In another embodiment of the above aspects, the temperature suitable for activity of the thermophilic hydrolytic enzymes is above about 90 ⁰ C. In a further embodiment of the above aspects, the polysaccharide is selected from the group consisting of cellulose, hemicellulose and mixtures thereof.

In one embodiment of the above aspects, the polysaccharide is a component of lignocellulosic biomass. The lignocellulosic biomass may be pre-treated with a supercritical solvent, acid hydrolysis or base hydrolysis.

In one embodiment of the above aspects, the mesophilic or thermophilic hydrolytic enzyme is a hydrolase.

In an additional embodiment of the above aspects, 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.

In another embodiment of the above aspects, the 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 βavothermus, 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. islandicum, 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,

Thermoanaerobacterium saccharolyticum, Thermotoga maritima, Thermotoga neapolitana, Thermotoga thermarum, Thermus aquaticus, Thermus thermophilus, Thermus ruber, Thermus filiformis, Thermothrix thiopara, Thermomicrobium roseum and Hydrogenobacter thermophilus. In one embodiment of the above aspects, the thermophilic hydrolytic enzyme is selected from the group consisting of β-glucosidase (BgIA) from Caldicellulosiruptor saccharolyticus TP8.3.3.1, Xylanase A (XynA) from Dictyoglomus thermophilum, bifunctional Cellulase B (CeIB) from Caldicellulosiruptor saccharolyticus, β-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 saccharolyticwn, xylanase (XynX) from Clostridium thermocellum, β-glycanases from Caldicellulosiruptor saccharolyticus, xylanase (XynB) from Dictyoglomus thermophilum strain Rt46B.l and xylanases (XynA, XynB, XynC and XynD) from Caldicellulosiruptor strain Rt69B.l.

In one embodiment of the above aspects, the thermophilic hydrolytic enzyme is selected from the group consisting of β-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, Xylosidase (XynD) from Cs. saccharolyticus, Cellulases (CeIE 1/2,

CelECterm and CelB5) from Caldicellulosiruptor Tok7B.l and Mannanase (ManA) from

Dictyoglomus thermophilum.

In a further embodiment of the above aspects, either or both of: (i) the mesophilic hydrolytic enzymes

(ii) the thermophilic hydrolytic enzymes are produced from a synthetic polynucleotide sequence.

In another embodiment of the above aspects, 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,

Geomyces, Hypocrea, Magnaporthe, Mucor, Neurospora, Ophiostoma, Penicillium,

Phoma, Phanerochaete and Emericella.

In another embodiment of the above aspects, 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,

Humicola, Chrysosporium, Doratomyces, Fusarium, Gliocladium, Geomyces, Hypocrea,

Magnaporthe, Mucor, Neurospora, Ophiostoma, Penicillium, Phoma, Phanerochaete and

Emericella fungal strains.

In one embodiment of the above aspects, 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:

1 ORFJ23283 Arabinofuranosidase (ABFI) 51.1/6.0 53/6.3 432 38

2 ORF_76210 Arabinofuranosidase (ABFII) 34.8/6.4 33/6.7 176 34

3 ORF_55319 Arabinofuranosidase (ABFIII) 53.1/5.7 55/5.5 168 16

4 ORF 54219 Candidate acetyl xylan esterase (AXE) 21.9/6.2 27/6.2 222 19

5 ORF 123989 Cellobiohydrolase I (Cel7A) 54.1/4.6 63/4.5 80 7

6 ORFJ 23989 Cellobiohydrolase I (Cel7A) 54.1/4.6 63/4.4 64 8,8

7 ORF l 23989 Cellobiohydrolase I (Cel7A) 54.1/4.6 63/4.6 86 9 8 ORF 123989 Cellobiohydrolase I (Cel7A) 54.1/4.6 57/4.7 107 13

9 ORF 72567 Cellobiohydrolase II (Cel6A) 49.6/5.1 56/5.2 210 24

10 ORF_72567 Cellobiohydrolase II (Cel6A) 49.6/5.1 58/5.0 153 11

11 ORF_72567 Cellobiohydrolase II (CelόA) 49.6/5.1 59/4.8 207 18

12 ORF 72567 Cellobiohydrolase II (CelόA) 49.6/5.1 58/6.0 165 17 13 ORF_72567 Cellobiohydrolase II (CelόA) 49.6/5.1 55/5.6 125 11

14 ORF_72567 Cellobiohydrolase II (CelόA) 49.6/5.1 55/5.4 77 4

15 ORF 72567 Cellobiohydrolase II (CelόA) 49.6/5.1 42/4.7 157 20

16 ORF_72567 Cellobiohydrolase II (CelόA) 49.6/5.1 38/4.9 279 17

17 ORF_72567 Cellobiohydrolase II (CelόA) 49.6/5.1 38/5.0 307 20 18 ORF 72567 Cellobiohydrolase II (CelόA) 49.6/5.1 30/5.1 279 17

19 ORF l 22081 Endoglucanase I (Cel7B) 48.2/4.7 55/4.6 57 9

20 ORF 120312 Endoglucanase II (Cel5A) 44.1/5.0 43/4.8 160 31

21 ORFJ 20312 Endoglucanase II (Cel5A) 44.1/5.0 48/4.6 64 12

22 ORFJ23232 Endoglucanase III (Cell2A) 25.1/6.7 25/5.7 185 19 23 ORFJ23232 Endoglucanase III (Cell2A) 25.1/6.7 26/57 185 19

24 ORF_49081 Xyloglucanase (Cel74A) 87.1/5.4 96/5.4 201 16

25 ORF 49081 Xyloglucanase (Cel74A) 36.2/8.7 96/5.3 520 26

26 ORF_49081 Xyloglucanase (Cel74A) 36.2/8.7 43/5.2 406 21

27 ORF 49081 Xyloglucanase (Cel74a) 36.2/8.7 35/6.0 354 12 28 ORF_27554 Candidate Endoglucanase (EGL) 36.2/8.7 36/5.5 87 15

29 ORFJ21127 Xylosidase I (BXLI) 87.2/5.5 97/5.6 208 12

30 ORFJ21127 Xylosidase I (BXLI) 87.2/5.5 97/5.7 203 15

31 ORF 74223 Xylanase I (XYNI) 24.6/5.0 21/4.6 85 16

32 ORFJ23818 Xylanase II (XYNII) 24.1/7.9 21/6.6 212 27 33 ORFJ 11849 Xylanase IV (XYNIV) 52.8/5.7 55/5.6 125 13

34 ORF_56996 Mannanase I (MANI) 40.2/5.1 53/5.1 147 17

35 ORF_76672 β-Glucosidase (BGLI) 78.4/6.4 81/6.7 440.

In another embodiment of the above aspects, 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 In one embodiment of the above aspects, 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.

In one embodiment of the above aspects, 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. s In a fifth aspect, 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, Geotrichum, Aspergillus, Penicillium, Fusarium, Saccharomyces,

Candida, Pachysolen and Pichia. The bacteria may be selected from the group consistingS of Zymomonas, Leuconostoc, Lactobacillus, Oenococcus, Leuconostoc and

Mycobacterium .

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. 0 In a sixth aspect the invention provides a fragment produced in accordance with the method of the first, second, third or fourth aspects.

In a seventh aspect the invention provides a fermented sugar product produced in accordance with method of the fifth aspect.

In an eighth 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.

In a ninth aspect 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.

In one embodiment of the ninth aspect, the mesophilic hydrolytic enzyme is used to hydrolyze said polysaccharide at a temperature of above about 10°C and below 60°C

In one embodiment of the ninth aspect, the mesophilic hydrolytic enzyme is used to hydrolyze said polysaccharide at a temperature of between about 30°C and below 60°C.

In one embodiment of the ninth aspect, the mesophilic hydrolytic enzyme is used to hydrolyze said polysaccharide at a temperature of between about 40 ⁰ C and below 60°C.

In another embodiment of the ninth aspect, 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.

In another embodiment of the ninth aspect, the thermophilic hydrolytic enzyme is used to hydrolyze said polysaccharide at a temperature of 60°C, or above 60 ⁰ C.

In additional embodiment of the ninth aspect, the thermophilic hydrolytic enzyme is used to hydrolyze the polysaccharide at a temperature of about 70°C, or above about 70°C.

In another embodiment of the ninth aspect, the thermophilic hydrolytic enzyme is used to hydrolyze the polysaccharide at a temperature of above about 80 ⁰ C.

In another embodiment the ninth aspect, the thermophilic hydrolytic enzyme is used to hydrolyze the polysaccharide at a temperature of above about 90 ⁰ C.

In another embodiment of the ninth aspect, the hydrolytic enzyme is a glycosylase selected from the group consisting of xylanases, glucosidases, cellulases, xylosidases, mannanases, glycoside hydrolases, dextranases, cellobiohydrolases and endoglucanases.

In one embodiment of the ninth aspect, the polysaccharide is selected from the group consisting of cellulose, hemicellulose and mixtures thereof.

Brief Description of the Drawings

A preferred embodiment of the present invention will now be described, by way of an example only, with reference to the accompanying drawings wherein:

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 λ ₅₄₀ ) are indicated on the y-axis. Chequered columns: absorbance at λ ₅₄₀ after 17 hour incubation; dark columns: absorbance at λ ₅₄₀ after a further 6 hour incubation.

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 λ ₅₄ o) are indicated on the z-axis, temperatures utilised in the assay are indicated on the x-axis. Dark columns: absorbance at λ ₅₄₀ after 16 hour incubation at 50°C; light columns: absorbance at λ 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 λs ₄ o) are indicated on the y-axis. Light columns: absorbance at λ ₅₄ o after 18 hour incubation; dark columns: absorbance at λ ₅₄₀ after a further 18 hour incubation. 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. Sample numbers (1-8) are indicated on the x-axis, absorbance values (measured at λ ₅₄ o) are indicated on the y-axis. Different shaded columns represent absorbance at λ 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.

Definitions

As used in this application, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a plant cell" also includes a plurality of plant cells.

As used herein, the term "comprising" means "including." Variations of the word

"comprising", such as "comprise" and "comprises," have correspondingly varied meanings. Thus, for example, a polynucleotide "comprising" a sequence encoding a protein may consist exclusively of that sequence or may include one or more additional sequences.

As used herein, the term "polysaccharide" encompasses any molecule comprising two or more monosaccharide units.

As used herein, the terms "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.

As used herein, the terms "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.

Any description of prior art documents herein, or statements herein derived from or based on those documents, is not an admission that the documents or derived statements are part of the common general knowledge of the relevant art in Australia or elsewhere.

For the purposes of description all documents referred to herein are incorporated by reference unless otherwise stated.

Detailed Description

As described herein, 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. In the second stage of the method, 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.

Advantageously, 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. For example, 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, raffϊnose, 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, isomaltose, mannobiose, kojibiose, rutinose, nigerose, and melibiose.

In a preferred embodiment, 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.

In a preferred embodiment of the invention, 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.

Examples of suitable woody plant matter include pine (e.g. Pinus radiata), birch, eucalyptus, beech, spruce, fir, cedar, poplar and aspen.

Examples of suitable fibrous plant matter 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.

In a preferred embodiment of the invention, the grass is switch grass (Panicum vergatum).

Examples of 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). In general, 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. For example, 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.

Additionally or alternatively, lignocellulosic biomass may be pre-treated using chemical methods. Chemical methods for the pre-treatment of lignocellulosic biomass are generally known in the art. For example, 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. Non- limiting examples of 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.

Additionally or alternatively, 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. pH 1.0 - 13.0) to the lignocellulosic matter, normally in conjunction with heat, in order to facilitate the solubilisation of hemicellulose and/or cellulose. Methods involving the acidic or alkaline hydrolysis of lignocellulosic matter are known in the art and are described, for example, in United States Patent 2218567, US Patent No. 2709699, US Patent No. 4708746, U.S.

Patent No. 4880473, US Patent No. 5410034, United States Patent No. 5424417 and

United States Patent No. 6022419. 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.

Additional methods or variations of the methods described above that may be used for the pre-treatment of lignocellulosic matter are described in United States Patent No. 4038481, United States Patent No. 4302252, United States Patent No. 6022419, United

States Patent No. 6228177 and United States Patent No. 6824599.

In a preferred embodiment, lignocellulosic biomass is fractionated into one or more components comprising purified or substantially purified hemicellulose and/or cellulose.

It is preferred that lignin is removed from lignocellulosic matter prior to enzymatic hydrolysis in accordance with the methods described herein. However, 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.

Two-stage enzyme hydrolysis

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.

In the context of this specification, 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. Accordingly, 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. For example, 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. For example, the 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.

Measurement of the release of hydrolysis products for determining optimal enzymatic activity can be performed using techniques generally known in the art. In general, 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 β" 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. For example, 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. 199: 68-74, whereby monosaccharides, disaccharides and oligosaccharides are first trapped on an AminoTrap-resin based column before being separated according to charge and molecular weight by competitive action of H ⁺ ions from NaOH buffers applied to the column.

Mesophilic and thermophilic hydrolytic enzymes used in the two-stage enzyme hydrolysis method may be applied to the polysaccharide separately or in combination.

In the first stage of the two-stage enzyme hydrolysis method, the polysaccharide is contacted with mesophilic hydrolytic enzymes, or a combination of mesophilic and thermophilic hydrolytic enzymes, thereby forming an enzyme/substrate mixture.

It will be understood that contacting the polysaccharide with mesophilic 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. Additionally or alternatively, 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. In general, 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.

In certain embodiments, 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.

In a preferred embodiment, the reaction temperature utilised during the first stage of the two-stage enzyme hydrolysis method is about 50°C. In the first stage of the two-stage enzyme hydrolysis method, 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. For example, the mixture may be incubated for a period of between about 1 hour and about 30 hours. Preferably, 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.

In the second stage of the two-stage enzyme hydrolysis method, 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.

It will be understood that contacting the polysaccharide with 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. In general, the reaction temperature utilised during the second stage of the hydrolysis method is 60°C, or above 60°C.

In certain embodiments, 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.

In a preferred embodiment, the reaction temperature utilised during the second stage of the hydrolysis method is about 60°C. In another preferred embodiment, the reaction temperature utilised during the second stage of the hydrolysis method is about 70°C.

In the second stage of the two-stage enzyme hydrolysis method, 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. For example, the mixture may be incubated for a period of between about 1 hour and about 40 hours. Preferably, 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.

In general, 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.

In certain embodiments, 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. For example, the mixture may be incubated for a period of between about 1 hour and about 40 hours. Preferably, 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.

Apart from the aforementioned reaction temperatures and incubation periods, 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.

In general, 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. Although the two-stage enzyme hydrolysis method preferably employs enzymes in dissolved state, it is equally possible to use enzymes immobilized on a solid support.

It will be recognised that 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. For example, hydrolysis products may be measured as reducing sugars assayed by the dinitrosalicyclic acid (DNS) method (see, for example, Bernfeld P. "Amylases a and β. " In: Methods in Enzymology, vol 1. Colowick, Kaplan (Eds) (1955), Academic, New York, pl49-158; Miller GL, "Use of

dinitrosalicylic acid reagent for determination of reducing sugar" Analytical Chemistry, (1959), 31, 426-428; Aibba et al. "Applied Environmental Microbiology", (1983) Vol. 46, p. 1059-1065). Briefly, this method provides a test for the presence of free carbonyl group (C=O) which is indicative of reducing sugars. This involves the oxidation of the aldehyde functional group present in, for example, glucose and the ketone functional group present in, for example, fructose. Simultaneously, 3,5-dinitrosalicylic acid (DNS) is reduced to 3- amino,5-nitrosalicylic acid under alkaline conditions, and would result in a yellow colouration of the solution, which can be measured spectrophotomerically at λ ₅₄₀ . At the same time, known amounts of sugars (e.g. glucose and xylose) are used as standards. The more sugar reducing ends present in the solution, the higher the yellow colour intensity.

Additionally or alternatively, hydrolysis products may be analysed using ion- exchange chromatography. For example, 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. 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 199: 68-74; whereby monosaccharides, disaccharides and oligosaccharides are firstly trapped on an AminoTrap-resin based column before being separated according to charge and molecular weight by competitive action of H ⁺ ions from NaOH buffers applied to the column.

Hydrolytic enzymes

Any enzyme capable of hydrolysing a polysaccharide may be used for the two-stage enzyme hydrolysis method described herein. In general, 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)

(http://www.chem.qmul.ac.uk/iubmb/) as of the filing date of this application. Preferably, the hydrolytic enzymes utilized in the methods described herein are those classified under class EC 3.2 (glycosylases) of the NC-IUBMB enzyme nomenclature.

In some embodiments, 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. In other embodiments, the enzymes that may be utilised are those classified under subclass EC 3.2.2

(Hydrolysing N-Glycosyl Compounds) of the NC-IUBMB nomenclature. In other embodiments, hydrolytic enzymes that may be utilized are those classified under subclass EC 3.2.3 (Hydrolysing 5-Glycosyl Compounds) of the NC-IUBMB nomenclature.

Examples of 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, β-glucosidases, β- xylosidases, mannanases, galactanases, dextranases, endoglucanases, exoglucanases, mannosidases, arabinosidases, and alpha-galactosidase. Specific examples of preferred thermophilic hydrolytic enzymes include, but are not limited to, β-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. saccharolyticus, Xylanases (xynE and xynl) from Cs. Saccharolyticus ,β-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, β-glycanases from Caldicellulosiruptor saccharolyticus, Xylosidase (XynD) from Cs. saccharolyticus, Cellulases (CeIE 1/2, CelECterm and CelB5) from Caldicellulosiruptor sp. Tok7B.l and xylanases (XynA, XynB, XynC and XynD) from Caldicellulosiruptor sp. strain Rt69B. l . In the first stage of the two-stage enzyme hydrolysis method described herein, 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. For example, mesophilic hydrolytic enzymes may be derived from mesophilic microorgansisms such as mesophilic fungi, mesophilic bacteria, and mesophilic yeasts. It will be understood that 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.

In one embodiment, mesophilic hydrolytic enzymes used in the two stage enzyme hydrolysis method are derived from a microorganism which endogenously expresses mesophilic hydrolytic enzymes. Preferably, the microorganism is a fungus.

In a preferred embodiment, the mesophilic hydrolytic enzymes are endogenous mesophilic enzymes derived from Trichoderma reesei. The endogenous enzymes may be one or more of:

1 ORFJ23283 Arabinofuranosidase (ABFI) 51.1/6.0 53/6.3 432 38

2 ORF_76210 Arabinofuranosidase (ABFII) 34.8/6.4 33/6.7 176 34

3 ORF 55319 Arabinofuranosidase (ABFIII) 53.1/5.7 55/5.5 168 16 4 ORF_54219 Candidate acetyl xylan esterase (AXE) 21.9/6.2 27/6.2 222 19

5 ORF 123989 Cellobiohydrolase I (Cel7A) 54.1/4.6 63/4.5 80 7

6 ORFJ23989 Cellobiohydrolase I (Cel7A) 54.1/4.6 63/4.4 64 8,8

7 ORF l 23989 Cellobiohydrolase I (Cel7A) 54.1/4.6 63/4.6 86 9

8 ORF 123989 Cellobiohydrolase I (Cel7A) 54.1/4.6 57/4.7 107 13 9 ORF_72567 Cellobiohydrolase II (Cel6A) 49.6/5.1 56/5.2 210 24

10 ORF 72567 Cellobiohydrolase II (Cel6A) 49.6/5.1 58/5.0 153 11

11 ORF 72567 Cellobiohydrolase II (Cel6A) 49.6/5.1 59/4.8 207 18

12 ORF 72567 Cellobiohydrolase II (Cel6A) 49.6/5.1 58/6.0 165 17

13 ORFJ72567 Cellobiohydrolase II (Cel6A) 49.6/5.1 55/5.6 125 11 14 ORF 72567 Cellobiohydrolase II (Cel6A) 49.6/5.1 55/5.4 77 4

15 ORF 72567 Cellobiohydrolase II (Cel6A) 49.6/5.1 42/4.7 157 20

16 ORF_72567 Cellobiohydrolase II (Cel6A) 49.6/5.1 38/4.9 279 17

17 ORF 72567 Cellobiohydrolase II (Cel6A) 49.6/5.1 38/5.0 307 20

18 ORF 72567 Cellobiohydrolase II (Cel6A) 49.6/5.1 30/5.1 279 17 19 ORF_122081 Endoglucanase I (Cel7B) 48.2/4.7 55/4.6 57 9

20 ORF 120312 Endoglucanase II (Cel5A) 44.1/5.0 43/4.8 160 31

21 ORF 120312 Endoglucanase II (Cel5A) 44.1/5.0 48/4.6 64 12

22 ORF 123232 Endoglucanase III (Cell2A) 25.1/6.7 25/5.7 185 19

23 ORF 123232 Endoglucanase III (Cell2A) 25.1/6.7 26/57 185 19 24 ORF 49081 Xyloglucanase (Cel74A) 87.1/5.4 96/5.4 201 16

25 ORF_49081 Xyloglucanase (Cel74A) 36.2/8.7 96/5.3 520 26

26 ORF 49081 Xyloglucanase (Cel74A) 36.2/8.7 43/5.2 406 21

27 ORF_49081 Xyloglucanase (Cel74a) 36.2/8.7 35/6.0 354 12

28 ORF_27554 Candidate Endoglucanase (EGL) 36.2/8.7 36/5.5 87 15

29 ORF 121127 Xylosidase I (BXLI) 87.2/5.5 97/5.6 208 12

30 ORF 121127 Xylosidase I (BXLI) 87.2/5.5 97/5.7 203 15

31 ORF 74223 Xylanase I (XYNI) 24.6/5.0 21/4.6 85 16

32 ORF 123818 Xylanase II (XYNII) 24.1/7.9 21/6.6 212 27 33 ORF l 11849 Xylanase IV (XYNIV) 52.8/5.7 55/5.6 125 13

34 ORF 56996 Mannanase I (MANI) 40.2/5.1 53/5.1 147 17

35 ORF_76672 β-Glucosidase (BGLI) 78.4/6.4 81/6.7 440.

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, Humicola {e.g. H. insolens and H. gήsea), Chrysosporium {e.g. C. lucknowense), Doratomyces {e.g. D. stemonitis), Fusarium, Gliocladium, Geomyces,

Hypocrea, Magnaporthe, Mucor, Neurospora, Ophiostoma, Penicillium, Phoma,

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.

In the second stage of the hydrolysis method described herein, 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. For example, 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.

In a preferred embodiment, the thermophilic hydrolytic enzymes are derived from thermophilic bacteria.

Non-limiting examples of suitable 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. emersoniϊ), 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. Non-limiting examples of suitable 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. islandicum, 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, Thermoanaerobacterium saccharolyticum, Thermotoga maritima, Thermotoga neapolitana, Thermotoga thermarum, Thermus aquaticus, Thermus thermophilus, Thermus ruber, Thermus filiformis, Thermothrix thiopara, Thermomicrobium roseum and Hydrogenobacter thermophilus.

In one embodiment of the above aspects, the thermophilic hydrolytic enzyme is selected from the group consisting of β-glucosidase (BgIA) from Caldicellulosiruptor saccharolyticus TP8.3.3.1, Xylanase A (XynA) from Dictyoglomus thermophilum, bifunctional Cellulase B (CeIB) from Caldicellulosiruptor saccharolyticus, β-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) from Clostridium

thermocellum, β-glycanases from Caldicellulosiruptor saccharolyticus, xylanase (XynB) from Dictyoglomus thermophilum strain Rt46B.l and xylanases (XynA, XynB, XynC and XynD) from Caldicellulosiruptor strain Rt69B.l.

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. Non-limiting examples of suitable microorganisms from which cryophilic hydrolytic enzymes may be derived include cryophilic bacteria, archaea and/or fungi.

In a preferred embodiment, 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. Accordingly, 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. Non limiting examples of 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. Preferably, the genetically modified form of T. reesei expresses thermophilic XynB. Preferably, 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).

Additionally or alternatively, hydrolytic enzymes suitable for use in the methods described herein may be produced using recombinant DNA technology. For example, 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. (Eds), John Wiley and Sons, Inc 2007), Molecular Cloning (Maniatis et al., Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y., 1982) and Current Protocols in Microbiology (Coico et al. (Eds), John Wiley and Sons, Inc, 2007).

In certain embodiments, 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. In a preferred embodiment, 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. 68:109; and Unites States Patent No. 4356270), or the diethylphosphoramidite method (see Beaucage et al. "Deoxynucleoside Phosphoramidites - A New Class of Key Intermediates for Deoxypolynucleotide Synthesis " (1981) Tetrahedron Letters, 22:1859- 1862). 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.

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).

By way of example, host cells may be transformed with vector constructs by the "heat shock" method. Under this 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.

Alternatively, 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.

Following entry of the vector construct, 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. For example, if the target vector contains one or more selectable markers, transformed host cells may be identified by expression of the marker or markers. Using the example of 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. In the case of host cell transformants expressing more than one selectable marker, 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. Examples of 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. For example, 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. The purification of secondary metabolites may also be "scaled-up" for large-scale production purposes.

In a preferred embodiment, hydrolytic enzymes for use in the two stage enzyme hydrolysis method are expressed and isolated from recombinant Trichoderma reesei host cells. In a particularly preferred embodiment, 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.

In one embodiment, the 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.

In another embodiment, hydrolytic enzymes for use in the methods described herein are produced synthetically (e.g. using a commercial facility).

Compositions

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

Nomenclature Committee of the International Union of Biochemistry and Molecular

Biology (NC-IUBMB) (http://www.chem.qmul.ac.uk/iubmb/) as of the filing date of this application. Preferably, the compositions of the invention comprise hydrolytic enzymes classified under class EC 3.2 (glycosylases) of the NC-IUBMB enzyme nomenclature.

In certain embodiments, 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. In other embodiments, the compositions comprise hydrolytic enzymes classified under subclass EC 3.2.2 (Hydrolysing iV-Glycosyl Compounds) of the NC-IUBMB nomenclature. In other embodiments, the compositions comprise hydrolytic enzymes classified under subclass EC 3.2.3 (Hydrolysing S-Glycosyl Compounds) of the NC-IUBMB nomenclature.

Additional examples of 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).

In one embodiment of the invention, the 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.

The compositions of the invention may be used for the enzymatic hydrolysis of a polysaccharide. In a preferred embodiment, the polysaccharide is cellulose, hemicellulose or a mixture thereof. In general, 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.

Temperatures and conditions suitable for hydrolysing polysaccharides using the compositions of the invention are described above in the section entitled "Two-stage enzyme hydrolysis".

For example, a temperature suitable for the activity of mesophilic hydrolytic enzymes may be above about 10°C and below 60°C. Preferably, 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. Preferably, 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. Examples of preferred 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, α-glycosidic bonds, β-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.

Accordingly, 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. Preferably, the fragment will be of a size suitable for fermentation, for example, by a microorganism such as a yeast, fungus or bacterium.

Examples of 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.

Examples of 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.

Examples of 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

(e.g. threose and erythrose) and ketotetroses (e.g. erythrulose), pentoses including aldopentoses (e.g. lyxose, ribose, arabinose, deoxyribose and xylose) and ketopentoses

(e.g. xylulose and ribulose), 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.

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. In the context of the present specification, 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. In general, 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. For example, 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.

In certain embodiments, 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.

In a preferred embodiment, 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. In another preferred embodiment, 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.

C. shehatae), Schizosaccharomyces (e.g. S. pombe), Pachysolen (e.g. P. tannophilus), and

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. fermeyitum, L.sanfranciscensis), Oenococcus (e.g. O. oeni), Leuconostoc (e.g. L. mesenteriode) and Mycobacterium (e.g. M. smegmatis).

Examples of fungi suitable for the fermentation of saccharides to produce mannitol include, but are not limited to, Candida (e.g. C. zeylannoide, C. lipolitica), Cryptococcus

(e.g. C. neoformans), 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).

Methods for the fermentation of polysaccharide fragments to produce mannitol are described, for example in United States Patent No. 6528290 and PCT publication No. WO/2006/044608. 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. guilliermondiϊ), Pichia , and Debaryomyces (e.g. D. hansenii).

Methods for the fermentation of xylitol from saccharides are described, for example, in U.S. Patent No. 5081026, U.S. Patent No. 5686277, U.S. Patent No. 5998181 and U.S. Patent No. 6893849.

In preferred embodiments of the invention, 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. In general, 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. For example, 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. Recombinant microorganisms capable of converting pentoses and hexoses to ethanol are described, for example, in PCT Publication No. WO 95/13362 and United States Patent No. 5000000, United States Patent No. 5028539, United States Patent No. 5424202, United States Patent No. 5482846, and United States Patent No. 5514583.

Culture conditions for sugar-fermenting microorganisms are generally known in the art, and are described in, for example, Bonifacino et al., (Eds) Current Protocols in Cell Biology (2007) John Wiley and Sons, Inc., and Coico et al., (Eds) Current Protocols in Microbiology (2007) John Wiley and Sons, Inc. Generally, 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. For example, 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). 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.

Examples

Example 1: Synthesis of genes encoding hydrolytic enzymes

The gene encoding the 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). The genes encoding the following thermophilic hydrolytic enzymes were used for hydrolysis experiments with their wild type codon usages intact: the β-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. Genet., 213: 84-92), the Cellulase/Cellobiohydrolase gene (celA) (see GenBank accession number L32742), Arabinofuranosidase gene (pcynF) (see GenBank accession number AF005383), Xylanase genes (xynE and xynl) (see GenBank accession numbers AF005383 and AF005382), and Xylosidase gene (xynD) (see GenBank accession number AF005383) from Cs. saccharolyticus, Cellulase genes (celEl/2 and celECterm) (see GenBank accession number AF078042) from Caldicellulosiruptor Tok7B.l (see also Gibbs et al., "Multidomain and multifunctional glycosyl hydrolases from the extreme thermophile

Caldicellulosiruptor isolate Tok7B.l ", (2000), Curr. Microbiol. 40: 333-340), and the 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.

Plasmids used for gene amplification/expression of 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. 185: 60-89), or pUC18/19 (see Yanish-Perron et al., "Improved Ml 3 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 andpUCW vectors ", 1985, Gene 33: 103-119).

Typically, 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. 5 The E. coli strains DH5α, and/or Topi OF' supplied by Invitrogen

(www.invitrogen.com/site/us/en/home.html, catalogue numbers 18258-012 and C3030- 03) were used as hosts for plasmid amplification and expression of gene products.

Following overnight incubations at 14°C, the ligation mixtures were introduced into competent E. coli host cells using the heat-shock method described by Inoue et al., "High0 efficiency transformation of Escherichia coli with plasmids", (1990), Gene, 96: 23-28. Transformed E. coli host cells were propagated on L-agar plates containing Ampicillin at 100 μg/mL.

Example 3: Expression of synthetic genes in T. reesei

A polynucleotide encoding the synthetic 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. reesei Rut-C30 using the protoplast transformation technique (as described in Penttila et al., "A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei", (1987), Gene, 61:155-164) or the biolistic bombardment delivery method (as described in Te'o et al., "Biolistic transformation of Trichoderma reesei using the Bio-Rad seven barrels Hepta Adaptor system ", (2003), J. Microbiol. Methods 51 :393-399).

Following transformation and depending on the selection marker used, cells were plated out onto potato dextrose agar (PDA) plates containing the antibiotic Hygromycin B (60 U/mL) or the antibiotic Phleomycin (90 U/ mL), or plated out onto minimal-medium based agar plates containing acetamide as a sole nitrogen source.

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.

Fermentation cultivations (IL - 10L) were performed using the same inducing medium as for 50 mL shake flasks. Typical batch fermentations were run for 6-7 days.

Example 4: Production and isolation of hydrolytic enzymes

Production of 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 ODβoo reached between 0.8-1.0. 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 β-D-1- thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM.

Following induction, cells were collected at -20,000 g for 30 minutes and the supernatant discarded. The cell pellets were resuspended in 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.

For production of hydrolytic enzymes from T. reesei (Rut-C30) and T. reesei-XyήB 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. Following a seven day cultivation at 28°C in the liquid medium, 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 dθ 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.

Example 5: Substrate preparation for enzyme-substrate hydrolysis

Separate enzyme hydrolysis assays were performed using three individual substrates: supercritical ethanol-treated P. radiata sawdust; liquor fraction containing hemicellulose from high temperature (195°C/5min)/pressure (1-2 /wι)-treated P. radiata sawdust; and P. radiata kraft pulp. Of these substrates, the liquor fraction containing hemicellulose from the high temperature/high pressure-treated P. radiata sawdust was soluble while each of the other two substrates was insoluble.

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.

Example 6: Two-stage enzyme hydrolysis assays

Separate two-stage enzyme hydrolysis assays were performed on the individual substrates referred to in Example 5 above. Hydrolytic enzyme concentrations used in the assays were in the range of -5-10 %

(v/v). 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

The release of hydrolysis products as reducing sugars was assayed as described in Bailey et al. "Inter laboratory testing of methods for assay ofxylanase activity", (1992), J. Biotechnol. 23: 257-270, using the appropriate sugars as standards with the following minor modifications. Briefly, 10-100 μL of sample were removed from tubes and added to 500-800 μL of the dinitrosalicyclic acid (DNS) and boiled for 5 minutes, collected, and the absorbance of a 50-100 μL portion of the resulting solution was measured at X _540. The mono and oligosaccharides released during hydrolysis were analysed using an ion chromatography-based separation by HPLC.

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,

ManA and BgIA.

Hemicellulose liquor sample (900 μl) prepared as indicated in Example 5 above was added to four tubes.

Hydrolytic enzymes were applied to each tube as follows:

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

BgIA.

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

Figure 1. The background absorbance values from reducing sugars in the substrate-only controls were subtracted from values obtained from substrate-enzyme samples before plotting the data.

Based on the results shown in Figure 1, an increase of- 17-22% in colour reaction, indicative of reducing sugars, was obtained from the T. reesej-XynB transformant and T. reesez-thermophilic enzyme combinations using the two-stage process (Figure 1, samples

2 and 4), when compared to T. reesei only (Figure 1, sample 1), and the thermophilic enzyme cocktail (Figure 1, sample 3). Without being bound to a particular mode of action, it is though that the higher increases in reducing sugars in samples 2 and 4

(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.

However, the expected total cellulase enzymatic activities from T. reesei hydrolytic enzymes is not apparent here (especially from RutC-30 only, tube 1) due to the low level of cellulose present in the hemicellulose soluble substrate.

Example 9: Two-stage enzyme hydrolysis assays using supercritical ethanol-treated P. radiata sawdust

A total of 100 mg of supercritical ethanol-treated P. radiata sawdust substrate was used per tube. About 2.5 mL of T. reesei-XynB culture supernatant/enzyme sample containing up to 500μg 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 500μg of total protein (was added to each of two tubes containing lOOmg of substrate (tubes 2 and 3).

Two additional samples were used as controls (tubes 1 and 4). 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 μL) 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.

Mono and oligosaccharides produced during the hydrolysis from enzyme-substrate and enzyme-only samples of supercritical ethanol-treated P. radiata sawdust were also analysed using an ion chromatography-based HPLC separation (see Example 7). HPLC analysis was not performed on the substrate only control, as the background reducing sugar level appeared low (Figure 2, sample 1). The results are summarised in Table 1 which shows a comparison of results obtained from glucose and xylose hydrolysis.

As shown in Table 1, relatively high amount of monosaccharides (eg. Glucose) is already present in the enzyme sample produced during a T.reesei-XynB transformant batch fermentation described in Example 4. However, a 1.28-fold increase in the amount of glucose following enzymatic hydrolysis was achieved from incubation of enzyme- substrate at 50 ⁰ C in comparison to the enzyme/substrate only control sample. A 1.34-fold increase in glucose hydrolysis product was obtained from the two-stage incubation of enzyme-substrate at 50 ⁰ C and 70 ⁰ C in comparison to the enzyme/substrate only control sample.

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.

Without being bound to a particlur mode of action, 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.

One feature of XynB, different to typical endo-acting xylanases is that it appears to be very effective on hydrolysing xylo-oligosccharides, but also appears to favour attacking longer oligosaccharides resulting in high levels of dimers, etc. These observations on xylo-oligosaccharide hydrolysis products by XynB were further supported in these HPLC analyses, whereby a clear increase in oligosaccharide production was observed in the enzyme-substrate samples following the 7O ⁰ C incubation when compared to 50°C incubation (Figure 5), contributing to the higher reducing sugar release observed during the DNS-based assay (Figure 2, sample 3, 70°C). In particular, 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. 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). Sample four: substrate only (no enzyme). Sample five: substrate only (no enzyme). Kraft pulp samples were used in the assay at 2.5% (w/v).

Samples one and four were incubated at 50°C for 36 hours.

Samples two and three were incubated at 70°C for 36 hrs

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.

Following hydrolysis, the appearances of the lysates were examined (photographic results not shown). The P. radiata kraft pulp appeared to be solubilised in samples containing T. reesei enzymes only (sample one) and T. reeseMhermophilic enzyme mixture combination (sample 3). There was no obvious change in the appearances of the substrates in samples containing thermophilic enzymes mixture only (sample 2) and in samples containing substrate-only controls (samples 4 and 5) except that the substrates appeared swollen, possibly due to the combination of H ₂ O and temperature.

The hydrolysates were analysed for the release of sugars as described in Example 7.

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.

Moreover, under these conditions an improvement of up to a 38% increase in the colour reaction, indicative of reducing sugars, was generated from the combined T.

reesez-thermophilic enzymes mixture (Figure 3, sample 3) when compared to the T. reesei enzymes only sample (Figure 3, sample 1).

No obvious change above the background (Figure 3, sample 5) was detected in the thermophilic enzymes mixture only sample (Figure 3, sample 2). For reproducibility, another enzyme hydrolysis experiment was performed on the P. radiata kraft pulp substrate and hydrolysed with different concentrations of hydrolytic enzymes. 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, CeIE-

Cterm, CeIA, ManA, XynE, XynF, XynD, Xynl and BgIA (5% v/v).

Sample three: endogenous mesophilic enzymes expressed by T. reesei Rut-C30 (10% v/v).

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).

Sample five: 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).

Sample seven: substrate only (no enzyme).

Sample eight: substrate only (no enzyme).

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.

It appeared that the combination of T. reesei and thermophilic hydrolytic enzymes (Figure 4a, samples 4 and 6) using the two-stage enzyme hydrolysis process still produced higher amounts of reducing ends indicative of reducing sugars, when compared

to using T. reesei (Figure 4a, samples 1 and 3) and the thermophilic hydrolytic enzymes (Figure 4a, samples 2 and 5) separately.

Even with a higher loading of thermophilic enzymes (Figure 4a, sample 5 compared to sample 2) no obvious change above background (substrate only: Figure 4a, sample 8) was evident, indicating that under these conditions the thermophilic enzyme mixture did not hydrolyze the kraft pulp.

An increase of up to 14-24% was generated in samples containing both T. reesei and thermophilic enzymes (Figure 4a, samples 4 and 6) when compared to using T. reesei enzymes only (Figure 4a, samples 1 and 3). Up to 5% increase in reducing sugars was generated in the sample treated with both

T. reesei and thermophilic enzymes at a combined total enzyme concentration of 5% (v/v) (Figure 4a, sample 4) when compared to the sample treated with T. reesei enzymes only at a total enzyme concentration of 5% (v/v) (Figure 4a, sample 3). However, in this case 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. As shown in Figure 6, 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) Furthermore, 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).

Example 11: Discussion

On the basis of the results provided in the Examples above, higher yields of reducing sugars (according to the DNS-colorimetric method) were produced from hemicellulose liquor of supercritical-ethanol-treated Pinus radiata sawdust (soluble substrate), supercritical ethanol-treated P. radiata sawdust (insoluble substrate) or P. radiata kraft pulp (insoluble substrate) using a combination of endogenous mesophilic hydrolytic enzymes (expressed by T. reesei Rut-C30) and thermophilic hydrolytic enzymes in a two-stage enzyme hydrolysis process, compared to using the mesophilic hydrolytic enzymes or the thermophilic hydrolytic enzymes separately. HPLC analyses

also demonstrated increase amounts of sugars were released when using the combination of mesophilic and thermophilic enzymes in the two-stage enzyme hydrolysis process.

From a commercially viable outlook, 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. Some 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. The temperature and pH ranges utilised in the two-stage hydrolysis assays described herein favoured the mesophilic and thermophilic hydrolytic enzymes utilised but it is contemplated also that the two-stage process can be applied to other enzyme types.

Furthermore, while most of the 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.

Incorporation by reference

This application claims priority from Australian provisional application number 2008903814 filed on 25 July 2008, the entire contents of which are incorporated herein by reference.