CELLULOLYTIC COMPOSITIONS COMPRISING MONOOXYGENASE POLYSACCHARIDE ENZYMES WITH IMPROVED ACTIVITY

Abstract

The invention refers to methods and compositions for stabilizing and increasing the activity of enzymatic mixtures comprising GH61 (PMO or polysaccharide monooxigenase) polypeptides used for the degradation of cellulosic material during the saccharification step of biofuel production processes. This improvement is achieved by the addition of a nickel cation to said enzymatic mixtures before and/or during the saccharification step. Thus, the invention provides compositions comprising PMOs, cellulolytic enzymes and a nickel cation, as well as methods for preparing said compositions and methods for producing fermentable sugars and bioproducts, preferably bioethanol, from cellulosic biomass in which said compositions are used.

Claims

1. A composition comprising at least one polysaccharide monooxygenase enzyme and a nickel cation.

2. The composition according to claim 1 wherein the nickel cation is present at a concentration of more than 0.0001 mM and less than 50 mM, preferably between 0.001 and 20 mM, more preferably between 0.001 and 5 mM, even more preferably between 0.05 and 5 mM, even more preferably between 0.05 and 0.5 mM and even more preferably between 0.075 and 0.125 mM.

3. The composition according to any of claim 1 or 2 wherein the nickel cation is in the form of a salt.

4. The composition according to claim 3 wherein the nickel salt is selected from nickel sulphate, nickel chloride, nickel nitrate, nickel acetate or nickel hydroxide, or any combination thereof.

5. The composition according to any of claims 1 to 4 wherein the polysaccharide monooxygenase enzyme is selected from PMO1, PMO2, PMO3 or any combination thereof.

6. The composition according to any of claims 1 to 5 wherein the composition further comprises cellulolytic enzymes.

7. The composition according to claim 6 wherein the cellulolytic enzymes are selected from endoglucanase, beta-glucosidase, cellobiohydrolase, beta-xylosidase, xyloglucanase, xylanase, arabinofuranosidase or any combination thereof.

8. The composition according to any of claims 1 to 7 wherein the polysaccharide monooxygenase enzyme and the cellulolytic enzymes are an enzymatic mixture secreted by Myceliophthora thermophila.

9. Use of the composition according to any of claims 1 to 8 for the degradation of cellulosic biomass.

10. Use according to claim 9 wherein the degradation of cellulosic biomass takes place in a bioproduct production process.

11. Use according to claim 10 wherein the bioproduct is a biofuel.

12. Use according to claim 11 wherein the biofuel is bioethanol or butanol.

13. A process for producing fermentable sugars comprising: a. Incubating cellulosic biomass with the composition according to any of claims 1 to 8, and b. Recovering the fermentable sugars obtained after the incubation of step (a).

14. A process for producing fermentable sugars comprising: a. Incubating cellulosic biomass with an enzymatic mixture comprising cellulolytic enzymes and at least one polysaccharide monooxygenase enzyme, b. Adding a nickel cation to the incubation of step (a), and c. Recovering the fermentable sugars obtained.

15. The process according to claim 14 wherein the polysaccharide monooxygenase enzyme is selected from PMO1, PMO2, PMO3 or any combination thereof.

16. The process according to any of claim 14 or 15 wherein the cellulolytic enzymes are selected from endoglucanase, beta-glucosidase, cellobiohydrolase, beta-xylosidase, xyloglucanase, xylanase, arabinofuranosidase or any combination thereof.

17. The process according to any of claims 14 to 16 wherein the enzymatic mixture used in step (a) is an enzymatic mixture secreted by Myceliophthora thermophila.

18. The process according to any of claims 14 to 17 wherein the nickel cation is added in step (b) at a concentration of more than 0.0001 mM and less than 50 mM, preferably between 0.001 and 20 mM, more preferably between 0.001 and 5 mM, even more preferably between 0.05 and 5 mM, even more preferably between 0.05 and 0.5 mM and even more preferably between 0.075 and 0.125 mM.

19. The process according to any of claims 14 to 18 wherein the nickel cation is added in step (b) in the form of a salt selected from nickel sulphate, nickel chloride, nickel nitrate, nickel acetate or nickel hydroxide, or any combination thereof.

20. A process for producing a bioproduct from cellulosic biomass comprising: a. Incubating cellulosic biomass with the composition according to any of claims 1 to 8, b. Fermenting the fermentable sugars obtained after the incubation of step (a) with at least one fermenting microorganism, and c. Recovering the bioproduct obtained after the fermentation step (b).

21. A process for producing a bioproduct from cellulosic biomass comprising: a. Incubating cellulosic biomass with an enzymatic mixture comprising cellulolytic enzymes and at least one polysaccharide monooxygenase enzyme, b. Adding a nickel cation to the incubation of step (a), c. Fermenting the fermentable sugars obtained with at least one fermenting microorganism, and d. Recovering the bioproduct obtained after the fermentation step (c).

22. The process according to claim 21 wherein the polysaccharide monooxygenase enzyme is selected from PMO1, PMO2, PMO3 or any combination thereof.

23. The process according to any of claim 21 or 22 wherein the cellulolytic enzymes are selected from endoglucanase, beta-glucosidase, cellobiohydrolase, beta-xylosidase, xyloglucanase, xylanase, arabinofuranosidase or any combination thereof.

24. The process according to any of claims 21 to 23 wherein the enzymatic mixture used in step (a) is an enzymatic mixture secreted by Myceliophthora thermophila.

25. The process according to any of claims 21 to 24 wherein the nickel cation is added in step (b) at a concentration of more than 0.0001 mM and less than 50 mM, preferably between 0.001 and 20 mM, more preferably between 0.001 and 5 mM, even more preferably between 0.05 and 5 mM, even more preferably between 0.05 and 0.5 mM and even more preferably between 0.075 and 0.125 mM.

26. The process according to any of claims 21 to 25 wherein the nickel cation is added in step (b) in the form of a salt selected from nickel sulphate, nickel chloride, nickel nitrate, nickel acetate or nickel hydroxide, or any combination thereof.

27. The process according to any of claims 20 to 26 wherein the bioproduct is biofuel.

28. The process according to claim 27 wherein the biofuel is bioethanol or butanol.

29. A process for the preparation of the composition according to any of claims 1 to 8 comprising adding a nickel cation to an enzymatic mixture comprising at least one polysaccharide monooxygenase enzyme.

30. The process according to claim 29 wherein the nickel cation is added at a concentration of more than 0.0001 mM and less than 50 mM, preferably between 0.001 and 20 mM, more preferably between 0.001 and 5 mM, even more preferably between 0.05 and 5 mM, even more preferably between 0.05 and 0.5 mM and even more preferably between 0.075 and 0.125 mM.

31. The process according to any of claim 29 or 30 wherein the nickel cation is added in the form of a salt selected from nickel sulphate, nickel chloride, nickel nitrate, nickel acetate or nickel hydroxide, or any combination thereof.

32. The process according to any of claims 29 to 31 wherein the polysaccharide monooxygenase enzyme is selected from PMO1, PMO2, PMO3 or any combination thereof.

33. The process according to any of claims 29 to 32 wherein the enzymatic mixture further comprises cellulolytic enzymes selected from endoglucanase, beta-glucosidase, cellobiohydrolase, beta-xylosidase, xyloglucanase, xylanase, arabinofuranosidase or any combination thereof.

34. The process according to any of claims 29 to 33 wherein the enzymatic mixture is an enzymatic mixture secreted by Myceliophthora thermophila.

Description

DESCRIPTION OF THE FIGURES

[0092] FIG. 1. Improvement of the yield of glucose (g/kg) by the enzymatic cocktail produced by M. thermophila C1 in the absence (0) or in the presence of different nickel concentrations (mM) on pretreated corn stover (PCS).

[0093] FIG. 2. Improvement of the yield of glucose and xylose (g/kg) released from pretreated corn stover biomass by the enzymatic cocktail produced by M. thermophila C1 in the absence (Control) or in the presence of different nickel salts.

[0094] FIG. 3. Improvement in the yield of glucose (g/kg) over biomass by a defined enzyme composition including endoglucanase, beta-glucosidase, cellobiohydrolases (Cellulase Mix) in the absence or in the presence of polysaccharide monooxygenases (PMO1 and/or PMO2) and in the absence or in the presence of nickel.

[0095] FIG. 4. Stability of several cellulolytic enzymes in the presence of different nickel concentrations (0). This figure represents the Tm values ( C.) at which 50% of the enzyme is denaturalizated. As the nickel concentration increases, PMOs become more stable as shown by the Tm increase indicating that a higher temperature is required to denature the PMO. Thermostability of other celulolytic enzymes is not affected by nickel addition.

[0096] FIG. 5. Improvement in the yield of glucose (g/kg) by the enzymatic cocktail produced by M. thermophila C1 in the absence (0) or in the presence of different concentrations (mM) of different divalent cations over biomass.

EXAMPLES

Example 1. Effect of Nickel Ion Concentration on Glucose Yield Released by C1 Enzyme Composition

[0097] The effect of nickel (II) ions on the saccharification performance of cellulase preparation from C1 on pretreated corn stover (hereinafter PCS) was evaluated according to the procedures described below. The cellulase preparation is designated hereinafter as the C1 composition.

[0098] The enzymatic mixture, C1 composition, produced by Myceliophthora thermophila C1 was obtained following the procedures previously described (Verdoes et al., 2007, Ind. Biotechnol. 3 (1) and Visser et al., 2011, Ind. Biotechnol., 7 (3)), using an industrial platform for enzyme production based on M. thermophila C1 developed by Dyadic Netherlands.

[0099] PCS obtained according to Nguyen et al. (1998, Appl. Biochem. Biotechnol. 70-72) was used as substrate for the hydrolysis reaction. The compositional analysis was performed using the procedures of NREL as Standard Biomass AnalyticalProcedures. This biomass was neutralized, lyophilized and milled.

[0100] C1 cellulase composition of Myceliophthora thermophila was used as the cellulase preparation. The hydrolysis of PCS was conducted in 10 ml plastic tubes with a reaction volume of 3.0 ml at 20% total solids adding 10 mg of protein per gram of glucan at 0-50 mM Nickel sulphate hexahydrated. Tubes were mixed and incubated at 50 C., pH 5, 250 rpm for 72 h. All experiments were performed at least in duplicate.

[0101] After hydrolysis, samples were filtered using a 0.22 m nylon filter and filtrates were analyzed for sugar content as described below. The sugar concentrations of samples, diluted to appropriate concentrations in 5 mM H.sub.2SO.sub.4, were measured using a 4.6250 mm AMINEX HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, Calif., USA) by elution with 5 mM H.sub.2SO.sub.4 at a flow rate of 0.6 ml per minute, and quantitated by integration of the glucose, cellobiose and xylose signals from refractive index detection (CHEMSTATION, AGILENT 1100 HPLC, Agilent Technologies, Santa Clara, Calif., USA) calibrated by pure sugar samples.

[0102] FIG. 1 shows the effect of different concentrations of nickel (mM) on glucose release (g/Km). It can be seen that the optimum nickel concentration was 0.125 mM (125 M).

Example 2. Comparison of Different Nickel Salts on Glucose Release

[0103] Different nickel salts were compared following the same procedure described in the example 1. Nickel was added as nickel sulphate hexahydrated, nickel chloride, nickel acetate tetrahydrated, nickel nitrate hexahydrated and nickel hydroxide to a final concentration of nickel ion of 125 M.

[0104] FIG. 2 shows the effect of nickel salts supplementation on glucose and xylose release. The supplementation of Nickel improved glucose release but xylose yield was not significantly affected. All nickel salts used produced the same glucose release improvement. The same effect was also obtained with nickel acetate tetahydrated (data not shown).

Example 3. Evaluation of Nickel Supplementation on Polysaccharide Monooxygenase Activity

[0105] The effect of nickel supplementation on several cellulolytic enzymes was compared following the same procedure described in the example 1. Here C1 composition was replaced by an enzymatic mixture containing the main celulases, where an endoglucanase, a beta-glucosidase, two kind of cellobiohydrolases (Type I and II) and two examples of polysaccharide monooxygenases, all of them obtained from Myceliophthora thermophila, were included in the cellulase preparation. The final dosage of the enzymatic mix was 8.5 mg of protein per gram of glucan.

[0106] The FIG. 3 shows the comparison of four defined compositions: (1) defined composition with endoglucanase, beta-glucosidase, both cellobiohydrolases and without polysaccharide monooxygenases (cellulase mix), (2) defined composition with cellulase mix plus PMO1, (3) cellulase mix supplemented with PMO2 and (4) cellulase mix supplemented with PMO1 and PMO2.

[0107] Supplementation of nickel at 125 M improved all defined compositions that contained polysaccharide monooxygenases (PMO1 or/and PMO2) but did not improve the defined composition without polysaccharide monooxygenases.

Example 4. Evaluation of Nickel Supplementation on Polysacharide Monooxygenase Stability

[0108] Stability of different PMOs was evaluated with thermo-fluorescence assay on Na-acetate buffer 200 mM, pH 5.0 and different concentrations of nickel. Experimental conditions were a lineal gradient of temperature 23-95 C. (0.8 C./min). Detection signal was measured with fluorescence of SYPRO orange protein gel stain (Sigma-Aldrich, St. Louis, Mo., USA) with and without nickel at different concentrations (0-200 M) added as nickel sulphate heptahydrated. Tm represents temperature values ( C.) at which 50% of the enzyme is denatured. PMO1 and PMO2 were obtained Myceliophthora thermophila while PMO3 was obtained from Penicilium sp.

[0109] As a general procedure to purify those PMOs, fungal cultures were centrifuged (21.000g, 40 min, 5 C.) to obtain cellulase enriched supernatants that were applied on a HiLoad 26/10 Q-Sepharose High Performance (53 ml) column pre-equilibrated with 50 mM Tris-HCl buffer, pH 7.0. After washing with the same buffer the bound protein was eluted with a 0-0.5 M NaCl gradient with a flow rate of 8 ml/min. PMOs enriched fractions were collected and loaded into a HiLoad 26/10 Phenyl-Sepharose High Performance column (53 ml) pre-equilibrated with 100 mM Na-Phosphate buffer, pH 7.0, 1M (NH.sub.4).sub.250.sub.4. The protein was eluted with a linear gradient of 100 mM Na-Phosphate buffer, pH 7.0 at a flow rate of 8 ml/min. Enriched fractions could also need an extra purification step with a HiPrep 26/10 desalting column equilibrated with 50 mM Na-Phosphate buffer, pH 7.0 or even a HiLoad 16/600 Superdex 75 pg (120 ml), this column was equilibrated with 50 mM Na-Phosphate buffer, pH 7.0.

[0110] FIG. 4 shows that average Tm of PMOs increased about 5 C. when Ni concetration was added. This indicates that as the nickel concentration increases, PMOs become more stable as shown by the Tm increase, indicating that a higher temperature is required to denature the PMO when nickel is present. This figure also evidences that thermostability of other celulolytic enzymes is not affected by nickel addition.

Example 5. Comparison of Nickel and Other Divalent Salts on the Saccharification of Pretreated Corn Stover

[0111] Nickel supplementation was compared with the supplementation of other divalent ions like magnesium or manganese at different concentrations following the same procedure as described in the example 1.

[0112] Nickel was added as nickel acetate tetrahydrated, magnesium as magnesium sulphate heptahydrated and manganese sulphate monohydrated.

[0113] The supplementation of nickel enhanced the glucose release (g/Kg) more than other divalent ions (FIG. 5).