PROCESS FOR ENZYMATIC PRODUCTION OF OXIDATION AND REDUCTION PRODUCTS OF MIXED SUGARS

Abstract

The present invention provides a process for obtaining n+a oxidation and reduction products from a mixture of n sugars selected from the group consisting of C5 and C6 sugars, wherein n is at least 2 and a is at least 1, wherein at least two of the sugars in the mixture are present at a non-equimolar ratio to each other, wherein, in a first stage, at least one of the sugars which are present at a non-equimolar ratio to each other is oxidized enzymatically and, at the same time, at least one of the other sugars present at a non-equimolar ratio to each other is reduced enzymatically, and wherein, in the first stage, a portion of at least one of the sugars present at a non-equimolar ratio to each other is not converted, and which is characterized in that, in at least a second stage, at least a portion of the sugar not converted in the first stage is oxidized enzymatically by half and, respectively, is reduced enzymatically by the remaining half.

Claims

1. A process for obtaining n+a oxidation and reduction products from a mixture of n sugars selected from the group consisting of C5 and C6 sugars, wherein n is at least 2 and a is at least 1, wherein at least two of the sugars in the mixture are present at a non-equimolar ratio to each other, wherein, in a first stage, at least one of the sugars which are present at a non-equimolar ratio to each other is oxidized enzymatically and, at the same time, at least one of the other sugars present at a non-equimolar ratio to each other is reduced enzymatically, and wherein, in the first stage, a portion of at least one of the sugars present at a non-equimolar ratio to each other is not converted, characterized in that, in at least a second stage, at least a portion of the sugar not converted in the first stage is oxidized enzymatically by half and, respectively, is reduced enzymatically by the remaining half.

2. A process according to claim 1, characterized in that sugar acids and sugar acid lactones, respectively, are obtained as oxidation products, and sugar alcohols are obtained as reduction products.

3. A process according to claim 1, characterized in that the mixture of sugars contains xylose and arabinose, with xylose being present in excess.

4. A process according to claim 3, characterized in that arabinose is oxidized to arabonic acid or to arabonic acid lactone, respectively, and a portion of the xylose is reduced to xylitol in the first stage, and, in the second stage, the unreacted xylose is oxidized completely or partly to xylonic acid or to xylonolactone by half and, respectively, the remaining half is reduced to xylitol.

5. A process according to claim 3, characterized in that arabonic acid which has formed and/or xylonic acid which has formed is/are processed further into -ketoglutaric acid.

6. A process according to claim 3, characterized in that the mixture additionally contains glucose.

7. A process according to claim 1, characterized in that the mixture contains glucose in excess relative to the other existing sugar(s), and sorbitol is obtained at least partly from the glucose.

8. A process according to claim 1, characterized in that, at least in one of the two stages, preferably at least in the second stage, particularly preferably both in the first and in the second stage, at least one redox cofactor and at least one enzyme dependent on said redox cofactor are present in the reaction mixture.

9. A process according to claim 8, characterized in that the redox cofactor(s) is/are regenerated by enzymatic reactions proceeding in parallel.

10. A process according to claim 1, characterized in that the first and the second stage are performed in a one-pot reaction.

11. A process according to claim 10, characterized in that the two stages proceed at least partly simultaneously.

12. A process according to claim 1, comprising the removal of accumulating sugar acids from the mixture.

13. A process according to claim 1, characterized in that the mixture containing the sugars has been obtained from a hemicellulose-containing material.

14. A process according to claim 13, characterized in that the hemicellulose-containing material has been obtained by pulping a lignocellulosic material.

15. A process according to claim 14, characterized in that the lignocellulosic material is a material selected from straw and/or husks.

16. A process according to claim 14, characterized in that the lignocellulosic material has been obtained by pulping with an alcohol.

17. A process according to claim 9, characterized in that the redox cofactor(s) is/are regenerated by enzymatic reactions both in the first and in the second stage.

18. A process according to claim 15, characterized in that the lignocellulosic material is selected from the group consisting of wheat straw, bagasse, energy grasses, elephant grass, switch grass, and lemmas.

19. A process according to claim 16, characterized in that the lignocellulosic material has been obtained by pulping with a C.sub.1-4 alcohol, water and an alkali.

Description

EXAMPLES

Example 1: Xylanase Treatment for Obtaining a Sugar Mixture from Biomass

[0110] Delignified pulp produced from straw was used. A description of the preparation of the pulp can be found in WO 2010/124312 A2 (Example 1). 10 g (dry weight) of the pulp was resuspended with distilled water to a consistency of 10%, and a pH-value of 4.9 was adjusted with H.sub.2SO.sub.4. 1000 l of Xylanase Ecopulp TX800A (Ecopulp Finland Oy) was added, and incubation took place at 50 C. for 16 h. A 1.5% sugar solution (w/v) is obtained which contains mainly glucose, xylose and arabinose at a ratio of about 2:10:1.

[0111] The following examples 2 to 5 serve for illustrating the possibilities for a selective enzymatic oxidation or reduction, respectively, of sugars from a sugar mixture.

Example 2: Glucose Oxidation with a Glucose Dehydrogenase (NADH-oxidase for Cofactor Recycling)

[0112] 18.5 mg NaHCO.sub.3 was added to a sugar mixture (approx. 500 l) containing glucose, xylose and arabinose (sugar concentration: approx. 1%). Thereupon, 30 l glucose dehydrogenase (activity of approx. 300 U/ml), 10 l NADH-oxidase (activity of approx. 1140 U/ml) and 2.5 l NADH (concentration: 100 mM) were added. The mixture was incubated at 25 C. for approx. 17 hours. 86% of the glucose was reacted to gluconic acid. The obtained solution was passed through a strong ion exchanger (Amberlyst A-26(OH), Alfa Aesar). Thereby, the resulting gluconic acid was separated completely from the mixture.

Example 3: Arabinose Oxidation with an Arabinose Dehydrogenase (NADH-oxidase for Cofactor Recycling)

[0113] 6.2 mg NaHCO.sub.3 was added to a sugar mixture (approx. 500 l) containing glucose, xylose and arabinose (sugar concentration: approx. 1%). Thereupon, 30 l arabinose dehydrogenase (activity of approx. 300 U/ml), 20 l NADH-oxidase (activity of approx. 1140 U/ml) and 2.5 l NADH (concentration: 100 mM) were added. The mixture was incubated at 25 C. for approx. 17 hours. 100% of the arabinose was reacted to arabonic acid. The obtained solution was passed through a strong ion exchanger (Amberlyst A-26(OH), Alfa Aesar). Thereby, the resulting arabonic acid was separated completely from the sugar mixture.

Example 4: Arabinose Oxidation with an Arabinose Dehydrogenase (Xylose Reductase for Cofactor Recycling)

[0114] 16.9 mg NaHCO.sub.3 was added to a sugar mixture (approx. 500 l) containing xylose and arabinose (xylose: approx. 10%, arabinose: approx. 1%). Thereupon, 30 l arabinose dehydrogenase (activity of approx. 300 U/ml), 30 l xylose reductase (activity of approx. 103 U/ml) and 2.5 l NADH (concentration: 100 mM) were added. The mixture was incubated at 30 C. for approx. 20 minutes. 100% of the arabinose was reacted to arabonic acid. Thereby, 10% of the xylose contained in the mixture was converted into xylitol. The obtained solution was passed through a strong ion exchanger (Amberlyst A-26(OH), Alfa Aesar). Thereby, the resulting arabonic acid was separated completely from the sugar mixture.

Example 5: Arabinose Oxidation with an Arabinose Dehydrogenase (Xylose Reductase for Cofactor Recycling), Xylose Reduction with a Xylose Reductase (Alcohol Dehydrogenase for Cofactor Recycling)

[0115] A sugar solution obtained from biomass (=xylan hydrolysate) was concentrated to a sugar concentration of approx. 63 g/l D-xylose and 7 g/l L-arabinose by evaporation, and pH=8.0 was adjusted with NaOH. 2.5 ml of 500 mM Tris-HCl buffer, pH=8.0, 200 U xylose reductase and 160 U arabinose dehydrogenase were added to 80 ml of said solution. In a 200 ml round-bottom flask, the solution was stirred with a magnetic stirrer (200 rpm) at 35 C. (water bath) for 20 minutes. The arabinose had been converted completely, and the solution now contained approx. 56 g/l D-xylose, approx. 7 g/l xylitol and approx. 7 g/l L-arabino-1,4-lactone/L-arabonic acid.

[0116] In said example, the employed cofactor was present in the employed enzyme lysates already to a sufficient extent and did not have to be added separately.

Example 6: Conversion of a Mixture of Xylose and Arabinose (according to the Invention)

[0117] 16.9 mg NaHCO.sub.3 was added to a sugar mixture (approx. 500 l) containing xylose and arabinose (xylose: approx. 10%, arabinose: approx. 1%). Thereupon, 30 l arabinose dehydrogenase (activity of approx. 300 U/ml with arabinose; the enzyme also exhibits a certain activity as a xylose dehydrogenase), 30 l xylose reductase (activity of approx. 103 U/ml) and 2.5 l NADH (concentration: 100 mM) were added. The mixture was incubated at 30 C. for approx. 72 hours. 100% of the arabinose was reacted to arabonic acid, 45% of the xylose was reacted to xylonic acid, and 55% of the xylose was reacted to xylitol. The obtained solution was passed through a strong ion exchanger (Amberlyst A-26(OH), Alfa Aesar). Thereby, the arabonic acid and the xylonic acid which resulted were separated completely from the mixture.

[0118] This example demonstrates an at least partly parallel course of the first stage and the second stage of the process according to the invention: The first stage is the oxidation of the arabinose and the reduction of an equimolar part of the xylose. The second stage, which proceeds at least partly in parallel thereto, comprises the oxidation of half of the unreacted xylose by means of the activity of the arabinose dehydrogenase and the reduction of the other half of the remaining xylose.

Example 7: Conversion of a Mixture of Xylose and Glucose (According to the Invention)

[0119] The reaction batch included the following components: 364 l dH.sub.2O, 2.5 l NADPH-solution (100 mM), 10 l D-glucose solution (50% w/v), 100 l D-xylose solution (50% w/v), 5 l glucose dehydrogenase (300 U/ml, measured with glucose), 19 l xylose reductase (160 U/ml), and 5.6 mg CaCO.sub.3. The glucose dehydrogenase which is used also exhibits a certain xylose dehydrogenase activity. The reaction was gently agitated at 35 C., and samples were taken at different points in time. By means of GC/MS, the content of sugars as well as of reaction products was determined. After 1 h, the glucose had, for the most part, been converted into gluconic acid. Likewise, a small part of the employed xylose (approx. 10%) had been oxidized to xylonic acid by this point in time. Stoichiometrically to the formed sugar acids, xylose had been reduced to xylitol after 1 h. Approximate composition of the reaction after 1 h: 10 mg/ml gluconic acid, 10 mg/ml xylonic acid, 20 mg/ml xylitol, 70 mg/ml xylose.

[0120] After 6 h, the xylose had been converted by approx. 90%. In comparison to the point in time after 1 h, the products xylonic acid and xylitol were thereby formed stoichiometrically. Approximate composition of the reaction after 6 h: 10 mg/ml gluconic acid, 40 mg/ml xylonic acid, 50 mg/ml xylitol, 10 mg/ml xylose.

Example 8: Analysis of the Reactions by Means of GC/MS

[0121] For analyzing the oxidation reactions on the GC/MS, substrates and products were derivatized. For this purpose, 4 l of the samples were transferred into a glass vial and dried in the Speedvac. For derivatization, 150 l pyridine and 50 l of a 99:1-mixture of N,O-bis(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane were then added. Derivatization took place at 60 C. for 16 h. Subsequently, the samples were analyzed by GC-MS. In doing so, the samples were separated via the separation column HP-5 ms (5% phenyl)methylpolysiloxane in a gas chromatograph and analyzed in the mass spectrometer GCMS QP2010 Plus of Shimadzu.

Non-Patent Literature

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