Method for producing fructose

Abstract

A method for the enzymatic production of D-fructose from D-glucose in a one-pot synthesis, wherein D-glucose is oxidized enzymatically to D-glucosone and D-glucosone is reduced enzymatically to D-fructose and the use of the D-fructose produced in this way for the production of furan derivatives.

Claims

1. A method for producing D-fructose from D-glucose, comprising: a) enzymatically oxidizing D-glucose to D-glucosone in a reaction vessel, and b) enzymatically reducing the D-glucosone to D-fructose in reaction vessel, wherein a redox cofactor is used in step b) and the redox cofactor is recycled by a cofactor regeneration system comprising a redox enzyme wherein both enzymatic reaction of steps a) and b) are carried out in the reaction vessel and without the D-glucosone being isolated.

2. A method according to claim 1, wherein the enzymatic oxidation of D-glucose to D-glucosone is catalyzed by a pyranose-2-oxidase.

3. A method according to claim 2, wherein nascent H.sub.2O.sub.2 is removed with the aid of a catalase.

4. A method according to claim 1, wherein a xylose reductase is used for the enzymatic reduction of D-glucosone to D-fructose.

5. A method according to claim 1, wherein the redox cofactor comprises NAD(P)H/NAD(P).sup.+ and is used for the reduction of D-glucosone to D-fructose, and wherein the cofactor regeneration system includes consuming a cosubstrate.

6. A method according to any one of claims 1, 2, 3, 4, or 5, wherein the reactions proceed according to the following Reaction Scheme 2: ##STR00006## in which LkADH denotes an alcohol dehydrogenase which is NADP(H)-dependent.

7. A method according to claim 1, wherein the produced D-fructose is converted further into furan derivatives.

8. A method according to claim 1, wherein redox cofactor NAD(P)H is used.

9. A method according to claim 1, wherein redox cofactor NAD(P)H is used and step b) uses an enzyme that is NADP(H)-dependent.

10. A method according to claim 1, wherein the redox enzyme is an alcohol dehydrogenase.

11. A method according to claim 1, wherein the redox enzyme is an alcohol dehydrogenase from Lactobacillus kefir.

12. A method according to claim 5, wherein the cosubstrate is selected from an alcohol, lactic acid and salts thereof, pyruvic acid and salts thereof, oxygen, hydrogen and/or formic acid and salts thereof.

13. A method according to claim 1, wherein a redox enzyme regenerating the redox cofactor is different from an enzyme for enzymatically reducing D-glucosone to D-fructose.

14. A method for producing D-fructose from D-glucose, comprising: a) enzymatically oxidizing D-glucose to D-glucosone in a single reaction vessel, and b) without removing or isolating any materials involved in step a) from the single reaction vessel, enzymatically reducing the D-glucosone to D-fructose in the single reaction vessel.

Description

EXAMPLE 1

(1) Bioconversion of D-Glucose into D-Glucosone Via Pyranose Oxidase, Using Catalase for Removing the H.sub.2O.sub.2 Formed Thereby

(2) A 0.5 ml batch contains 2.5% (w/v) D-glucose and 1 U of pyranose-2-oxidase (Sigma Aldrich). For converting the H.sub.2O.sub.2 formed in this reaction, 50 U of catalase (Sigma Aldrich) is used which converts the nascent H.sub.2O.sub.2 into H.sub.2O+O.sub.2. The reaction is carried out in a Tris-HCl buffer (50 mM, pH 7.0) at 30 C. under continuous shaking (850 rpm). An open system is used in order to achieve a sufficient supply of oxygen. After 48 h, 99% of the D-glucose had been converted into D-glucosone.

EXAMPLE 2

(3) Bioconversion of D-Glucosone into D-Fructose Via Xylose Reductase, Using an Alcohol-Dehydrogenase Dependent Cofactor Regeneration System

(4) A 0.5 ml batch contains 2.5% (w/v) D-glucosone and 10 U of the recombinant xylose reductase from Candida tropicalis (overexpressed in E. coli BL21 (DE3)). For the regeneration of NADPH, 10 U of the recombinant alcohol dehydrogenase from Lactobacillus kefir (overexpressed in E. coli BL21 (DE3)) and initially 5% (w/v) 2-propanol are used. The reaction is carried out without addition of NADPH. The cofactor is provided by the cell extract of the E. coli BL21 (DE3) used for the expression of the xylose reductase and the alcohol dehydrogenase. The reaction is carried out in a Tris-HCl buffer (50 mM, pH 7.0) at 30 C. and under continuous shaking (850 rpm). An open system is used in order to enable the evaporation of acetone and to shift the reaction toward D-fructose. 2.5% (w/v) IPA is additionally dosed in after 6 h, 5% IPA (w/v) after 18 h and 2.5% (w/v) IPA after 24 h. After 48 h, 90% of the D-glucosone had been converted into D-fructose.

EXAMPLE 3

(5) Bioconversion of D-Glucose into D-Glucosone and Further into D-Fructose in a One-pot Synthesis (Two Consecutive Steps without the Intermediate being Isolated), Using an Alcohol-Dehydrogenase Dependent Cofactor Regeneration System

(6) A 0.5 ml batch contains 2.5% (w/v) D-glucose and 1 U of pyranose-2-oxidase (Sigma Aldrich). For converting the H.sub.2O.sub.2 formed in this reaction, 50 U of catalase is used which converts the nascent H.sub.2O.sub.2 into H.sub.2O+O.sub.2. The reaction is carried out in a Tris-HCl buffer (50 mM, pH 7.0) at 30 C. under continuous shaking (850 rpm). Furthermore, an open system is used in order to achieve a sufficient supply of oxygen. After 24 h, the reaction mixture is heated to 65 C. for 10 minutes in order to inactivate the enzymes. Subsequently, 10 U of the recombinant xylose reductase from Candida tropicalis (overexpressed in E. coli BL21 (DE3)) is added to the reaction mixture. For the regeneration of NADPH, 10 U of the recombinant alcohol dehydrogenase from Lactobacillus kefir (overexpressed in E. coli BL21 (DE3)) and initially 5% (w/v) 2-propanol are used. The reaction is carried out without addition of NADPH. The cofactor is provided by the cell extract of the E. coli BL21 (DE3) used for the expression of the recombinant xylose reductase and the recombinant alcohol dehydrogenase. The reaction is carried out at 30 C. and under continuous shaking (850 rpm). An open system is used in order to enable the evaporation of acetone and to shift the reaction toward D-fructose. 2.5% (w/v) IPA is additionally dosed in after 6 h, 5% (w/v) IPA after 18 h and 2.5% (w/v) IPA after 24 h. After 48 h, 91% of the employed D-glucose had been converted into D-fructose.