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EP-4739785-A2 - PROCESS FOR THE PRODUCTION OF AN AQUEOUS SOLUTION CONTAINING ALLOSE

EP4739785A2EP 4739785 A2EP4739785 A2EP 4739785A2EP-4739785-A2

Abstract

A process for producing an aqueous solution containing allose by forming D-psicose in vitro from D-fructose, which is dissolved in an aqueous solution, by treating the dissolved D-fructose with an epimerase, whereupon the D-psicose is reduced in vitro to allitol by treating the D-psicose with an NAD(P)H-dependent oxidoreductase, resulting in the formation of oxidized cofactor NAD(P) + , and then the allitol is enzymatically oxidized to obtain allose.

Inventors

  • STAUNIG, Nicole

Assignees

  • Annikki GmbH

Dates

Publication Date
20260513
Application Date
20240708

Claims (15)

  1. 1. Process for the preparation of an aqueous solution containing allose, in which D-psicose is formed from D-fructose, which is dissolved in an aqueous solution, by treatment with an epimerase in vitro, after which the D-psicose is reduced to allitol by treatment with an NAD(P)H-dependent oxidoreductase in vitro to form oxidized cofactor NAD(P) + , after which the allitol is enzymatically oxidized to allose.
  2. 2. Process according to claim 1, characterized in that the epimerase and the NAD(P)H-dependent oxidoreductase are deactivated or removed from the aqueous solution by ultrafiltration before the allitol is enzymatically oxidized to allose.
  3. 3. Process according to one of claims 1 or 2, characterized in that the oxidized cofactor NAD(P) + formed by the reduction is reduced by means of an alcohol dehydrogenase and a secondary alcohol to form a ketone.
  4. 4. Process according to claim 3, characterized in that the secondary alcohol is 2-propanol.
  5. 5. Process according to one of claims 1 to 4, characterized in that it is carried out as a one-pot reaction without isolation of intermediate products.
  6. 6. Process for preparing an aqueous solution containing allose by oxidizing allitol, characterized in that the oxidation is carried out enzymatically.
  7. 7. Process according to one of claims 1 to 6, characterized in that the enzymatic oxidation of allitol to allose is carried out with a corresponding NAD(P) + -dependent oxidoreductase with formation of reduced cofactor NAD(P)H.
  8. 8. Method according to one of claims 1 to 7, characterized in that the enzymes are present as a lysate of the corresponding cells producing them.
  9. 9. The method according to claim 7, characterized in that the reduced cofactor NAD(P)H formed by the oxidation is oxidized to NAD(P) + by means of an NAD(P)H oxidase and oxygen.
  10. 10. The method according to any one of claims 1 to 9, characterized in that the enzymatic oxidation of the allitol to allose is carried out with an NAD(P) + -dependent oxidoreductase which comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence which has an identity to SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17 or SEQ ID No. 23 of at least 75%, ii) an amino acid sequence which is encoded by a nucleic acid which has an identity to SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 24 of at least 75%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 24.
  11. 11. The method according to claim 3 or 4, characterized in that the alcohol dehydrogenase for reducing the cofactor NAD(P) + comprises or consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 19 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 20 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 20.
  12. 12. The method according to claim 9, characterized in that the NAD(P)H oxidase for oxidizing NAD(P)H to NAD(P) + comprises or consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 21 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 22 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 22.
  13. 13. Use of a NAD(P) + -dependent oxidoreductase comprising an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17 or SEQ ID No. 23 of at least 75%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18 or SEQ ID No. 24 of at least 75%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18 or SEQ ID No. 24, for the oxidation of allitol to allose.
  14. 14. Use of an alcohol dehydrogenase for the reduction of NAD(P) + to NAD(P)H, which comprises or consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 19 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID No. 20, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 20.
  15. 15. Use of a HjO-forming NAD(P)H oxidase for the oxidation of NAD(P)H to NAD(P) + which comprises or consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 21 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 22 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 22.

Description

Process for the preparation of an aqueous solution containing allose The present invention relates to a process for producing an aqueous solution of the rare monosaccharide allose. Background of the invention p-Allose The aldohexose D-allose is the C3 epimer of D-glucose and is a so-called rare sugar due to its low natural occurrence. It can be found, for example, in certain seagrasses (Kannan et al., 2012) or as a component of glycosides in the plant Protect rubropilosa (sugar bush) native to South Africa (Perold et al., 1973). D-allose is a sweet-tasting sugar (80% sweetness compared to sucrose), but unlike sucrose, it has hardly any calories (Mooradian et al., 2017). In addition, D-allose has a wealth of other positive properties such as antitumor (e.g. in bladder cancer (Tohi et al., 2022)), antioxidant (Ishihara et al., 2011) or anti-inflammatory (Gao et al., 2011) effects. Further examples and other physiological effects of D-allose are given in review articles (Chen et al., 2018; Lim & Oh, 2011; Mijailovic et al., 2021). Since the demand for D-allose cannot be covered by natural resources due to the wide range of possible applications, synthetic methods (chemical or biocatalytic) have been and are being developed to produce D-allose starting from more common (and therefore cheaper) monosaccharides such as D-fructose or D-glucose. US 9109266 B2 describes the conversion of D-fructose to D-allose using NaOH or strongly basic ion exchange resins (according to the mechanism of the Lobry-de-Bruyn-Alberda-van-Ekenstein rearrangement), which, however, leads to a large number of by-products such as D-glucose, D-mannose, D-altose and D-psicose, which must be separated. US 5433793 discloses the ammonium molybdate-catalyzed isomerization of D-glucose to D-allose, which is carried out at 130 °C in an acetic acid aqueous medium. This allows approximately 10 weight percent of D-allose (based on the dry mass) to be obtained, but isolation of the product requires activated carbon filtration, ion exchange and simulated moving bed chromatography. Jumde et al. (2016) presented a chemical synthesis route starting from D-glucose, which involves a palladium-catalyzed regioselective oxidation at the C3 position and a stereoselective reduction to D-allose using borohydride-based reagents. WO 1997/042339 A1 describes a process for converting sucrose to D-allose and the corresponding sugar alcohol allitol. By fermentation with Agrobacterium tumefaciens, sucrose is oxidized to 3-ketosucrose, which is converted to allosucrose by hydrogenation (on Raney nickel). The cleavage of allosucrose can be carried out using acid catalysis (cation exchanger) or enzymatically using invertase or ß-fructosidase. The separation of the D-allose/D-fructose mixture can be carried out either by removing the D-fructose by yeast fermentation or by chromatographic methods (ion exchanger). Allitol can be produced by hydrogenation of the D-allose (on Raney nickel). Chemical processes such as those mentioned above are generally characterized by low conversions, complex reaction steps with poor atom economy and/or formation of by-products and are therefore completely unsuitable for the production of D-allose on an industrial scale. The inefficient chemical synthesis routes have now been replaced by more efficient biocatalytic processes. Starting from D-glucose, three enzymatic steps lead to D-allose. D-glucose is first isomerized with a glucose/xylose isomerase (Gl/Xl) to D-fructose (Nam, 2022), which is epimerized with a 3-ketose epimerase (D-tagatose-3-epimerase (DTE), D-psicose/allulose-3-epimerase (DPE/DAE) or L-ribulose-3-epimerase (LRE)) to D-psicose, also known as D-allulose (Zhang et al., 2016; Jiang et al., 2020). The final step (D-psicose D-allose) is accomplished by the action of various isomerases - L-rhamnose isomerase (L-RI, EC 5.3.1.14), D-ribose-5-phosphate isomerase (RPI, EC 5.3.1.6), D-galactose-6-phosphate isomerase (GaPI, EC 5.3.1.26) and D-glucose-6-phosphate isomerase (Gl PI, EC 5.3.1.9) (Chen et al., 2018; Lim & Oh, 2011). L-rhamnose isomerases, by far the most important enzyme class for the isomerization of D-psicose and D-allose (Chen et al., 2018), are described, for example, in EP 1589102 Bl, EP 1788089 Bl, EP 1860195 Bl, US 7205141 B2, US 7501267 B2, US 7691619 B2 and US 8748589 B2. The conversion of D-fructose to D-psicose using ketose-3-epimerase does not proceed to completion, but rather an equilibrium ratio is formed between the two epimers. Depending on the reaction conditions (temperature between 40 and 70 °C, pH between 6 and 11), this is between 80:20 and 62.5:37.5 (D-fructose: D-psicose) (Zhang et al., 2016; Jiang et al., 2020). The subsequent isomerization is also an equilibrium reaction in which the equilibrium lies on the side of D-psicose - depending on the conditions, the mass ratio is between 77:23 and 62.5:37.5 (D-psicose: D-allose) (Chen et al., 2018; Lim & Oh, 2011). Lee et al. (2018) described a method for