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EP-4739786-A1 - PROCESS FOR THE PRODUCTION OF AN AQUEOUS SOLUTION CONTAINING L-SORBOSE

EP4739786A1EP 4739786 A1EP4739786 A1EP 4739786A1EP-4739786-A1

Abstract

A process for producing an aqueous solution containing L-sorbose by reducing D-glucose, which is dissolved in an aqueous solution, in vitro to D-sorbitol by treating the dissolved D-glocose with an NAD(P)H-dependent oxidoreductase, whereupon the D-sorbitol is oxidized in vitro with an NAD(P)+-dependent oxidoreductase to obtain L-sorbose, and then the two oxidoreductases are separated out.

Inventors

  • STAUNIG, Nicole
  • DUPONT, MARIA

Assignees

  • Annikki GmbH

Dates

Publication Date
20260513
Application Date
20240708

Claims (11)

  1. 1. Process for the preparation of an aqueous solution containing L-sorbose, in which D-glucose, which is dissolved in an aqueous solution, is reduced to D-sorbitol by treatment with an NAD(P)H-dependent oxidoreductase in vitro, after which the D-sorbitol is oxidized to L-sorbose with an NAD(P) + -dependent oxidoreductase in vitro, after which the two oxidoreductases are separated.
  2. 2. Process according to claim 1, characterized in that the oxidized cofactor NAD(P) + produced by the reduction is reduced by means of an alcohol dehydrogenase and a secondary alcohol to form a ketone.
  3. 3. Process according to claim 2, characterized in that the secondary alcohol is 2-propanol.
  4. 4. Process according to one of claims 1 to 3, characterized in that the reduced cofactor NAD(P)H produced by the oxidation is oxidized to NAD(P) + by means of an NAD(P)H oxidase and oxygen.
  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. Method according to one of claims 1 to 5, characterized in that the oxidoreductases are present as lysate of the corresponding cells producing them.
  7. 7. The method according to any one of claims 1 to 6, characterized in that the NAD(P)H-dependent oxidoreductase for reducing D-glucose to D-sorbitol is a xylose reductase and preferably comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 1 or 3 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 2 or 4 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. 2 or 4.
  8. 8. The method according to any one of claims 1 to 7, characterized in that the NAD(P) + - dependent oxidoreductase for the oxidation of D-sorbitol to L-sorbose is a mannitol dehydrogenase and preferably comprises an amino acid sequence which is selected from the group consisting of: i) an amino acid sequence which has an identity to SEQ ID No. 5 of at least 80%, ii) an amino acid sequence which is encoded by a nucleic acid which has an identity to SEQ ID No. 6 of at least 80%, and iii) an amino acid sequence which is 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. 6.
  9. 9. The method according to any one of claims 4 to 8, 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. 7 or SEQ ID No. 9 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 8 or SEQ ID No. 10 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. 8 or SEQ ID No. 10.
  10. 10. Use of a NAD(P)H-dependent oxidoreductase for the reduction of D-glucose to D-sorbitol, wherein the NAD(P)H-dependent oxidoreductase comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 1 or 3 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 2 or 4 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. 2 or 4.
  11. 1. Use of a NAD(P) + -dependent oxidoreductase for the oxidation of D-sorbitol to L-sorbose, wherein the NAD(P) + -dependent oxidoreductase comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 5 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 6 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. 6.

Description

Process for the preparation of an aqueous solution containing L-sorbose The present invention relates to the preparation of an aqueous solution of L-sorbose. Background of the invention The monosaccharide L-sorbose belongs to the group of ketohexoses and is a C5 epimer of D-fructose. L-sorbose can be used as a low-calorie sweetener (Shintani, 2019) and also serves as a starting material for the enzymatic synthesis of rare sugars such as L-tagatose (Itoh et al., 1996) or L-galactose (Leang et al., 2004) and sugar alcohols such as L-iditol (Vongsuvanlert & Tani, 1988). A recent study demonstrated the antitumor properties of L-sorbose, which induces apoptosis of cancer cells (Xu et al., 2023). In addition, L-sorbose is an intermediate in the Reichstein synthesis of vitamin C (L-ascorbic acid). The route starts from D-glucose, which is first reduced to the sugar alcohol D-sorbitol (see below). D-sorbitol is then microbially oxidized to L-sorbose. Treatment with KMnO 4 as an oxidizing agent yields 2-keto-L-gulonic acid (using acetal protecting groups), which is converted to L-ascorbic acid by adding acid (Reichstein & Grüssner, 1934). L-sorbose can also be oxidized to 2-keto-L-gulonic acid microbially or with molecular oxygen on Pt- or Pd-based catalysts (Sugisawa et al., 1990; Bronnimann et al., 1994). Several chemical processes are known for the production of L-sorbose. For example, D-glucose can be isomerized to L-sorbose using Pure Silicate Zeolite Beta with Lewis acidic Ti 4+ centers. However, the observed yields of L-sorbose are max. 12% (w/w) when using a 1% (w/w) D-glucose solution and a reaction temperature of 100 °C. In addition, D-fructose and D-mannose are found as by-products (Gounder & Davis, 2013). This method was also patented in US 9255120 B2 and US 11292806 B2. L-sorbose can also be obtained by electrochemical oxidation of D-sorbitol on Pt electrodes with p-block metals (Bi, Sb, Pb, Sn, In) as promoters, but D-fructose is formed as a byproduct (Kwon et al., 2014). For the microbial oxidation of D-sorbitol, mainly acetic acid bacteria such as Gluconobacter or Acetobacter are used (Shintani, 2019; Zebiri et al., 2011; WO 2000/049133 Al; EP 0233050 Bl; EP 0273648 Bl; EP 0955358 Bl; EP 1153120 Al; EP 0199548 A2; US 6664082 Bl; US 4945048; JP H07102127 B2; KR 820001130 Bl). US 4904588 A describes various Gluconobacter (sub)oxydans strains that can completely oxidize up to 300 g/l D-sorbitol within 24 h (Gluconobacter suboxydans strains) or 200 g/l D-sorbitol within 40 h (Gluconobacter oxydans strains) to L-sorbose. Liu et al. (2022) used an engineered G. oxydons strain to convert 300 g/l D-sorbitol to 298.61 g/l L-sorbose in 100 h. The enzymes responsible for the oxidation of D-sorbitol to L-sorbose are the membrane-bound D-sorbitol dehydrogenase (mSLDH; EC 1.1.99.21), which has either pyrroloquinoline quinone (PQQ) or flavin adenine dinucleotide (FAD) as a cofactor (Toyama et al., 2005; Soemphol et al., 2007; Yang et al., 2008), the NADP-dependent D-sorbitol dehydrogenase from G. oxydans (GoSLDH) (Kim et al., 2019), the NADP-dependent L-sorbose reductase (SR; EC 1.1.1.289) (Sugisawa et al., 1991; Shinjoh et al., 2002; Kubota et al., 2011) and mannitol-2-dehydrogenase (MDH; EC 1.1.1.67) from Pseudomonas fluorescens (Kavanagh et al., 2002) or Aspergillus fumigatus (Krahulec et al., 2011). KR 102060253 Bl describes a recombinant E. coli strain expressing GoSLDH and a NAD(P)H oxidase (for cofactor regeneration) from Lactobacillus reuteri, which converted 10 mM (1.82 g/l) D-sorbitol to 92%. The enzymatic oxidation of D-sorbitol can also be carried out in cell-free processes (in vitro). WO 2014/171635 Al describes a GoSLDH with which 10 mM (1.82 g/l) D-sorbitol was converted to 7.5 mM L-sorbose within 3 h. Kim et al. (2016) also used a GoSLDH from G. oxydans G624 to oxidize 50 mM (9.11 g/l) D-sorbitol to L-sorbose (47% conversion in 3 h). In both described processes, the cofactor NADP was used in stoichiometric amounts, i.e. no regeneration system for the cofactor was used. EP 0955358 A2 describes genetically modified Gluconobacter or Acetofaocter mutants lacking L-sorbose reductase activity. With the SR3 mutant, 80 g/l D-sorbitol was oxidized to 75 g/l L-sorbose within 24 hours, with the L-sorbose concentration in the medium falling to 70 g/l after 5 days. In the comparison strain (G. suboxydans IFO 3291) with intact L-sorbose reductase activity, only about 40 g/l L-sorbose was found after 24 hours, most of which was then further metabolized. When using an mSLDH (from G. suboxydans IFO 3255), an electron acceptor such as phenazine methosulfate (7.7 mM) must be added for the oxidation of D-sorbitol (38 mM = 6.95 g/l) (formation rate of L-sorbose: 1.3 mg/h), as described in EP 0728840 Bl and US 5747301. The sugar alcohol D-sorbitol also occurs naturally in small amounts, e.g. in the fruits of the rowan tree (Sorbus aucuparia L.) (Lohmar, 1957), but is mostly produced chemically by hydrogenation on nicke