EP4680760A2 - Procédé de production d'une solution aqueuse contenant du d-psicose - Google Patents

Procédé de production d'une solution aqueuse contenant du d-psicose

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Publication number
EP4680760A2
EP4680760A2 EP24710783.2A EP24710783A EP4680760A2 EP 4680760 A2 EP4680760 A2 EP 4680760A2 EP 24710783 A EP24710783 A EP 24710783A EP 4680760 A2 EP4680760 A2 EP 4680760A2
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EP
European Patent Office
Prior art keywords
seq
acid sequence
amino acid
nucleic acid
psicose
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EP24710783.2A
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German (de)
English (en)
Inventor
Nicole STAUNIG
Maria Dupont
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Annikki GmbH
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Annikki GmbH
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Publication date
Priority claimed from EP23162092.3A external-priority patent/EP4431614A1/fr
Priority claimed from EP23173451.8A external-priority patent/EP4464786A1/fr
Priority claimed from EP23219873.9A external-priority patent/EP4446422A3/fr
Application filed by Annikki GmbH filed Critical Annikki GmbH
Publication of EP4680760A2 publication Critical patent/EP4680760A2/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01001—Alcohol dehydrogenase (1.1.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01009—D-Xylulose reductase (1.1.1.9), i.e. xylitol dehydrogenase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01047—Glucose 1-dehydrogenase (1.1.1.47)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01056—Ribitol 2-dehydrogenase (1.1.1.56)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01067—Mannitol 2-dehydrogenase (1.1.1.67)
    • C—CHEMISTRY; METALLURGY
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    • C12Y—ENZYMES
    • C12Y106/00—Oxidoreductases acting on NADH or NADPH (1.6)
    • C12Y106/03—Oxidoreductases acting on NADH or NADPH (1.6) with oxygen as acceptor (1.6.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y501/00—Racemaces and epimerases (5.1)
    • C12Y501/03—Racemaces and epimerases (5.1) acting on carbohydrates and derivatives (5.1.3)

Definitions

  • the present invention relates to a process for preparing an aqueous solution containing D-psicose.
  • the monosaccharide D-psicose also known as D-allulose, is a ketohexose that is rarely found in nature (Zhang et al., 2016). It has been detected in the leaves of rosemary willows (Itea sp.) (Hough & Stacey, 1966), but is also found in processed foods such as confectionery and seasoning sauces, where it is formed from D-fructose, its Ca epimer, under the influence of heat (Oshima et al., 2006).
  • D-psicose is interesting for the food industry due to its sweet taste. Compared to sucrose, D-psicose has a relative sweetening power of 70%, but has a low energy content (0.2 kcal/g), which corresponds to a calorie reduction of about 95% (relative to sucrose) (Jiang et al., 2020).
  • D-psicose is recognized as a Generally Recognized as Safe (GRAS) sweetener by the US Food and Drug Administration (FDA), but is not yet approved in the EU (Ahmed et al., 2022).
  • GRAS Generally Recognized as Safe
  • D-psicose also has positive effects on lipid metabolism and carbohydrate metabolism (e.g. antidiabetic) and has anti-inflammatory and antioxidant effects (Zhang et al., 2016; Jiang et al., 2020; Chen et al., 2022).
  • D-psicose Due to its low natural occurrence, D-psicose is largely produced synthetically (chemically or biotechnologically).
  • D-fructose to D-psicose can be carried out by boiling in pyridine under reflux followed by removal of the other hexoses by yeast fermentation, but only 6.8% of the theoretical yield of D-psicose is achieved (Doner, 1979).
  • Another method involves the epimerization of D-fructose with molybdate ions as catalyst, whereby only 0.5% of the D-fructose is converted to D-psicose (Bilik & Tihlärik, 1974).
  • Ketose 3-epimerases can be divided into three groups depending on their substrate specificity: 1) D-tagatose 3-epimerase (DTE), 2) D-psicose 3-epimerase (DPE) or D-allulose 3-epimerase (DAE), and 3) L-ribulose 3-epimerase (LRE) (Zhang et al., 2016; Jiang et al., 2020).
  • DTE D-tagatose 3-epimerase
  • DPE D-psicose 3-epimerase
  • DAE D-allulose 3-epimerase
  • LRE L-ribulose 3-epimerase
  • the equilibrium during epimerization can be influenced not only by temperature or pH, but also by the addition of (toxic) borate. Due to the preferential formation of a D-psicose-borate complex, the equilibrium is shifted towards D-psicose (Kim et al., 2008; Lim et al., 2009).
  • EP 3643786 A2 and US 11028420 B2 describe the chromatographic separation of the D-psicose-borate complex using simulated moving bed (SMB) chromatography.
  • EP 3395952 Bl and US 10550414 B2 disclose that the conversion during epimerization using DPE can be increased to up to 67% with the addition of sodium aluminate and up to 52% with potassium iodate (comparison: 25% without the addition of aluminate or iodate).
  • Zhu et al. presented a system consisting of two enzymes (exo-inulase from Bacillus velezenis and DAE from Ruminococcus sp.) that can convert inulin from Helianthus tuberosus L. (Jerusalem artichoke) into a syrup consisting of D-glucose, D-fructose and D-psicose (1:3:1).
  • Li et al. (2021a) used a system consisting of invertase, D-glucose isomerase and immobilized DAE from Pirellula sp.
  • SH-Sr6A to convert sucrose, D-glucose and D-fructose (from fruit juices) into D-psicose. 16 - 19% D-psicose (based on the total carbohydrate content) could be enriched in the juices.
  • Patel et al. (2018) used a Smt3-DPE (fusion protein) immobilized on magnetic iron oxide nanoparticles to produce D-psicose from D-fructose in fruit pomace washing solutions.
  • the immobilized epimerase was able to convert 20% of the D-fructose and was separated with a magnet after completion of the reaction.
  • the D-fructose/D-psicose mixtures resulting from the epimerization of D-fructose can be separated either by chromatographic methods or by the "biological method” (fermentation of the excess D-fructose to e.g. ethanol) (Jiang et al., 2020).
  • US 2021/0189441 Al describes the separation of a D-fructose/D-psicose mixture, whereby the D-fructose is converted into L-lactic acid by a probiotic microorganism (Lactobacillus or Saccharomyces).
  • EP 3423460 Bl describes a process for purifying a D-fructose/D-psicose mixture and for obtaining highly pure D-psicose.
  • EP 3553069 Al and Van Duc Long et al. (2009) describe a method for separating D-psicose and D-fructose based on SMB chromatography open.
  • D-psicose via the epimerase route has the following disadvantages: 1) position of the equilibrium on the side of D-fructose, 2) addition of (partly toxic) metal ions as cofactors for many epimerases, 3) low activity and long-term stability of the epimerases and 4) complex separation of the product mixture.
  • thermodynamically unfavorable epimerization is to use enzyme cascades with phosphorylated intermediates.
  • the last step, dephosphorylation, is irreversible and thus drives the cascade (Li et al., 2021b).
  • a cascade described in almost identical form by Li et al. (2021b) as well as in US 11168342 B2 and US 10907182 B2 has D-glucose-l-phosphate (G1P) as the central intermediate.
  • G1P is first converted to D-glucose-6-phosphate (G6P) by phosphoglucomutase and then further to D-frucose-6-phosphate (F6P) by glucose-6-phosphate isomerase.
  • F6P is then epimerized to D-psicose-6-phosphate by D-allulose-6-phosphate epimerase, which is then dephosphorylated to D-psicose by D-allulose-6-phosphate phosphatase.
  • G1P can be produced directly by the action of phosphorylases on maltose and amylodextrins (obtained by the hydrolysis of starch), cellodextrins (obtained by the hydrolysis of cellulose) or sucrose using phosphate. Since the terminal sugar monomers of the oligo- and polysaccharides cannot be phosphorylated by the corresponding phosphorylases, the polyphosphate glucokinase (D-glucose G6P) or polyphosphate fructokinase (D-fructose F6P), whereby additional polyphosphates must be added as a phosphate source to increase the yields (US 11168342 B2; US 10907182 B2). Starch serves as the substrate for the cascade of Li et al. (2021b), which is converted to D-psicose with yields of 79% (at 50 g/l substrate concentration; reaction time 24 h).
  • WO 2016/201110 Al describes a method for producing D-psicose from dihydroxyacetone and D-glyceraldehyde using fructose-6-phosphate aldolase and DTE (intermediate D-fructose).
  • Xiao et al. coupled the epimerization of D-fructose with the conversion of D-psicose to D-psicose-l-phosphate using L-rhamnulose kinase (using adenosine triphosphate (ATP)) to shift the equilibrium of epimerization. Subsequent cleavage of the phosphate group by an acid phosphatase yields D-psicose (99% conversion of 20 mM D-fructose). However, ATP must be regenerated with a polyphosphate kinase with the addition of polyphosphate (Xiao et al., 2019).
  • a major disadvantage of routes with phosphorylated intermediates is the use of expensive, energy-rich phosphate compounds such as polyphosphate or ATP in stoichiometric amounts to introduce the phosphate groups.
  • energy-rich phosphate compounds such as polyphosphate or ATP in stoichiometric amounts to introduce the phosphate groups.
  • phosphorylases By using phosphorylases, this problem can be overcome. can be partially bypassed, but terminal monosaccharides cannot be phosphorylated without the aid of high-energy phosphate compounds.
  • the remaining phosphate compounds and phosphate ions must be removed after the reaction has ended.
  • the unfavorable equilibrium of the epimerization of D-fructose to D-psicose can also be favorably influenced by downstream redox reactions.
  • DTE ribitol dehydrogenase
  • FDH formate dehydrogenase
  • D-fructose can be converted in vitro to the sugar alcohol allitol (Takeshita et al., 2000).
  • Allitol can then be converted back to D-psicose through an oxidation step.
  • This can be achieved microbially, for example, with Enterobacter aerogenes IK7 (complete oxidation of 100 g/l allitol in 24 h) or Bacillus pallidus Y25 (48% conversion of 50 g/l allitol in 48 h) (Gullapalli et al., 2007; Poonperm et al., 2007).
  • the achiral sugar alcohol allitol forms an interface between the D- and L-hexoses in the so-called Izumoring strategy for the bioproduction of rare sugars (Izumori, 2006; Hassanin et al., 2017). Thus, it can also serve as a precursor for the production of other rare monosaccharides.
  • the interior of the cells provides a suitable microenvironment for the enzymes and also allows cofactor regeneration (NAD(P) + /NAD(P)H),
  • a working group led by Wang et al. (2022, 2023) is also working on the enzymatic biotransformation of sugars, investigating the biotransformations in vitro and in vivo.
  • the aim is to develop an economical production method that can be carried out on an industrial scale.
  • the object is achieved according to the invention by forming a first D-psicose from D-fructose, which is dissolved in an aqueous solution, by treating it with an epimerase in vitro, after which the first D-psicose is reduced to allitol by treating it with a corresponding NAD(P)H-dependent oxidoreductase in vitro and, after deactivation and/or ultrafiltration of the epimerase, is treated with a corresponding NAD(P) + -dependent oxidoreductase to form D-psicose, after which the deactivated epimerase and the oxidoreductases are removed.
  • it is also possible to immobilize it on or in a carrier material and to filter it out of the aqueous solution together with the carrier.
  • the process according to the invention is therefore not carried out by fermentation, but the enzymes are contained as such in the aqueous solution. According to the invention, the process is therefore carried out in vitro.
  • NAD(P) + -dependent oxidoreductase for the formation of D-psicose from allitol 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. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No. 12 of at least 80%, ii) an amino acid sequence which is encoded by a nucleic acid which has an identity to SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9 or SEQ ID No.
  • nucleic acid 11 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9 or SEQ ID No. 11, or a functional fragment thereof.
  • a "functional fragment" of this NAD(P) + -dependent oxidoreductase comprises an N-terminally and/or C-terminally truncated variant of the oxidoreductase having the amino acid sequence SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No. 12, which has at least 50%, preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, enzyme activity compared to the non-truncated oxidoreductase.
  • a preferred variant of the process according to the invention consists in that the oxidized cofactor NAD(P) + formed by the reduction of D-psicose to allitol is reduced by means of an alcohol dehydrogenase (ADH) and a secondary alcohol to form a ketone, wherein the secondary alcohol is preferably D-glucose or 2-propanol (isopropanol).
  • ADH alcohol dehydrogenase
  • 2-Propanol is a very cheap hydrogen donor for the regeneration of NAD(P)H and the oxidation product acetone is easily separated due to its volatility (Xu et al., 2021).
  • Acetone recovered from the exhaust gas stream can be hydrogenated back to 2-propanol using heterogeneous catalysis (Al-Rabiah et al., 2022), either in the gas phase or in solution, present as 2-propanol/acetone/water mixtures or as acetone/water mixtures, whereby hydrogen from sustainable sources (“green hydrogen”) could be increasingly used in the future.
  • heterogeneous catalysis Al-Rabiah et al., 2022
  • the oxidized cofactor NAD(P) + formed by the reduction is reduced by means of a glucose dehydrogenase and D-glucose to form D-gluconate.
  • a glucose dehydrogenase to regenerate NAD(P)H is particularly advantageous because in the course of the reduction of NAD(P) + D-gluconate is formed from D-glucose, which can be obtained from the reaction mixture and used in a wide variety of areas (e.g. in metal pickling agents, in medicines and as a stabilizer in foodstuffs, etc.).
  • glucose dehydrogenase makes it possible to use a mixture comprising D-fructose and D-glucose as a substrate for the production of allitol or D-psicose without additional D-glucose being added to the reaction mixture and without D-glucose being isomerized to D-fructose beforehand.
  • Mixtures of D-fructose and D-glucose can be produced, for example, by hydrolysis of sucrose.
  • a glucose dehydrogenase derived from Priestia megaterium and comprising an amino acid sequence available under the NCBI accession number MDQ0804260.1 is particularly preferred.
  • a further preferred variant of the process according to the invention comprises the use of a formate dehydrogenase (FDH) to regenerate the oxidized cofactor NAD(P) + produced by the reduction by means of a formate dehydrogenase and formate (eg sodium formate) with the formation of CO2.
  • FDH formate dehydrogenase
  • a further preferred variant of the process according to the invention is characterized in that it is carried out as a one-pot reaction without isolation of intermediate products.
  • the enzymes are preferably used as a lysate of the corresponding cells that produce them.
  • the enzymes are expressed individually in suitable E. co// production strains. This allows the enzyme ratios to be optimized and this is thus independent of the expression level in the overall construct compared to Wang et al. (2023).
  • the enzymes (epimerase and reductase and/or dehydrogenase) of the first step (D-fructose allitol) are deactivated by heat and/or removed by ultrafiltration to prevent the formation of byproducts by the enzymes. Without the appropriate treatment, much of the D-psicose produced by the oxidation would be converted back to D-fructose by the epimerase.
  • NAD nicotinamide-based cofactors
  • the particularly preferred concentration of D-fructose is 50 - 250 g/l.
  • the particularly preferred temperature range for the first step is between 25 and 45 °C, and for the second step (oxidation) between 20 and 30 °C.
  • the preferred pH range for both steps is between 7 and 8.5.
  • the enzymes are present in a suspension and/or in the homogenate and/or in the lysate of the corresponding cells that produce them, with lysates being particularly preferred.
  • suspension means a suspension of resting cells. These are harvested after cultivation (separated from the nutrient medium) and used as a paste or suspended in a suitable buffer system. In contrast to fermentative processes, which also work with whole cells, the resting cells can no longer grow due to the lack of carbon sources and nutrients, but only serve to convert substrates (Lin & Tao, 2017).
  • Homogenate in this context means a physically and/or chemically treated suspension (e.g. treated by pressure, lysozyme or ultrasound), whereby the cell components are released from the cells. A lysate is obtained when the insoluble cell components of the homogenate are removed, for example by filtration or centrifugation (see Production of enzymes & Preparation of lysates for details).
  • the enzymes can also be modified at the N-terminus with a water-soluble polymer such as polyethylene glycol, immobilized in or on a solid matrix, or be part of a fusion protein.
  • a water-soluble polymer such as polyethylene glycol
  • the enzymes can be in powder form, in lyophilized or spray-dried form.
  • D-psicose is particularly preferably present in an aqueous solution, from which solid D-psicose can be obtained, for example, by spray drying (US 2019/0315790 Al; Kawakami et al., 2013; Kawakami et al., 2014).
  • the D-psicose is present in a syrup, the syrup being prepared by concentrating the filtrate described above or by dissolving crystalline D-psicose, which can be prepared by the process according to the invention, in water.
  • the syrup according to the invention preferably has a total solids content of about 50% by weight to about 90% by weight.
  • the D-psicose content in the syrup according to the invention is about 80% by weight to about 99% by weight based on the dry substance.
  • a further aspect of the present invention relates to a syrup comprising D-psicose, which can be prepared by the process according to the invention.
  • a syrup comprising D-psicose, which can be prepared by the process according to the invention.
  • only enzymes from the enzyme groups epimerases and oxidoreductases are used for the conversion of the starting material, with one or more of these enzymes being selected from each of these groups.
  • the epimerase used in the process can originate from one of the groups EC 5.1.3.30 (D-psicose-3-epimerase) or EC 5.1.3.31 (D-tagatose-3-epimerase/L-ribulose-3-epimerase), the former being particularly preferred.
  • the enzymes used for the reduction of D-psicose and the oxidation of allitol belong to the group of oxidoreductases (see Table 1 for details).
  • the alcohol dehydrogenase (ADH) used for cofactor regeneration can come from one of the groups EC 1.1.1.1 (NAD-dependent ADH) and EC 1.1.1.2 (NADP-dependent ADH).
  • the NAD(P)-dependent alcohol dehydrogenase for cofactor regeneration preferably 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. 18 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 17 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 17.
  • Particularly suitable for cofactor regeneration in general is an alcohol dehydrogenase whose amino acid sequence is at least 80% identical to SEQ ID No. 18 or which is encoded by a nucleic acid which has an identity to SEQ ID No. 17 of at least 80% or binds under stringent conditions to a nucleic acid molecule with the nucleic acid sequence SEQ ID No. 17, or a functional fragment of this alcohol dehydrogenase.
  • a "functional fragment" of the alcohol dehydrogenase comprises an N-terminally and/or C-terminally truncated variant of the alcohol dehydrogenase with the amino acid sequence SEQ ID No.
  • the alcohol dehydrogenase for cofactor regeneration mentioned here preferably comprises an amino acid sequence which has an identity to SEQ ID No. 18 of at least 80%, even more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%.
  • the alcohol dehydrogenase according to the invention for cofactor regeneration particularly preferably comprises the amino acid sequence SEQ ID No. 18 or consists of this.
  • the alcohol dehydrogenase for cofactor regeneration preferably comprises an amino acid sequence which is encoded by a nucleic acid which has an identity to SEQ ID No. 17 of at least 80%, even more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%.
  • the nucleic acid which comprises the inventive Alcohol dehydrogenase for cofactor regeneration encodes or consists of the nucleic acid sequence SEQ ID No. 17.
  • a further aspect of the present invention relates to the use of an alcohol dehydrogenase for cofactor regeneration, wherein the alcohol dehydrogenase comprises or consists of 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. 18 of at least 80%, ii) an amino acid sequence which is encoded by a nucleic acid which has an identity to SEQ ID No. 17 of at least 80%, and iii) an amino acid sequence which is encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 17, or a functional fragment thereof.
  • the alcohol dehydrogenases disclosed here can be used for the regeneration of NAD(P) + or NAD(P)H, ie for the reduction of NAD(P) + or for the oxidation of NAD(P)H, in a wide variety of enzymatic reactions.
  • the use of the alcohol dehydrogenases according to the invention is particularly preferred in the cofactor regeneration of NAD(P) + , which is formed during the reduction of D-psicose to allitol by means of an NAD(P)H-dependent oxidoreductase.
  • the cofactor NAD(P) + which is formed during the reduction of D-psicose to allitol, is reduced to NAD(P)H by means of formate and a formate dehydrogenase with the formation of CO2 (cofactor regeneration).
  • a formate dehydrogenase which comprises or consists of the amino acid sequence SEQ ID No. 2, or a functional fragment of this formate dehydrogenase.
  • the formate dehydrogenase preferably used is preferably encoded by the nucleic acid sequence SEQ ID No. 1.
  • a "functional fragment" of the formate dehydrogenase comprises an N-terminally and/or C-terminally truncated variant of the formate dehydrogenase with the amino acid sequence SEQ ID No.
  • the formate dehydrogenase used for cofactor regeneration 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. 2 of at least 80%, ii) an amino acid sequence which is encoded by a nucleic acid which has an identity to SEQ ID No. 1 of at least 80%, and (iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 1, or a functional fragment thereof.
  • the formate dehydrogenase preferably comprises an amino acid sequence which has an identity to SEQ ID No. 2 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100%.
  • the formate dehydrogenase preferably comprises an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 1 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100%.
  • a further aspect of the present invention relates to the use of a formate dehydrogenase for cofactor regeneration or a functional fragment thereof, wherein the formate dehydrogenase 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. 2 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 1 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 1.
  • the glucose dehydrogenase (GDH) used for cofactor regeneration can come from one of the groups EC 1.1.1.47 (glucose-l-dehydrogenase), EC 1.1.1.118 (glucose-l-dehydrogenase (NAD + )), EC 1.1.1.119 (glucose-l-dehydrogenase (NADP + )) or EC 1.1.1.360 (glucose/galactose-l-dehydrogenase).
  • the NAD(P)-dependent glucose dehydrogenase for cofactor regeneration preferably 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. 20 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 19, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID NO: 19.
  • glucose dehydrogenase whose amino acid sequence is at least 80% identical to SEQ ID No. 20 or which is encoded by a nucleic acid which has an identity to SEQ ID No. 19 of at least 80% or binds under stringent conditions to a nucleic acid molecule with the nucleic acid sequence SEQ ID No. 19, or a functional fragment of this glucose dehydrogenase.
  • a "functional fragment" of the glucose dehydrogenase comprises an N-terminal and/or C-terminal truncated variant of the glucose dehydrogenase with the amino acid sequence SEQ ID No.
  • the glucose dehydrogenase for cofactor regeneration mentioned here preferably comprises an amino acid sequence which has an identity to SEQ ID No. 20 of at least 80%, even more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%.
  • the glucose dehydrogenase according to the invention for cofactor regeneration particularly preferably comprises the amino acid sequence SEQ ID No. 20 or consists of this.
  • the glucose dehydrogenase for cofactor regeneration preferably comprises an amino acid sequence which is encoded by a nucleic acid which has an identity to SEQ ID No. 19 of at least 80%, even more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%.
  • the nucleic acid which encodes the glucose dehydrogenase for cofactor regeneration according to the invention comprises or consists of the nucleic acid sequence SEQ ID No. 19.
  • a further aspect of the present invention relates to the use of a glucose dehydrogenase for cofactor regeneration, wherein the glucose dehydrogenase 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. 20 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 19 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 19, or a functional fragment thereof.
  • the NAD(P)H oxidase used for cofactor regeneration can originate from one of the groups EC 1.6.3.1 (NAD(P)H oxidase (H 2 O 2 -forming)), EC 1.6.3.2 (NAD(P)H oxidase (H 2 O-forming)), EC 1.6.3.3 (NADH oxidase (H 2 O 2 -forming)) and EC 1.6.3.4 (NADH oxidase (H 2 O-forming)), with the H 2 O-forming classes being particularly preferred.
  • a particularly preferred H 2 O-forming NAD(P)H oxidase preferably comprises or consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence having at least 80% identity to SEQ ID No. 14 or SEQ ID No. 16, ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID No. 13 or SEQ ID No. 15, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 13 or SEQ ID No. 15, or a functional fragment thereof.
  • a "functional fragment" of this NAD(P)H oxidase comprises an N-terminally and/or C-terminally truncated variant of the NAD(P)H oxidase having the amino acid sequence SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No. 12, which has at least 50%, preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, enzyme activity compared to the non-truncated NAD(P)H oxidase.
  • the preferably used F O-forming NAD(P)H oxidase preferably comprises or consists of an amino acid sequence which has an identity to SEQ ID No. 16 or SEQ ID No. 14 of at least 80%, even more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%.
  • the F O-forming NAD(P)H oxidase particularly preferably comprises or consists of the amino acid sequence SEQ ID No. 16 or SEQ ID No. 14.
  • the F O-forming NAD(P)H oxidase preferably comprises an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 15 or SEQ ID No. 13 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100%.
  • the nucleic acid encoding the F O-forming NAD(P)H oxidase comprises or consists of the nucleic acid sequence SEQ ID No. 15 or SEQ ID No. 13.
  • a further aspect of the present invention relates to the use of a F O-forming NAD(P)H oxidase for cofactor regeneration (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. 16 or SEQ ID No. 14 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 15 or SEQ ID No. 13 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 15 or SEQ ID No. 13, or a functional fragment thereof.
  • the enzymatic strategy presented here in combination with cofactor regeneration enables a biocatalytic, environmentally friendly and highly efficient production process for the production of D-psicose.
  • the NAD(P)H-dependent oxidoreductase for reducing the first D-psicose to allitol preferably 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. 4, SEQ ID No. 10 or SEQ ID No. 12 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 3, SEQ ID No. 9 or SEQ ID No.
  • nucleic acid 11 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 3, SEQ ID No. 9 or SEQ ID No. 11.
  • Particularly suitable for reducing the first D-psicose to allitol or D-psicose to allitol in general is an oxidoreductase whose amino acid sequence is at least 80% identical to SEQ ID No. 4, SEQ ID No. 10 or SEQ ID No. 12 or which is encoded by a nucleic acid which has an identity to SEQ ID No. 3, SEQ ID No. 9 or SEQ ID No. 11 of at least 80% or which binds under stringent conditions to a nucleic acid molecule with the nucleic acid sequence SEQ ID No. 3, SEQ ID No. 9 or SEQ ID No. 11.
  • This oxidoreductase can also, surprisingly, be used to oxidize allitol to D-psicose.
  • the NAD(P) + -dependent oxidoreductase for the formation of D-psicose from allitol preferably comprises or consists of 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. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No. 12 of at least 80%, ii) an amino acid sequence which is encoded by a nucleic acid which has an identity to SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9 or SEQ ID No.
  • the oxidoreductases mentioned here for the reduction of D-psicose to allitol and/or for the oxidation of allitol to D-psicose preferably comprise an amino acid sequence which has an identity to SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No. 12 of at least 80%, even more preferably of 85%, even more preferably of 90%, even more preferably of 95%, even more preferably of 98%, even more preferably of 99%, in particular of 100%.
  • the oxidoreductase according to the invention for the reduction of D-psicose to allitol and/or for the oxidation of allitol to D-psicose comprises the amino acid sequence SEQ ID No. 4, SEQ ID No. 10 or SEQ. ID No. 12 or consists of this
  • the oxidoreductase for the oxidation of allitol to D-psicose comprises one of the amino acid sequences SEQ ID No. 6 or SEQ ID No. 8 or consists of this.
  • the oxidoreductases for reducing D-psicose to allitol and/or for oxidizing allitol to D-psicose preferably comprise an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9 or SEQ ID No. 11 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100%.
  • the nucleic acid which encodes the oxidoreductase according to the invention for the reduction of D-psicose to allitol and/or for the oxidation of allitol to D-psicose comprises or consists of the nucleic acid sequence SEQ ID No. 3, SEQ ID No. 9 or SEQ ID No. 11, and which encodes the oxidoreductase for the oxidation of allitol to D-psicose comprises or consists of the nucleic acid sequence SEQ ID No. 5 or SEQ ID No. 7.
  • identity refers to the percentage of identical nucleotide or amino acid matches between at least two nucleotide or amino acid sequences aligned using a standardized algorithm. Such an algorithm can, in a standardized and reproducible manner, insert gaps in the compared sequences to optimize the alignment between two sequences, thus achieving a more meaningful comparison of the two sequences.
  • Percent identity between sequences can be determined using one or more computer algorithms or programs known in the art or described herein.
  • the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990) provided by the National Center for Biotechnology Information (NCBI) is used to determine identity.
  • the BLAST suite of software includes several programs, including a tool called "BLAST 2 Sequences” which is used for direct pairwise comparison of two nucleotide or amino acid sequences. "BLAST 2 Sequences” can also be accessed and used interactively on the Internet via the NCBI World Wide Web site.
  • the oxidoreductases for reducing D-psicose to allitol and/or for oxidizing allitol to D-psicose preferably comprise an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11.
  • stringent conditions refer to conditions under which so-called specific hybrids, but not non-specific hybrids, are formed.
  • stringent conditions comprise hybridization in 6xSSC (sodium chloride/sodium citrate) at 45°C and then washing with 0.2 to 1xSSC, 0.1% SDS at 50 to 65°C; or such conditions may include hybridization in 1xSSC at 65 to 70°C and then washing with 0.3xSSC at 65 to 70°C.
  • Hybridization may be carried out by conventionally known methods such as those described by J. Sambrook et al. in Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989).
  • One aspect of the present invention relates to the use of an oxidoreductase for reducing D-psicose to allitol and/or for oxidizing allitol to D-psicose, wherein the oxidoreductase comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 4, SEQ ID No. 10 or SEQ ID No. 12 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 3, SEQ ID No. 9 or SEQ ID No.
  • nucleic acid 11 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 3, SEQ ID No. 9 or SEQ ID No. 11.
  • a further aspect of the present invention relates to the use of an oxidoreductase for the oxidation of allitol to D-psicose, wherein the oxidoreductase 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. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No. 12 of at least 80%, ii) an amino acid sequence which is encoded by a nucleic acid which has an identity to SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9 or SEQ ID No.
  • nucleic acid 11 of at least 80%, and (iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9 or SEQ ID No. 11.
  • the oxidoreductases according to the invention require appropriate cofactors, as mentioned above.
  • D-Psicose was purchased from TCI and Hunan Garden Naturals Inc. (China), allitol was from TCI, D-fructose, NADPH tetrasodium salt, and methanol were from PanReac AppliChem (ITW Reagents), D-glucose, sodium gluconate, IPTG (isopropyl-ß-D-thiogalactopyranoside) were from Sigma-Aldrich, potassium dihydrogen phosphate, di-potassium hydrogen phosphate, NAD + , NADH disodium salt, NADP + - disodium salt, and sodium dodecyl sulfate (SDS) were from Carl Roth, and triethanolamine (TEA) was from Chem-Lab NV.
  • the gene to be expressed was first amplified in a PCR using the genomic DNA or its synthetic equivalent adapted to the codon usage of E. coli as a template together with specific oligonucleotides that additionally carry recognition sequences for restriction endonucleases and isolated from the reaction mixture. After nucleic acid digestion with the restriction enzymes Sphl and Hindlll, the gene fragment coding for the target enzyme was ligated into the backbone of the expression vector pQE70-Kan cut with Sphl and Hindlll. The ligation product was transformed into chemically competent E. coli cells ToplOF and the resulting colonies were used for plasmid isolation and restriction analysis.
  • the result of the cloning step was verified by restriction enzyme digestion and DNA sequencing.
  • the resulting construct carries the target gene under the IPTG-inducible T5 promoter.
  • the resulting expression plasmid was transformed into the competent expression cells RB791. After 24 h of incubation at 37 °C, the resulting colonies were inoculated into LB medium for expression tests.
  • the cell pellet prepared according to the above procedure was weighed into a suitable container and mixed with buffer and lysozyme (final concentration 0.5 mg/ml) (e.g. triethanolamine (TEA) - HCl) and dissolved with stirring.
  • buffer and lysozyme final concentration 0.5 mg/ml
  • the mass fraction of biomass is usually 20%, the rest is made up of the buffer.
  • the resulting homogenate was centrifuged for 10 min at 4 °C and 16000 rpm (Eppendorf Centrifuge 5417R) to separate the insoluble cell fragments and obtain the lysate.
  • a Dionex ICS6000 system with AS-AP autosampler was used to quantify D-gluconic acid/D-gluconate using HPAEC (High Performance Anion Exchange Chromatography). The measurement was carried out using conductivity detection (CD) coupled to a Dionex AERS 500 electrolytically regenerated suppressor in external water mode.
  • CD conductivity detection
  • a Dionex lonPac ASll-HC-4pm column with a corresponding pre-column and a NaOH gradient was used to separate the analytes.
  • the mobile phase was also pretreated with a Dionex ATC Anion Trap Column.
  • Enzyme activities in the lysates were determined using a Shimadzu UV-1900 spectrophotometer. The formation or consumption of NAD(P)H was monitored at a wavelength of 340 nm via the change in absorption. The measurements were carried out with 0.2 mM cofactor (NAD(P) + or NAD(P)H). For this purpose, 20 ⁇ l of a 10 mM stock solution of the cofactor was placed in a cuvette (Greiner bio-one semi-micro cuvette made of polystyrene) and the desired pH value was adjusted with 100 mM TEA-HCl buffer (870 ⁇ l).
  • the reaction was carried out in a Labfors 5 table bioreactor (Infors AG). A glass reactor (volume 3.4 l) with a stirrer and pH electrode was used as the vessel. The pH was controlled by adding 1M NaOH or 1M H2SO4.
  • samples were continuously taken from the reactor solution and analyzed as follows: 100 ⁇ l of the reactor solution were mixed with 200 ⁇ l of methanol and incubated in the Eppendorf Thermomixer at 60 °C and 1200 rpm for 15 min. The sample was briefly centrifuged centrifuged, mixed with 700 pl deionized water, vortexed and then centrifuged for 5 min at max. g. 200 pl of the supernatant were transferred to an HPLC vial with insert and measured by HPLC (RI detection).
  • the entire reactor contents were then heated to 70 °C for 60 min (deactivation of D-psicose-3-epimerase) and after cooling to 24 °C, 25 ml of xylitol dehydrogenase lysate, 10 kU NADH oxidase lysate and 10 ml of a 10 mM NAD + solution were added.
  • the filtrate was concentrated to a syrup with a D-psicose concentration of 520 g/l using a rotary evaporator, whereby any remaining acetone and 2-propanol were also separated.
  • Example 1 shows that the epimerase can be denatured by heat (in a one-pot process) and that the resulting precipitate does not interfere with the further reaction.
  • the reaction was carried out in a Multifors table bioreactor (Infors AG).
  • a glass reactor (volume 1 1) with a stirrer and pH electrode was used as the vessel.
  • the pH was controlled by adding SM NaOH or IM H2SO4. Initially, 17.5 g D-fructose and 17.5 g D-glucose (final concentration 50 g/l each), 217.8 ml deionized water and 26.5 ml of a 500 mM potassium phosphate buffer (pH 7.5) were placed in the reactor and brought to 35 °C while stirring.
  • samples were continuously taken from the reactor solution and analyzed as follows: 100 ⁇ l of the reactor solution were mixed with 200 ⁇ l of methanol and incubated in the Eppendorf Thermomixer at 60 °C and 1200 rpm for 15 min. The sample was briefly centrifuged in a centrifuge, mixed with 700 ⁇ l of deionized water, vortexed and then centrifuged for 5 min at max. g. 200 ⁇ l of the supernatant were transferred to an HPLC vial with insert and measured using HPLC (RI detection). For the HPAEC measurements (conductivity detection), the clear supernatant was diluted 1:250.
  • the entire reactor content was then heated to 70 °C for 60 min (deactivation of D-psicose-3-epimerase) and after cooling to 30 °C, 17.5 ml of xylitol dehydrogenase lysate, 7 kU NADH oxidase lysate and 3.5 ml of a 10 mM NAD + solution were added.
  • the reactor contents were heated to 70 °C, the pH was adjusted to 4 and stirred for 30 min at 70 °C.
  • the enzymes were filtered off through a glass frit (P3).
  • the reaction was carried out in a Multifors table bioreactor (Infors AG).
  • a glass reactor (volume 1 1) with a stirrer and pH electrode was used as the vessel.
  • the pH was controlled by adding 5M NaOH or 6M H2SO4. Initially, 70 ml of a D-fructose solution (500 g/l), 26.3 ml of deionized water and 43.8 ml of 8 M sodium formate were placed in the reactor and brought to 37 °C while stirring.
  • samples were continuously taken from the reactor solution and analyzed as follows: 100 ⁇ l of the reactor solution were mixed with 200 ⁇ l of methanol and incubated in the Eppendorf Thermomixer at 60 °C and 1200 rpm for 15 min. The sample was briefly centrifuged in a centrifuge, mixed with 700 ⁇ l of deionized water, vortexed and then centrifuged for 5 min at max. g. 200 ⁇ l of the supernatant was transferred to an HPLC vial with insert and measured using HPLC (RI detection).
  • the entire reactor content was then heated to 70 °C for 60 min (deactivation of D-psicose-3-epimerase) and after cooling to 24 °C, 25 ml of xylitol dehydrogenase lysate, 10 kll of NADH oxidase lysate and 10 ml of a 10 mM NAD + solution were added.

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Abstract

La présente invention concerne un procédé de préparation d'une solution aqueuse contenant du D-psicose, selon lequel on forme un premier D-psicose à partir de D-fructose, présent à l'état dissous dans une solution aqueuse, par traitement avec une épimérase in vitro, puis on réduit le premier D-psicose par traitement avec une oxydoréductase NAD(P)H-dépendante correspondante in vitro pour obtenir de l'allitol et on le traite avec une oxydoréductase NAD(P)+-dépendante correspondante après désactivation et/ou ultrafiltration de l'épimérase pour former du D-psicose, et on élimine ensuite l'épimérase désactivée et les oxydoréductases.
EP24710783.2A 2023-03-15 2024-03-15 Procédé de production d'une solution aqueuse contenant du d-psicose Pending EP4680760A2 (fr)

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EP23162092.3A EP4431614A1 (fr) 2023-03-15 2023-03-15 Procédé de préparation de solutions aqueuses contenant de la d-psicose ou de la l-psicose
EP23173451.8A EP4464786A1 (fr) 2023-05-15 2023-05-15 Procédé de préparation de solutions aqueuses contenant de la d-psicose ou de la l-psicose
EP23219873.9A EP4446422A3 (fr) 2023-03-15 2023-12-22 Procédé de préparation d'une solution aqueuse contenant de la l-psicose
EP23219887.9A EP4520839A3 (fr) 2023-03-15 2023-12-22 Procédé de préparation d'une solution aqueuse contenant de la d-psicose
PCT/EP2024/057013 WO2024189215A2 (fr) 2023-03-15 2024-03-15 Procédé de production d'une solution aqueuse contenant du d-psicose

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JP3711296B2 (ja) 1995-08-25 2005-11-02 株式会社林原生物化学研究所 Ｌ−プシコースの製造方法
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KR101723007B1 (ko) 2016-02-29 2017-04-04 씨제이제일제당(주) 고순도 d-사이코스를 제조하는 방법
KR101966530B1 (ko) 2016-06-30 2019-04-08 씨제이제일제당 (주) 신규 내열성 과당-6-인산-3-에피머화 효소 및 이를 이용한 알룰로스 제조방법
JP2019534021A (ja) 2016-11-11 2019-11-28 ファイファー・ウント・ランゲン・ゲゼルシャフト・ミット・ベシュレンクテル・ハフツング・ウント・コンパニー・コマンディートゲゼルシャフト Ｄ−アルロースの合成
KR102065155B1 (ko) 2016-12-08 2020-02-11 주식회사 삼양사 사이코스의 제조방법
KR102581107B1 (ko) 2016-12-14 2023-09-21 보너모스, 인코포레이티드 D-알룰로스의 효소적 생산
KR101981430B1 (ko) 2017-06-23 2019-05-23 씨제이제일제당 (주) D-사이코스 붕산염 착물로부터 크로마토그래피를 이용한 d-사이코스의 생산 방법 및 d-사이코스를 포함하는 조성물
CN109306365A (zh) * 2017-07-26 2019-02-05 保龄宝生物股份有限公司 一种真空喷雾干燥制备d-阿洛酮糖的方法
US11401536B2 (en) 2018-05-31 2022-08-02 Ngee Ann Polytechnic D-psicose production using probiotic microorganisms
CN109022521B (zh) 2018-09-18 2023-05-05 上海立足生物科技有限公司 一种由淀粉制备d-阿洛酮糖的方法
CN109022520B (zh) 2018-09-18 2023-05-05 上海立足生物科技有限公司 一种阿洛酮糖的生产工艺
KR20250162615A (ko) * 2023-03-15 2025-11-18 안니키 게엠베하 D-프시코스를 함유하는 수용액을 제조하는 방법
EP4695410A2 (fr) * 2023-04-11 2026-02-18 Annikki GmbH Procédé de production d'allitol

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