Method for preparing aqueous solution containing D-psicose

Abstract

The present invention relates to a method for preparing an aqueous solution containing D-psicose by forming a first D-psicose from D-fructose present in the aqueous solution by in vitro treatment with an epimerase, then reducing the first D-psicose to alliitol by in vitro treatment with a corresponding NAD (P) H-dependent oxidoreductase, and after inactivating and / or ultra-filtering the epimerase, adding the corresponding NAD (P) + dependent oxidoreductase to form D-psicose, and then removing the inactivated epimerase and oxidoreductase.

Description

Technical Field

[0001] This invention relates to a method for preparing an aqueous solution containing D-allulose. Background Technology

[0002] D-Allulose

[0003] D-psicose, also known as D-allulose, is a ketohexose rarely found in nature (Zhang et al., 2016). It is primarily detected in the leaves of *Sweetspires* (*Itea* sp.) (Hough & Stacey, 1966), but is also present in processed foods such as candies and spice sauces, where it is formed from D-fructose (its C3 epimer) under heating (Oshima et al., 2006).

[0004] D-Allulose has attracted attention from the food industry due to its sweetness. Compared with sucrose, D-Allulose has a relative sweetening capacity of 70%, but a lower energy content (0.2 kcal / g), which corresponds to a calorie reduction of about 95% (compared to sucrose) (Jiang et al., 2020).

[0005] In the United States, D-allulose has been designated as a Generally Recognized As Safe (GRAS) sweetener by the U.S. Food and Drug Administration (FDA); however, it has not yet been approved in the European Union (Ahmed et al., 2022).

[0006] In addition, D-allulose has positive effects on lipid and carbohydrate metabolism (e.g., anti-diabetic effects), and also has anti-inflammatory and antioxidant effects (Zhang et al., 2016; Jiang et al., 2020; Chen et al., 2022).

[0007] Due to the scarcity of D-allulose in nature, it is mainly produced through synthesis (chemical or biotechnology).

[0008] The epimerization of D-fructose to D-allulose can be achieved by reflux boiling in pyridine followed by yeast fermentation to remove other hexoses, but only 6.8% of the theoretical yield of D-allulose can be obtained (Doner, 1979). Another method involves the epimerization of D-fructose using molybdate ions as a catalyst, but only 0.5% of D-fructose is converted to D-allulose (Bílik & Tihlárik, 1974). Currently, inefficient chemical synthesis routes have been replaced by more efficient biotechnological processes.

[0009] In 1993, Izumori et al. described a ketose-3-epimerase from *Pseudomonas cichorii* ST-24 for the production of D-allulose from D-fructose (Izumori et al., 1993), which was also granted a patent (EP 0592202B1). Based on their substrate specificity, ketose-3-epimers can be divided into three groups: 1) D-tagatose-3-epimerase (DTE); 2) D-allulose-3-epimerase (DPE) or D-allulose-3-epimerase (DAE); and 3) L-ribulose-3-epimerase (LRE) (Zhang et al., 2016; Jiang et al., 2020).

[0010] However, the conversion of D-fructose to D-allulose via ketose-3-epimerase is not complete; rather, an equilibrium is formed between the two epimers. Depending on the reaction conditions (temperature 40 to 70°C, pH 6 to 11), this equilibrium is 80:20 to 62.5:37.5 (D-fructose:D-allulose). Many epimerases also require divalent metal ions such as Mn. 2+ or Co 2+ (Toxic) as a cofactor (Zhang et al., 2016; Jiang et al., 2020).

[0011] The equilibrium during epimerization can be affected not only by temperature or pH, but also by the addition of borates (which are toxic). The equilibrium shifts towards D-allulose due to the preferential formation of the D-allulose-borate complex (Kim et al., 2008; Lim et al., 2009). EP 3643786 A2 and US11028420 B2 describe the chromatographic separation of the D-allulose-borate complex using simulated moving bed (SMB) chromatography. EP 3395952 B1 and US10550414B2 disclose that during epimerization using DPE, the addition of sodium aluminate can increase the conversion to up to 67%, and the addition of potassium iodate can increase the conversion to up to 52% (compared to 25% without the addition of aluminate or iodate).

[0012] Zhu et al. (2020) proposed a system consisting of two enzymes (an exo-inulinase from *Bacillus velezenis* and a DAE from *Ruminococcus* sp.) to convert inulin from *Helianthus tuberosus* L. (Jerusalem artichoke) into a syrup composed of D-glucose, D-fructose, and D-allulose (1:3:1). Li et al. (2021a) used a system consisting of an invertase, a D-glucose isomerase, and an immobilized DAE from *Pirellula* sp. SH-Sr6A to convert sucrose, D-glucose, and D-fructose (from fruit juice) into D-allulose. They were able to enrich the fruit juice with 16-19% D-allulose (based on total carbohydrate content).

[0013] In a study, Juneja et al. (2019) analyzed the techno-economic aspects of a modified corn dry milling process that, in addition to producing ethanol, used a modified yeast strain (by expressing DPE) to produce D-allulose from corn flour. The authors calculated that one ton of corn yielded 390.4 L of ethanol and 75.3 kg of D-allulose, with a minimum selling price of US$1.29 / kg for D-allulose produced using this process (compared to a market price of US$10-20 / kg in 2018). WO 2020 / 057560A1 and WO 2020 / 057561 A1 describe the production of D-allulose from starch via saccharification, enzymatic isomerization, and epimerization.

[0014] Patel et al. (2018) used Smt3-DPE (fusion protein) immobilized on magnetic iron oxide nanoparticles to produce D-allulose from D-fructose derived from pomace washing liquid. The immobilized epimerase was able to convert 20% of the D-fructose, and the reaction was separated using a magnet after completion.

[0015] Yang et al. (2018) transferred the DPE gene from *Agrobacterium tumefaciens* into the thermostable bacterium *Kluyveromyces marxianus*. As a result, at 55°C, 190 g / l D-allulose could be produced from 750 g / l D-fructose within 12 hours, with the remaining D-fructose being fermented into ethanol by *Kluyveromyces marxianus*. Dedania et al. (2020) immobilized DPE from *Agrobacterium tumefaciens* onto titanium dioxide nanoparticles and were able to convert 36% of the D-fructose into D-allulose. Furthermore, the immobilized enzyme could be reused up to nine times.

[0016] The D-fructose / D-allulose mixture generated during D-fructose epimerization can be separated by chromatography or a “biological” method (fermenting excess D-fructose into, for example, ethanol) (Jiang et al., 2020). US2021 / 0189441 A1 describes the separation of the D-fructose / D-allulose mixture in which D-fructose is converted to L-lactic acid by probiotics (Lactobacillus or Saccharomyces). EP 3423460 B1 describes a process for purifying the D-fructose / D-allulose mixture and obtaining high-purity D-allulose. EP 3553069 A1 and Van Duc Long et al. (2009) disclose a method for separating D-allulose and D-fructose based on SMB chromatography.

[0017] However, the production of D-allulose via epimerase has the following drawbacks: 1) the equilibrium position is biased towards the D-fructose side; 2) many epimerases require the addition of (partially toxic) metal ions as cofactors; 3) epimerases have low activity and poor long-term stability; and 4) the separation of product mixtures is complicated.

[0018] One option to avoid thermodynamically unfavorable epimerization is to use a phosphorylation intermediate in the enzyme cascade. The final dephosphorylation step is irreversible and thus drives the cascade (Li et al., 2021b).

[0019] The nearly identical cascade reaction described in Li et al. (2021b) and US11168342 B2 and US10907182 B2 shows D-glucose-1-phosphate (G1P) as the central intermediate. G1P is first converted to D-glucose-6-phosphate (G6P) by phosphoglucose mutase, and then further converted to D-fructose-6-phosphate (F6P) by glucose-6-phosphate isomerase. F6P is then epimerized to D-allulose-6-phosphate by D-allulose-6-phosphate epimerase, which is subsequently dephosphorylated to D-allulose by D-allulose-6-phosphate phosphatase.

[0020] G1P can be generated directly, for example, by phosphorylating enzymes acting on maltose and starch dextrin (obtained from starch hydrolysis), cellodextrin (obtained from cellulose hydrolysis), or sucrose, while consuming phosphate. Since the terminal sugar monomers of oligosaccharides and polysaccharides cannot be phosphorylated by their corresponding phosphorylases, glucokinase (D-glucose → G6P) or fructose phosphokinase (D-fructose → F6P) must be used, in which case additional polyphosphates must be added as a phosphate source to increase yield (US11168342B2; US10907182 B2). Li et al. (2021b) used starch as a substrate for a cascade reaction, which was converted to D-allulose, with a yield of 79% (substrate concentration 50 g / L; reaction time 24 hours).

[0021] Wang et al. (2020) also developed an enzyme cascade reaction for the production of D-allulose from starch, where starch is converted to glyceraldehyde-3-phosphate and dihydroxyacetone phosphate in several steps. Under the activity of L-fuco-1-phosphate aldolase (FucA), dihydroxyacetone phosphate is converted together with D-glyceraldehyde to D-allulose-1-phosphate, which is then dephosphorylated to D-allulose by phosphatase (95% yield at 15.2 mM titer). Glyceraldehyde-3-phosphate is further converted to 2-deoxy-D-ribose (Wang et al., 2020).

[0022] This literature also describes an enzyme cascade starting with glycerol. Glycerol is converted to dihydroxyacetone phosphate (glycerol is phosphorylated by acid phosphatase and subsequently oxidized by glycerol phosphate oxidase) and D-glyceraldehyde (glycerol is oxidized by allitol oxidase), which in turn serve as substrates for aldolases (e.g., FucA). After phosphate group cleavage, a mixture of D-sorbose and D-allulose is obtained, which can be separated by chromatography (Li et al., 2020). On the other hand, WO 2016 / 201110 A1 describes a method for producing D-allulose from dihydroxyacetone and D-glyceraldehyde using fructose-6-phosphate aldolase and DTE (intermediate D-fructose).

[0023] Xiao et al. linked the epimerization of D-fructose with the conversion of D-allulose to D-allulose-1-phosphate using L-rhamnokinase (which consumes adenosine triphosphate (ATP)) to shift the epimerization equilibrium. Subsequently, the phosphate group was cleaved by acid phosphatase to yield D-allulose (99% conversion rate for 20 mM D-fructose). However, polyphosphate regeneration was required using polyphosphatase to add polyphosphate (Xiao et al., 2019).

[0024] A major drawback of the phosphorylation intermediate route is the need for stoichiometric amounts of expensive and high-energy phosphate compounds (such as polyphosphates or ATP) to introduce the phosphate group. This problem can be partially avoided by using phosphorylases, but terminal monosaccharides cannot be phosphorylated without the aid of high-energy phosphate compounds. Furthermore, residual phosphate compounds and phosphate ions must be removed after the reaction is complete.

[0025] Fermentation processes for producing D-allulose are also known. Zhang et al. (2021) proposed a fermentation process based on the co-culture of engineered Bacillus subtilis and Escherichia coli for the joint production of D-allulose (titer 11.7 g / L; conversion rate: 69.5%) and lipase. US2017 / 0298400 A1 describes the expression of DPE (e.g., from Agrobacterium tumefaciens) in various microorganisms. EP 3088515 B1 and US 9701953 B2 describe the production of D-allulose by Ensifer adhaerens strains. EP 2470668 B1 discloses the immobilization of a DPE-expressing GRAS microorganism (Corynebacterium glutamicum KCCM 11046) on a sodium alginate carrier.

[0026] Garlicitol as an intermediate

[0027] The equilibrium of D-fructose epimerization to D-allulose is unfavorable, but can be favorably affected by downstream redox reactions. D-fructose can be converted to allitol in vitro by conjugating DTE with ribitol dehydrogenase (RDH; EC 1.1.1.56) and formate dehydrogenase (FDH; a regenerating enzyme that acts as a cofactor for nicotinamide adenine dinucleotide NADH) (Takeshita et al., 2000).

[0028] Allitol can then be converted back to D-allulose via an oxidation step. This can be achieved, for example, by microbial methods using *Enterobacter aerogenes* IK7 (which completely oxidizes 100 g / L allitol within 24 hours) or *Bacillus pallidus* Y25 (which converts 50 g / L allitol to 48% within 48 hours) (Gullapalli et al., 2007; Poonperm et al., 2007).

[0029] Due to its symmetry, the achiral allitinol forms a link between D-hexoses and L-hexoses in the so-called Izumoring strategy for the bioproduction of rare sugars (Izumori, 2006; Hassanin et al., 2017). Therefore, it can also serve as a precursor for the production of other rare monosaccharides.

[0030] Theoretical papers by Hold et al. (2009) and Siedentop et al. (2021) explored the optimization of enzyme cascade reactions. They described how all components and multiple parameters must be considered, especially those related to cascade design, the enzyme itself, reaction conditions and environment, and process design, and that it is impossible to predict which parameters will succeed. The synthesis of D-allulose was not specifically mentioned in the papers.

[0031] Chen et al. (2022) shifted to the fermentation pathway of D-allulose via whole-cell biocatalysts (“in vivo”) and concluded that this is the only pathway with the potential for economical and industrial-scale production of D-allulose (with different optimization schemes). The advantages of whole-cell biocatalysts are evident:

[0032] (1) Cells containing enzymes are easier to obtain than the enzymes themselves, and the purification of enzymes is usually very laborious.

[0033] (2) The cell interior provides a suitable microenvironment for enzymes and also allows for the regeneration of cofactors (NAD(P)). + / NAD(P)H);

[0034] (3) The cell wall and cell membrane protect enzymes from the effects of the reaction medium environment;

[0035] (4) Co-localization of multiple enzymes in the cell is beneficial to local enzyme concentration and reduces the diffusion of intermediates in cascade reactions.

[0036] Therefore, the author believes that "microbial cell factories" are the best way to produce D-allulose on a large scale, so that ordinary consumers can also enjoy this rare sugar in the near future.

[0037] A team led by Wang et al. (2022, 2023) is also studying the enzymatic biotransformation of sugars, investigating both in vitro and in vivo biotransformations. Their goal is to develop economical production methods that can be implemented on an industrial scale.

[0038] To produce D-allulose from D-fructose, Wang et al. (2023) described an in vivo method consisting of two whole-cell biocatalysts from *E. coli*. In the first step (conversion of D-fructose to allitol), *E. coli* cells containing DPE from *Clostridiales*, RDH from *Providencia alcalifaciens*, FDH from *Starkeya*, and another DPE from *Rhizobium straminoryzae* were used. In this manner, D-fructose (500 mM = 90 g / L) was converted to 452 mM allitol (conversion rate 90.4%) over 12 hours at 37 °C and pH 6 using 1000 mM sodium formate (based on 2 equivalents of D-fructose). Approximately 30 mM of D-sorbitol was formed as a byproduct. Cells were separated by centrifugation, and proteins leaking from the allitol-containing supernatant were inactivated by heating. Then, Escherichia coli cells containing RDH from *Rubrivivax* sp. and NADH oxidase from *Streptococcus pyogenes* were added to the allicin solution. At pH 7, allicin (452 ​​mM) was converted to D-allulose (450 mM) within 24 hours.

[0039] Wang et al. (2023) further described that, to their knowledge, the 90% conversion rate is the highest achieved to date in the production of D-allulose from D-fructose, and announced their intention to further optimize the in vivo pathway, as the theoretical conversion rate of the proposed two-step process is 100%. One drawback of Wang et al.'s process is the formation of the byproduct D-sorbitol, which is formed by the reduction of D-fructose.

[0040] The purpose of this invention is to further improve the two-step process for the formation of D-allulose from D-fructose and to provide a method for preparing an aqueous solution containing D-allulose, which can be particularly carried out in a one-pot process. Invention Details

[0042] According to the present invention, this objective is achieved by: treating D-fructose present in aqueous solution in vitro with an epimerase to form a first D-allulose, then reducing the first D-allulose to allitol in vitro with a corresponding NAD(P)H-dependent oxidoreductase, and adding the corresponding NAD(P)H after inactivation of the epimerase and / or ultrafiltration. +The epimerase is activated to form D-allulose, followed by removal of the inactivated epimerase and oxidoreductase. In addition to inactivating the epimerase, it can also be immobilized on or within a carrier material, and then filtered out of the aqueous solution along with the carrier.

[0043] Surprisingly, it has been shown that almost no undesirable D-sorbitol formation occurs in the method according to the invention, and the yield of D-allulose can even be easily increased further toward 100%.

[0044] Therefore, the process according to the invention is not carried out by fermentation, but rather the enzyme itself is contained in an aqueous solution. According to the invention, the process is therefore carried out in vitro.

[0045] A preferred variant of the method of the present invention is characterized by NAD(P) used for the formation of D-allulose from allicinol. + Redox enzymes contain amino acid sequences selected from the following groups:

[0046] i) An amino acid sequence that has at least 80% identity with SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:12.

[0047] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11, and

[0048] iii) An amino acid sequence encoded by a nucleic acid bound under stringent conditions to a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11, or

[0049] Its functional segments.

[0050] The NAD(P) + The “functional fragment” of the dependent oxidoreductase includes an N-terminal and / or C-terminal truncated variant of the oxidoreductase having the amino acid sequence of 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%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95% of the enzyme activity compared to the untruncated oxidoreductase.

[0051] The method according to the invention is schematically illustrated in the accompanying drawings.

[0052] A preferred variant of the method of the present invention involves the reduction of D-allulose to allitol to form the oxidized cofactor NAD(P). + The ketone is formed by reduction via alcohol dehydrogenase (ADH) and a secondary alcohol, preferably D-glucose or 2-propanol (isopropanol). 2-Propanol is a very inexpensive hydrogen donor for NAD(P)H regeneration, and the oxidation product acetone is easily separated due to its volatility (Xu et al., 2021). Acetone recovered from the waste gas stream can be heterogeneously catalytically rehydrogenated to 2-propanol in the gas phase or in solution as a 2-propanol / acetone / water mixture or an acetone / water mixture (Al-Rabiah et al., 2022), and the application of hydrogen from sustainable sources (“green hydrogen”) is likely to become increasingly widespread in the future.

[0053] Cofactor regeneration using ADH is known, for example, from EP 2812439 B1, or has been described in Xu et al. (2021).

[0054] In another preferred embodiment of the invention, the oxidized cofactor NAD(P) is formed through reduction. + It is reduced by glucose dehydrogenase and D-glucose to form D-glucose. The use of glucose dehydrogenase for NAD(P)H regeneration is particularly advantageous because NAD(P)H... + During reduction, D-glucose forms D-gluconate, which can be obtained from the reaction mixture and used in various fields (e.g., in metal pickling agents, pharmaceuticals, and as a food stabilizer). Furthermore, the use of glucose dehydrogenase allows the production of allitol or D-allulose using a mixture of D-fructose and D-glucose as a substrate without adding additional D-glucose to the reaction mixture or pre-isomerizing D-glucose to D-fructose. For example, a mixture of D-fructose and D-glucose can be produced by the hydrolysis of sucrose. Particularly preferred is the use of glucose dehydrogenase derived from *Priestia megaterium*, the amino acid sequence of which is available under NCBI accession number MDQ0804260.1.

[0055] Another preferred variant of the method according to the invention includes the use of formate dehydrogenase (FDH) to regenerate the oxidized cofactor NAD(P) generated by reduction via formate dehydrogenase and formate (e.g., sodium formate). + And form CO2.

[0056] Another preferred variant of the method of the present invention is characterized by a one-pot reaction that does not require separation of any intermediate products.

[0057] In the method of the present invention, the enzymes are preferably used in the form of lysates of the corresponding cells that produce them. Unlike the method of whole-cell biocatalysts of E. coli with co-expressed recombinant enzymes described by Wang et al. (2023), the enzymes of the present invention are expressed separately in suitable E. coli producing strains.

[0058] This allows for optimization of enzyme proportions, and therefore, compared to Wang et al. (2023), this method is independent of expression levels throughout the construct.

[0059] Before the final step (oxidation), the enzymes (epomerases, reductases, and / or dehydrogenases) from the first step (D-fructose → allitol) are inactivated by heating and / or removed by ultrafiltration to prevent the formation of enzymatic byproducts. Without proper treatment, most of the D-allulose produced by oxidation will be converted back to D-fructose.

[0060] Regeneration of nicotinamide-based cofactors (NAD or NADP) occurs via NAD(P)-dependent alcohol dehydrogenase, glucose dehydrogenase, or formate dehydrogenase in the case of D-allulose reduction to allitol, and via NAD(P)H oxidase to form H2O in the case of oxidation reaction (second step).

[0061] The preferred concentration of D-fructose is 50-250 g / L.

[0062] The particularly preferred temperature range for the first step (epimerization and reduction) is 25 to 45°C, and the particularly preferred temperature range for the second step (oxidation) is 20 to 30°C.

[0063] The preferred pH range for both steps is 7 to 8.5.

[0064] In another preferred embodiment of the method of the present invention, the enzymes are present in the suspension and / or homogenate and / or lysate of the corresponding cells that form them, wherein the lysate is particularly preferred.

[0065] In this paper, suspension refers to a suspension of resting cells. These cells are harvested after culture (isolated from the growth medium) and used as a paste or suspended in a suitable buffer system. Unlike fermentation methods that also use whole cells, resting cells cannot continue to grow due to the lack of carbon source and nutrients and can only be used for substrate transformation (Lin & Tao, 2017). In this paper, homogenate refers to a suspension that has undergone physical and / or chemical treatment (e.g., by pressure, lysozyme, or ultrasound) to release cellular components from the cells. When insoluble cellular components in the homogenate are removed (e.g., by filtration or centrifugation), lysates are obtained (see Enzyme Production and Lysate Preparation for details).

[0066] In another variant, the enzyme may also be modified at the N-terminus with a water-soluble polymer (e.g., polyethylene glycol), immobilized in or on a solid matrix, or become part of a fusion protein.

[0067] In another variant, the enzyme can exist in powder form, lyophilized form, or spray-dried form.

[0068] After enzymatic separation, D-allulose is most preferably present in an aqueous solution. For example, solid D-allulose can be obtained from the aqueous solution by spray drying (US2019 / 0315790 A1; Kawakami et al., 2013; Kawakami et al., 2014).

[0069] Because of the high purity of the resulting solution, the filtrate can be concentrated to obtain D-allulose in crystalline or syrup form.

[0070] In another preferred embodiment, D-allulose is present in the syrup, wherein the syrup is prepared by concentrating the aforementioned filtrate or by dissolving crystalline D-allulose (which can be prepared by the method according to the invention) in water. The syrup according to the invention preferably has a total solids content of about 50% to about 90% by weight. The syrup according to the invention contains about 80% to about 99% by weight, on a dry matter basis.

[0071] Therefore, another aspect of the present invention relates to a syrup containing D-allulose, which can be produced by the method according to the present invention.

[0072] In a particularly preferred embodiment of the method, the starting material is converted using only enzymes from the epimerase and oxidoreductase groups, wherein one or more of these enzymes are selected from each of these groups.

[0073] The epimer used in this method can be from either EC 5.1.3.30 (D-allulose-3-epimerase) or EC 5.1.3.31 (D-tagatose-3-epimerase / L-ribulose-3-epimerase), with the former being particularly preferred.

[0074] The enzymes used to reduce D-allulose and oxidize allitol are from the oxidoreductase group (see Table 1 for details).

[0075] The alcohol dehydrogenase (ADH) used for cofactor regeneration can belong to either EC 1.1.1.1 (NAD-dependent ADH) or EC 1.1.1.2 (NADP-dependent ADH).

[0076] NAD(P)-dependent alcohol dehydrogenases used for cofactor regeneration preferably contain, or consist of, an amino acid sequence selected from, or composed of:

[0077] i) An amino acid sequence that is at least 80% identical to SEQ ID NO:18,

[0078] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO:17, and

[0079] iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO:17 under strict conditions.

[0080] Generally, alcohol dehydrogenases particularly suitable for cofactor regeneration are those whose amino acid sequence has at least 80% identity with SEQ ID NO:18, or is encoded by a nucleic acid having at least 80% identity with SEQ ID NO:17, or is encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO:17, or is a functional fragment of the alcohol dehydrogenase. The “functional fragment” of an alcohol dehydrogenase includes N-terminal and / or C-terminal truncated variants of the alcohol dehydrogenase having the amino acid sequence of SEQ ID NO:18, which have at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95% of the enzymatic activity compared to the untruncated alcohol dehydrogenase.

[0081] SEQ ID NO:17:

[0082]

[0083] SEQ ID NO:18:

[0084]

[0085] The alcohol dehydrogenase for cofactor regeneration described herein preferably comprises an amino acid sequence having at least 80% identity with SEQ ID NO:18, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and particularly 100% identity. Particularly preferably, the alcohol dehydrogenase for cofactor regeneration according to the invention comprises or is composed of the amino acid sequence of SEQ ID NO:18.

[0086] Alternatively, the alcohol dehydrogenase for cofactor regeneration preferably comprises an amino acid sequence encoded by a nucleic acid having at least 80% identity, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and particularly 100% identity with SEQ ID NO:17. Particularly preferably, the nucleic acid encoding the alcohol dehydrogenase for cofactor regeneration according to the invention comprises or is composed of the nucleic acid sequence of SEQ ID NO:17.

[0087] Another aspect of the invention relates to the use of alcohol dehydrogenases for cofactor regeneration, wherein the alcohol dehydrogenase comprises, or is composed of, an amino acid sequence selected from, the group consisting of:

[0088] i) An amino acid sequence that is at least 80% identical to SEQ ID NO:18,

[0089] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO:17, and

[0090] iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO:17 under stringent conditions, or

[0091] Its functional segments.

[0092] The alcohol dehydrogenase disclosed in this article can be used for NAD(P) in a variety of enzymatic reactions. + Or the regeneration of NAD(P)H, i.e., for the purpose of NAD(P) + The reduction or oxidation of NAD(P)H. Particularly preferred is the use of the alcohol dehydrogenase of the present invention for NAD(P) reduction. + The regeneration of cofactors, namely NAD(P) + It is formed during the reduction of D-allulose to allitol by NAD(P)H-dependent oxidoreductase.

[0093] NAD(P) produced during the reduction of D-allulose to allitol + The cofactor is reduced to NAD(P)H by formate and formate dehydrogenase, and CO2 is generated (cofactor regeneration).

[0094] Particularly preferred are formate dehydrogenases containing or composed of the amino acid sequence of SEQ ID NO:2, or functional fragments thereof. The preferred formate dehydrogenase used is preferably encoded by the nucleic acid sequence of SEQ ID NO:1. The “functional fragment” of the formate dehydrogenase comprises a truncated variant of the formate dehydrogenase having the amino acid sequence of SEQ ID NO:2 at its N-terminus and / or C-terminus, which has at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95% of the enzymatic activity compared to the untruncated formate dehydrogenase.

[0095] SEQ ID NO:1:

[0096]

[0097] SEQ ID NO:2:

[0098]

[0099] According to another preferred embodiment of the invention, the formate dehydrogenase for cofactor regeneration comprises an amino acid sequence selected from the group consisting of:

[0100] i) An amino acid sequence that is at least 80% identical to SEQ ID NO:2,

[0101] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO:1, and

[0102] iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO:1 under stringent conditions, or

[0103] Its functional segments.

[0104] The formate dehydrogenase preferably contains an amino acid sequence that is at least 80% identical to SEQ ID NO:2, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and especially 100% identical.

[0105] Alternatively, the formate dehydrogenase preferably comprises an amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO:1, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and particularly 100% identity.

[0106] Another aspect of the invention relates to the use of formate dehydrogenase or a functional fragment thereof for cofactor regeneration, wherein the formate dehydrogenase comprises, or is composed of, an amino acid sequence selected from, the group consisting of:

[0107] i) An amino acid sequence that is at least 80% identical to SEQ ID NO:2,

[0108] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO:1, and

[0109] iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO:1 under strict conditions.

[0110] The glucose dehydrogenase (GDH) used for cofactor regeneration can be derived from EC 1.1.1.47 (glucose-1-dehydrogenase) and EC 1.1.1.118 (glucose-1-dehydrogenase (NAD)). +EC 1.1.1.119 (glucose-1-dehydrogenase (NADP)) + ()) or one of EC 1.1.1.360 (glucose / galactose-1-dehydrogenase).

[0111] NAD(P)-dependent glucose dehydrogenases used for cofactor regeneration preferably contain, or consist of, an amino acid sequence selected from, or composed of:

[0112] i) An amino acid sequence that has at least 80% identity with SEQ ID NO:20,

[0113] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO:19, and

[0114] iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO:19 under strict conditions.

[0115] Generally, glucose dehydrogenases particularly suitable for cofactor regeneration are glucose dehydrogenases whose amino acid sequence has at least 80% identity with SEQ ID NO:20, or is encoded by a nucleic acid having at least 80% identity with SEQ ID NO:19, or is encoded by a nucleic acid that binds to a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO:19 under stringent conditions, or is a functional fragment of the glucose dehydrogenase. The “functional fragment” of glucose dehydrogenase includes N-terminal and / or C-terminal truncated variants of glucose dehydrogenase having the amino acid sequence of SEQ ID NO:18, which have at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95% of the enzymatic activity compared to untruncated alcohol dehydrogenases.

[0116] SEQ ID NO:19:

[0117]

[0118] SEQ ID NO:20:

[0119]

[0120] The glucose dehydrogenase for cofactor regeneration described herein preferably comprises an amino acid sequence having at least 80% identity with SEQ ID NO:20, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and particularly 100% identity. Particularly preferably, the glucose dehydrogenase for cofactor regeneration of the present invention comprises or is composed of the amino acid sequence of SEQ ID NO:20.

[0121] Alternatively, the glucose dehydrogenase for cofactor regeneration preferably comprises an amino acid sequence encoded by a nucleic acid having at least 80% identity, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and particularly 100% identity with SEQ ID NO:19. Particularly preferably, the nucleic acid encoding the glucose dehydrogenase of the present invention for cofactor regeneration comprises or is composed of the amino acid sequence of SEQ ID NO:19.

[0122] Another aspect of the invention relates to the use of glucose dehydrogenase for cofactor regeneration, wherein said glucose dehydrogenase comprises, or is composed of, an amino acid sequence selected from the group consisting of:

[0123] i) An amino acid sequence that has at least 80% identity with SEQ ID NO:20,

[0124] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO:19, and

[0125] iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO:19 under stringent conditions, or

[0126] Its functional segments.

[0127] NAD(P)H oxidase for cofactor regeneration (see...) Figure 1 The enzyme can be derived from one of the following groups: EC 1.6.3.1 (NAD(P)H oxidase (forms H2O2)), EC 1.6.3.2 (NAD(P)H oxidase (forms H2O)), EC 1.6.3.3 (NADH oxidase (forms H2O2)), and EC 1.6.3.4 (NADH oxidase (forms H2O)), wherein the group that forms H2O is particularly preferred.

[0128] Particularly preferred NAD(P)H oxidases that form H2O preferably contain, or consist of, an amino acid sequence selected from, the group consisting of:

[0129] i) An amino acid sequence that has at least 80% identity with SEQ ID NO:14 or SEQ ID NO:16,

[0130] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO:13 or SEQ ID NO:15, and

[0131] iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO:13 or SEQ ID NO:15 under stringent conditions, or

[0132] Its functional segments.

[0133] The “functional fragment” of the NAD(P)H oxidase includes an N-terminal and / or C-terminal truncated variant of the NAD(P)H oxidase having the amino acid sequence of 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%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95% of the enzyme activity compared to the untruncated NAD(P)H oxidase.

[0134] SEQ ID NO:13:

[0135]

[0136] SEQ ID NO:14:

[0137]

[0138] SEQ ID NO:15:

[0139]

[0140] SEQ ID NO:16:

[0141]

[0142] The preferred NAD(P)H oxidase for forming H2O preferably comprises, or consists of, an amino acid sequence having at least 80% identity with, 85% more, 90% more, 95% more, 98% more, 99% more, and particularly 100% identity with, SEQ ID NO:16 or SEQ ID NO:14. Particularly preferably, the NAD(P)H oxidase for forming H2O comprises, or consists of, the amino acid sequence of SEQ ID NO:16 or SEQ ID NO:14.

[0143] Alternatively, the NAD(P)H oxidase that forms H2O preferably has an amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO:15 or SEQ ID NO:13, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and particularly 100% identity. Particularly preferably, the nucleic acid encoding the NAD(P)H oxidase that forms H2O comprises, or is composed of, the nucleic acid sequence of SEQ ID NO:15 or SEQ ID NO:13.

[0144] Another aspect of the invention relates to NAD(P)H oxidase, which forms H2O, for cofactor regeneration (NAD(P)H to NAD(P)). + The NAD(P)H oxidase that forms H2O comprises, or is composed of, an amino acid sequence selected from, the group consisting of:

[0145] i) An amino acid sequence that has at least 80% identity with SEQ ID NO:16 or SEQ ID NO:14,

[0146] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO:15 or SEQ ID NO:13, and

[0147] iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO:15 or SEQ ID NO:13 under stringent conditions, or

[0148] Its functional segments.

[0149] By combining cofactor regeneration, the enzymatic strategy proposed in this paper allows for a biocatalytic, environmentally friendly, and efficient production method for D-allulose.

[0150] The NAD(P)H-dependent oxidoreductase for reducing the first D-allulose to allitol preferably comprises, or is composed of, an amino acid sequence selected from, the group consisting of:

[0151] i) An amino acid sequence that has at least 80% identity with SEQ ID NO:4, SEQ ID NO:10, or SEQ ID NO:12.

[0152] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO:3, SEQ ID NO:9, or SEQ ID NO:11, and

[0153] iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO:3, SEQ ID NO:9 or SEQ ID NO:11 under strict conditions.

[0154] Particularly suitable for the reduction of first D-allulose to allitol, or generally for the reduction of D-allulose to allitol, is an oxidoreductase whose amino acid sequence has at least 80% identity with SEQ ID NO:4, SEQ ID NO:10, or SEQ ID NO:12, or is encoded by a nucleic acid having at least 80% identity with SEQ ID NO:3, SEQ ID NO:9, or SEQ ID NO:11, or is encoded by a nucleic acid bound to a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO:3, SEQ ID NO:9, or SEQ ID NO:11 under stringent conditions. Furthermore, this oxidoreductase can surprisingly be used to oxidize allitol to D-allulose.

[0155] NAD(P) used in the formation of allitol from D-allulose. + The oxidoreductase-dependent enzyme preferably comprises, or consists of, an amino acid sequence selected from, the group consisting of:

[0156] i) An amino acid sequence that has at least 80% identity with SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:12.

[0157] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11, and

[0158] iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11 under strict conditions.

[0159] SEQ ID NO:3:

[0160]

[0161] SEQ ID NO:4:

[0162]

[0163] SEQ ID NO:5:

[0164]

[0165] SEQ ID NO:6:

[0166]

[0167] SEQ ID NO:7:

[0168]

[0169] SEQ ID NO:8:

[0170]

[0171] SEQ ID NO:9:

[0172]

[0173] SEQ ID NO:10:

[0174]

[0175] SEQ ID NO:11:

[0176]

[0177] SEQ ID NO:12:

[0178]

[0179] The oxidoreductases described herein for reducing D-allulose to allitol and / or for oxidizing allitol to D-allulose preferably contain amino acid sequences having at least 80% identity with SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and particularly 100% identity. Particularly preferably, the oxidoreductases of the present invention for reducing D-allulose to allitol and / or for oxidizing allitol to D-allulose contain or consist of the amino acid sequences of SEQ ID NO:4, SEQ ID NO:10, or SEQ ID NO:12, and the oxidoreductases for oxidizing allitol to D-allulose contain or consist of one of the amino acid sequences of SEQ ID NO:6 or SEQ ID NO:8.

[0180] Alternatively, the oxidoreductase for reducing D-allulose to allitol and / or for oxidizing allitol to D-allulose preferably comprises an amino acid sequence encoded by a nucleic acid having at least 80% identity, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and particularly 100% identity with SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11. Particularly preferably, the nucleic acid encoding the oxidoreductase of the present invention for reducing D-allulose to allitol and / or oxidizing allitol to D-allulose comprises or is composed of the nucleic acid sequence of SEQ ID NO:3, SEQ ID NO:9, or SEQ ID NO:11, and the nucleic acid encoding the oxidoreductase for oxidizing allitol to D-allulose comprises or is composed of the nucleic acid sequence of SEQ ID NO:5 or SEQ ID NO:7.

[0181] As used in this paper, “identity” refers to the percentage of identical nucleotides or amino acids between at least two nucleotide or amino acid sequences aligned using a normalization algorithm (“alignment”). Such algorithms can insert gaps in the sequences being compared in a normalized and reproducible manner to optimize the alignment between the two sequences, thereby enabling a more meaningful comparison.

[0182] The percentage identity between sequences can be determined using one or more computer algorithms or programs known in the art or described herein. According to the present invention, identity is determined using the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990) provided by the National Center for Biotechnology Information (NCBI). The BLAST software suite contains several programs, including a tool called “BLAST 2Sequences”, which is used for direct pairwise comparisons between two nucleotide or amino acid sequences. “BLAST 2Sequences” can also be interactively retrieved and used on the Internet via the NCBI World Wide Web page. The blastn program (for nucleotide sequences) defaults to a word length (W) of 11, an expected value (E) of 10, M = 5, N = -4, and double-strand comparison. For amino acid sequences, the blastp program uses a word length of 3, an expected value (E) of 10, a BLOSUM62 scoring matrix (Henikoff & Henikoff, 1989), an alignment (B) of 50, an expected value (E) of 10, M = 5, and N = -4 by default.

[0183] Alternatively, the oxidoreductase used to reduce D-allulose to allitol and / or oxidize allitol to D-allulose preferably comprises an amino acid sequence encoded by a nucleic acid bound under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11. The stringent conditions used herein refer to conditions that form so-called specific hybrids but not non-specific hybrids. For example, stringent conditions include hybridization at 45°C in 6xSSC (sodium chloride / sodium citrate), followed by washing with 0.2 to 1xSSC, 0.1% SDS at 50 to 65°C; or, such conditions may include hybridization at 65 to 70°C in 1xSSC, followed by washing with 0.3xSSC at 65 to 70°C. Hybridization can be performed 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).

[0184] One aspect of the present invention relates to the use of an oxidoreductase for reducing D-allulose to allitol and / or oxidizing allitol to D-allulose, wherein said oxidoreductase comprises an amino acid sequence selected from the group consisting of:

[0185] i) An amino acid sequence that has at least 80% identity with SEQ ID NO:4, SEQ ID NO:10, or SEQ ID NO:12.

[0186] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO:3, SEQ ID NO:9, or SEQ ID NO:11, and

[0187] iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO:3, SEQ ID NO:9 or SEQ ID NO:11 under strict conditions.

[0188] Another aspect of the invention relates to the use of an oxidoreductase for oxidizing allitol to D-allulose, said oxidoreductase comprising an amino acid sequence selected from the group consisting of:

[0189] i) An amino acid sequence that has at least 80% identity with SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:12.

[0190] ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11, and

[0191] iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11 under strict conditions.

[0192] Depending on the reaction (reduction or oxidation), the oxidoreductase of the present invention requires a corresponding cofactor, as described above.

[0193] Material

[0194] D-Allulose was purchased from TCI and Hunan Garden Naturals Inc. (China); Allicin was purchased from TCI; D-Fructose, NADPH tetrasodium salt, and methanol were purchased from PanReac AppliChem (ITWReagents); D-Glucose, sodium gluconate, and IPTG (isopropyl-β-D-thiogalactoside) were purchased from Sigma-Aldrich; Potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and NADPH were also purchased from Sigma-Aldrich. + Disodium NADH, NADP + Disodium salt and sodium dodecyl sulfate (SDS) were purchased from Carl Roth; triethanolamine (TEA) was purchased from Chem-Lab NV.

[0195] Enzyme production and preparation of lysates

[0196] General information on recombinase expression in Escherichia coli

[0197] For the production of recombinases in *E. coli* strains, genomic DNA or its synthetic equivalent adapted to *E. coli* codons is first used as a template. A specific oligonucleotide carrying an additional restriction endonuclease recognition sequence is then added to amplify the gene to be expressed via PCR, and the DNA is isolated from the reaction mixture. After digestion with restriction enzymes Sphl and HindIII, the gene fragment encoding the target enzyme is ligated to the Sphl and HindIII cleavage backbone of the expression vector pQE70-Kan. The ligation product is transformed into chemically competent Top10F *E. coli* cells, and the resulting colonies are used for plasmid isolation and restriction analysis.

[0198] The cloning results were verified by restriction enzyme digestion and DNA sequencing. The resulting construct carried the target gene located under the IPTG-inducible T5 promoter.

[0199] To overexpress this enzyme in *E. coli*, the resulting expression plasmid was transformed into competent expression cells RB791. After incubation at 37°C for 24 hours, the resulting colonies were inoculated into LB medium for expression assay.

[0200] The next day, the optical density OD was inoculated. 550 The expression culture was 0.02 mg / L and cultured with shaking at 37°C until the OD value reached 0.02 mg / L. 550 It reached 0.3. Subsequently, the temperature was lowered to 25°C, and when OD... 550 When the concentration reached 0.5, the culture was induced with 0.1 mM IPTG. After 22 hours, the culture was harvested (separated from the medium as a cell pellet by centrifugation), and the expression of the recombinase was analyzed using SDS gel electrophoresis and activity assays (use test or optical enzymatic assay).

[0201] Cell lysates were prepared by ultrasonic disruption.

[0202] To prepare the cell suspension, the cell pellet prepared according to the above method is weighed into a suitable container and mixed with buffer and lysozyme (final concentration of 0.5 mg / ml) (e.g., triethanolamine (TEA)-HCl), and stirred to dissolve. The biomass mass fraction is typically 20%, with the remainder being buffer.

[0203] Cell disruption was performed using a Branson Sonifier 450. The suspension was treated three times, each time with 15 ultrasonic pulses (device settings: timer = 15; duty cycle = 50; output control = 3–5).

[0204] The resulting homogenate was centrifuged at 16,000 rpm for 10 minutes at 4°C (Eppendorf Centrifuge 5417R) to separate insoluble cell debris and obtain lysates.

[0205] Table 1. Enzymes and donor organisms used in the examples (SDR = oxidoreductases from the short-chain dehydrogenase / reductase family).

[0206]

[0207]

[0208] Analytical methods

[0209] High performance liquid chromatography

[0210] D-allulose, D-fructose, D-glucose, and allitol were quantitatively determined by HPLC (high performance liquid chromatography) using an Agilent HPLC 1260 Infinity II Series system. Detection was performed using a refractive index detector (RI detection). For measurements, a Phenomenex Rezex RPM-monosaccharide Pb+2 (8%) column with a suitable pre-column was used, eluted isocratically with ultrapure water.

[0211] High-performance anion exchange chromatography

[0212] D-gluconic acid / D-gluconate was quantitatively determined using HPAEC (High Performance Anion Exchange Chromatography) with a Dionex ICS6000 system equipped with an AS-AP autosampler. Conductivity detection (CD) was performed using a Dionex AERS 500 electrolytic regeneration suppressor coupled to an external water mode. Analytes were separated using a Dionex lonPac AS11-HC-4 μm column with appropriate pre-column and NaOH gradient. The mobile phase was additionally pretreated with a Dionex ATC anion capture column.

[0213] Enzyme activity assay (optical enzymatic assay)

[0214] Enzyme activity in the lysates was determined using a Shimadzu UV-1900 spectrophotometer. For this purpose, the formation or consumption of NAD(P)H was monitored at 340 nm by changes in absorbance. Measurements were performed using 0.2 mM cofactor (NAD(P)H). + Alternatively, NAD(PH) can be used. For this purpose, 20 μl of 10 mM cofactor stock solution is placed in a cuvette (a Greiner Bio-One semi-micro cuvette made of polystyrene) and adjusted to the desired pH with 870 μl of 100 mM TEAHCl buffer. 10 μl of lysate (diluted or undiluted) and 100 μl of substrate solution are added to the cuvette, and the measurement begins immediately. Measurements are performed at 25°C by default. The enzyme activity of the lysate can be measured using the extinction coefficient of NADH / NADPH at 340 nm (ε = 6220 L mol). -1 cm -1 Measurements are expressed in U / ml (based on the volume of lysate) or U / g (based on the biomass used for production). Here, 1 U represents the conversion of 1 μmol of substrate per minute (1 U = 1 μmol / min = 1.67 × 10⁻⁶). -8 kat).

[0215] The following examples will describe preferred variations of the method of the present invention in more detail. The pyrolytes used in these examples were all prepared according to the steps described above.

[0216] Example 1

[0217] Production of D-allulose from D-fructose – Cofactor regeneration using ADH and 2-propanol

[0218] The reaction was carried out in a Labfors 5 benchtop bioreactor (Infors AG). A glass reactor (3.4 L volume) equipped with a stirrer and pH electrode was used as the vessel. The pH was controlled by adding 1 M NaOH or 1 M H₂SO₄.

[0219] At the beginning, 50 ml of D-fructose solution (500 g / L), 246.1 ml of deionized water and 96 ml of 200 mM TEA-HCl buffer (pH 8) were placed in the reactor and the temperature was raised to 35 °C with stirring.

[0220] To initiate the reaction, add 25 ml of D-allulose 3-epimerase lysate. Then, add 25 ml of SDRI lysate, 4 kU of alcohol dehydrogenase lysate, and 10 ml of 10 mM NAD. + Solution and 40 ml of 2-propanol.

[0221] During the operation, samples were continuously taken from the reactor solution and analyzed as follows: 100 μl of reactor solution was mixed with 200 μl of methanol and incubated in an Eppendorf Thermomixer at 60 °C and 1200 rpm for 15 minutes. The sample was briefly centrifuged, mixed with 700 μl of deionized water, vortexed, and then centrifuged at maximum centrifugal force for 5 minutes. 200 μl of the supernatant was transferred to an HPLC vial with an insert and measured by HPLC (RI detection).

[0222] After running for 23 hours, add 15 ml of isopropanol; after 50 hours, add 20 ml; and after 77 hours, add 15 ml.

[0223] After 74 hours, 5 ml of D-allulose 3-epimerase lysate was added, and after 77 hours, 15 ml of SDR I lysate and 2.4 kU of alcohol dehydrogenase lysate were added.

[0224] After 97 hours, 94% of the D-fructose (50 g / L) had been converted into allicin.

[0225] The entire reactor contents were then heated to 70°C and maintained for 60 minutes (D-allulose 3-epimerase inactivation). After cooling to 24°C, 25 ml of xylitol dehydrogenase lysate, 10 kU of NADH oxidase lysate, and 10 ml of 10 mM NAD were added. + Solution.

[0226] Allicin was completely oxidized to D-allulose within 3 hours. The reactor contents were heated to 70°C, the pH was adjusted to 4, and the mixture was stirred at 70°C for 30 minutes. Enzyme was filtered using glass frit (P3).

[0227] In this way, 94% of the D-fructose is converted into D-allulose. D-sorbitol is undetectable.

[0228] The filtrate was concentrated in a rotary evaporator to a syrup with a D-allulose concentration of 520 g / L, while any residual acetone and isopropanol were separated.

[0229] Example 1 shows that epimerases can be denatured by heating (in a one-pot process), and the resulting precipitate does not interfere with further reactions.

[0230] Example 2

[0231] Production of D-allulose from D-fructose – Cofactor regeneration using GDH and D-glucose

[0232] The reaction was carried out in a Multifors benchtop bioreactor (Infors AG). A glass reactor (1 L volume) equipped with a stirrer and pH electrode was used as the vessel. The pH was controlled by adding 5 M NaOH or 1 M H₂SO₄.

[0233] Initially, 17.5g of D-fructose and 17.5g of D-glucose (final concentration of 50g / l each), 217.8ml of deionized water and 26.5ml of 500mM potassium phosphate buffer (pH 7.5) were placed in the reactor, and the temperature was raised to 35°C with stirring.

[0234] To initiate the reaction, 17.5 ml of D-allulose-3-epimerase lysate was added. Then, 24.5 ml of SDR I lysate, 0.4 kU of glucose dehydrogenase lysate, and 3.5 ml of 10 mM NAD+ solution were added.

[0235] During the operation, samples were continuously taken from the reactor solution and analyzed as follows: 100 μl of reactor solution was mixed with 200 μl of methanol and incubated in an Eppendorf Thermomixer at 60 °C and 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 700 μl of deionized water, vortexed, and then centrifuged at maximum centrifugal force for 5 min. 200 μl of the supernatant was transferred to an HPLC vial with an insert and measured by HPLC (RI detection). For HPAEC measurements (conductivity detection), the clarified supernatant was diluted 1:250.

[0236] Sixteen hours later, only allicin and D-gluconate were detected in the reactor.

[0237] The entire reactor contents were then heated to 70°C and held for 60 minutes (to inactivate D-allulose 3-epimerase). After cooling to 30°C, 17.5 ml of xylitol dehydrogenase lysate, 7 kU of NADH oxidase lysate, and 3.5 ml of 10 mM NAD+ solution were added.

[0238] Allicin is completely oxidized to D-allulose within 27 hours.

[0239] Thus, 17.5g of D-fructose can be 100% oxidized to D-allulose (17.8g in solution). D-sorbitol and D-fructose are undetectable in the resulting solution.

[0240] Heat the reactor contents to 70°C, adjust the pH to 4, and stir the mixture at 70°C for 30 minutes. Filter the enzyme using glass frit (P3).

[0241] Example 3

[0242] Production of D-allulose from D-fructose – Cofactor regeneration using FDH and sodium formate

[0243] The reaction was carried out in a Multifors benchtop bioreactor (Infors AG). A glass reactor (1 L volume) equipped with a stirrer and pH electrode was used as the vessel. The pH was controlled by adding 5 M NaOH or 6 M H₂SO₄.

[0244] Initially, 70 ml of D-fructose solution (500 g / L), 26.3 ml of deionized water, and 43.8 ml of 8 M sodium formate solution were placed in the reactor and stirred and heated to 37 °C.

[0245] To initiate the reaction, add 25 ml of D-allulose-3-epimerase lysate. Then add 35 ml of SDRI lysate, 7 kU of formate dehydrogenase lysate, and 17.5 ml of 10 mM NAD. + Solution.

[0246] During the operation, samples were continuously taken from the reactor solution and analyzed as follows: 100 μl of reactor solution was mixed with 200 μl of methanol and incubated in an Eppendorf Thermomixer at 60 °C and 1200 rpm for 15 minutes. The sample was briefly centrifuged, mixed with 700 μl of deionized water, vortexed, and then centrifuged at maximum centrifugal force for 5 minutes. 200 μl of the supernatant was transferred to an HPLC vial with an insert and measured by HPLC (RI detection).

[0247] After 40 hours, 95% of the D-fructose (100g / l) was converted into allicin.

[0248] The entire reactor contents were then heated to 70°C and held for 60 minutes (to inactivate D-allulose 3-epimerase). After cooling to 24°C, 25 ml of xylitol dehydrogenase lysate, 10 kU of NADH oxidase lysate, and 10 ml of 10 mM NAD were added. + Solution.

[0249] Allicin was completely oxidized to D-allulose within 4 hours. The reactor contents were heated to 70°C, the pH was adjusted to 4, and the mixture was stirred at 70°C for 30 minutes. Enzyme was filtered using glass frit (P3).

[0250] In this way, 97% of D-fructose was converted to D-allulose. Trace amounts of D-fructose were still detected in the reaction solution, but D-sorbitol was not detected.

[0251] References:

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Claims

1. A method for preparing an aqueous solution containing D-allulose, comprising: treating D-fructose present in the aqueous solution in vitro with an epimerase to form a first D-allulose; then reducing the first D-allulose to allitol in vitro with a corresponding NAD(P)H-dependent oxidoreductase; and adding the corresponding NAD(P)H after inactivating the epimerase and / or ultrafiltration. + The enzymes are dependent on oxidoreductases to form D-allulose, which are then removed by inactivating epimerases and oxidoreductases.

2. The method according to claim 1, characterized in that, The NAD(P) used in the formation of D-allulose from allicinol. + Redox enzymes contain amino acid sequences selected from the following groups: i) An amino acid sequence that has at least 80% identity with SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:

12. ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11, and iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11 under strict conditions.

3. The method according to claim 1 or 2, characterized in that, The oxidized cofactor NAD(P) formed through this reduction... + It is reduced by alcohol dehydrogenase and secondary alcohols to form ketones.

4. The method according to claim 3, characterized in that, The secondary alcohol is D-glucose or 2-propanol.

5. The method according to any one of claims 1 to 4, characterized in that, The method is carried out in a one-pot reaction without the need to separate any intermediate products.

6. The method according to any one of claims 1 to 5, characterized in that, The enzymes exist in the form of lysates from the corresponding cells that produce them.

7. The method according to any one of claims 1 to 6, characterized in that, The NAD(P)H-dependent oxidoreductase used to reduce the first D-allulose to allitol comprises an amino acid sequence selected from the group consisting of: i) An amino acid sequence that has at least 80% identity with SEQ ID NO:4, SEQ ID NO:10, or SEQ ID NO:

12. ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO:3, SEQ ID NO:9, or SEQ ID NO:11, and iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO:3, SEQ ID NO:9 or SEQ ID NO:11 under strict conditions.

8. The method according to any one of claims 3 to 8, characterized in that, The alcohol dehydrogenase comprises, or consists of, an amino acid sequence selected from, the group consisting of: i) An amino acid sequence that is at least 80% identical to SEQ ID NO:18, ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO:17, and iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO:17 under strict conditions.

9. The method according to any one of claims 1 to 8, characterized in that, The oxidized cofactor NAD(P) formed through this reduction... + It is reduced by glucose dehydrogenase and D-glucose to form D-glucose.

10. The method according to claim 9, characterized in that, The glucose dehydrogenase comprises, or consists of, an amino acid sequence selected from, the group consisting of: i) An amino acid sequence that has at least 80% identity with SEQ ID NO:20, ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO:19, and iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO:19 under strict conditions.

11. The method according to any one of claims 1 to 10, characterized in that, The oxidized cofactor NAD(P) produced by the reaction is described above. + Formate is reduced by formate dehydrogenase and formate salts to form CO2.

12. The method according to claim 11, characterized in that, The formate dehydrogenase comprises, or consists of, an amino acid sequence selected from, the group consisting of: i) An amino acid sequence that is at least 80% identical to SEQ ID NO:2, ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO:1, and iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO:1 under strict conditions.

13. Use of an aqueous solution that can be prepared by any one of claims 1 to 12 in the preparation of a syrup containing D-allulose.

14. The use of oxidoreductase in the formation of D-allulose from allicin, characterized in that, The oxidoreductase comprises an amino acid sequence selected from the group consisting of: i) An amino acid sequence that has at least 80% identity with SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:

12. ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11, and iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11 under strict conditions.

15. Use of alcohol dehydrogenase for cofactor regeneration, wherein said alcohol dehydrogenase comprises, or consists of, an amino acid sequence selected from, the group consisting of: i) An amino acid sequence that is at least 80% identical to SEQ ID NO:18, ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO:17, and iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO:17 under strict conditions.

16. Use of glucose dehydrogenase for cofactor regeneration, wherein said glucose dehydrogenase comprises, or is composed of, an amino acid sequence selected from, the group consisting of: i) An amino acid sequence that has at least 80% identity with SEQ ID NO:20, ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO:19, and iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO:19 under strict conditions.

17. Use of formate dehydrogenase or a functional fragment thereof for cofactor regeneration, wherein said formate dehydrogenase comprises, or is composed of, an amino acid sequence selected from, the group consisting of: i) An amino acid sequence that is at least 80% identical to SEQ ID NO:2, ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO:1, and iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO:1 under strict conditions.

18. Use of NAD(P)H oxidase for H2O formation in cofactor regeneration, said NAD(P)H oxidase comprising, or consisting of, an amino acid sequence selected from, the group consisting of: i) An amino acid sequence that has at least 80% identity with SEQ ID NO:14 or SEQ ID NO:16, ii) An amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO:13 or SEQ ID NO:15, and iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO:13 or SEQ ID NO:15 under strict conditions.

Images