Method for preparing an aqueous solution containing L-psicose

Abstract

The present invention relates to a method for producing a first D-psicose from D-fructose present in an aqueous solution by treatment with an epimerase in vitro, and then reducing the first D-psicose to allitol by treatment with a respective NAD(P)H-dependent oxidoreductase in vitro, and, after inactivation of the epimerase and / or ultrafiltration, reducing the first D-psicose to allitol, and then reducing the first D-psicose to allitol by treatment with a respective NAD(P)H-dependent oxidoreductase in vitro to form L-psicose. + The present invention relates to a method for preparing an aqueous solution containing L-psicose by adding a dependent oxidoreductase and then removing the inactivated epimerase and oxidoreductase.

Description

[Technical Field]

[0001] The present invention relates to a method for preparing an aqueous solution containing L-psicose. [Background technology]

[0002] D-Psicose The monosaccharide D-psicose, also known as D-allulose, is a ketohexose rarely found in nature (Zhang et al., 2016). It has been detected, inter alia, in the leaves of sweet spirea (Itea sp.) (Hough & Stacey, 1966), but is also found in processed foods such as confectionery and spice sauces, where it is formed from its C3 epimer, D-fructose, under the influence of heat (Oshima et al., 2006).

[0003] D-psicose is of interest to the food industry due to its sweetness. Compared to sucrose, D-psicose has 70% of the relative sweetening power but a lower energy content (0.2 kcal / g), which corresponds to approximately a 95% calorie reduction (compared to sucrose) (Jiang et al., 2020).

[0004] In the United States, D-psicose is recognized as a generally recognized as safe (GRAS) sweetener by the U.S. Food and Drug Administration (FDA), but it is not yet approved in the EU ( Ahmed et al., 2022 ).

[0005] Furthermore, D-psicose has positive effects on lipid and carbohydrate metabolism (e.g., antidiabetic), and is anti-inflammatory and antioxidant (Zhang et al., 2016; Jiang et al., 2020; Chen et al., 2022).

[0006] Due to its rarity in nature, D-psicose is primarily produced synthetically using biotechnology.

[0007] In 1993, Izumori et al. described a ketose-3-epimerase from Pseudomonas cichorii ST-24 for producing D-psicose from D-fructose (Izumori et al., 1993) and patented it (EP0592202B1). Ketose-3-epimerases can be divided into three groups based 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).

[0008] However, the conversion of D-fructose to D-psicose via ketose-3-epimerase does not proceed completely, and an equilibrium between the two epimers is formed. Depending on the reaction conditions (temperature between 40 and 70 °C, pH between 6 and 11), the ratio is between 80:20 and 62.5:37.5 (D-fructose:D-psicose). Many epimerases also use divalent metal ions, such as Mn, as cofactors. 2+ or Co 2+ (toxicity) (Zhang et al., 2016; Jiang et al., 2020).

[0009] However, the production of D-psicose via the epimerase pathway has the following drawbacks: 1) the position of the equilibrium on the D-fructose side, 2) the addition of (partially 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.

[0010] The unfavorable position of the equilibrium for the epimerization of D-fructose to D-psicose can be favorably influenced, for example, by downstream redox reactions. By combining DTE with ribitol dehydrogenase (RDH; EC 1.1.1.56) and formate dehydrogenase (FDH; as a regenerating enzyme for the nicotinamide adenine dinucleotide NADH cofactor), D-fructose can be converted to the sugar alcohol allitol in vitro (Takeshita et al., 2000).

[0011] Due to its symmetry, the achiral allitol sugar alcohol forms the interface between D- 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.

[0012] Theoretical papers by Hold et al. (2009) and Siedentop et al. (2021) address the optimization of enzyme cascades. They note that all components and multiple parameters must be taken into account, particularly those related to the cascade design, the enzymes themselves, the reaction conditions and environment, and process design, and that it is impossible to predict which will be successful. The synthesis of L-psicose is not specifically mentioned.

[0013] Chen et al. (2022) focused on a fermentation route via whole-cell biocatalysis ("in vivo") for the production of D-psicose and concluded that this is the only route with extensive optimization and has the potential for economical and industrial-scale D-psicose production in the future. The advantages of whole-cell biocatalysis are clear: (1) Cells containing enzymes are more readily available than the enzymes themselves, which are often laborious to purify; (2) The interior of the cell provides a suitable microenvironment for the enzyme and also allows for the regeneration of cofactors (NAD(P) + / NAD(P)H) is also possible, (3) Cell walls and membranes protect enzymes from the environment of the reaction medium; (4) Colocalization of multiple enzymes within cells favors local enzyme concentrations and reduces the diffusion of intermediates in cascade reactions.

[0014] As a result, the authors see "microbial cell factories" as the best opportunity to produce D-psicose on a large scale, so that ordinary consumers can enjoy this rare sugar in the near future.

[0015] A team led by Wang et al. (2022, 2023) is also working on the enzymatic biotransformation of sugars, investigating in vitro and in vivo biotransformations. Their goal is to develop an economical production method that can be carried out on an industrial scale.

[0016] For the production of D-psicose from D-fructose, Wang et al. (2023) described an in vivo method consisting of two whole-cell biocatalysts: a DPE from Clostridiales, a RDH from Providencia alcalifaciens, a FDH from Starkeya, and an additional DPE from Rhizobium straminoryzae. In this way, D-fructose (500 mM = 90 g / L) was converted to 452 mM allitol (90.4% conversion) within 12 hours at 37 °C and pH 6 using 1000 mM sodium formate (2 equivalents relative to D-fructose). Approximately 30 mM D-sorbitol was formed as a by-product. The cells were separated by centrifugation, and proteins leaked into the allitol-containing supernatant were inactivated by heat. E. coli cells containing RDH from Rubrivivax sp. and NADH oxidase from Streptococcus pyogenes were then added to the allitol solution. Allitol (452 ​​mM) was oxidized to D-psicose (450 mM) at pH 7 within 24 hours.

[0017] L-Psicose Unlike its enantiomer, D-psicose, L-psicose does not occur naturally. L-psicose serves as a starting material for the production of L-fructose (Itoh & Izumori, 1996), L-tagatose (Rao et al., 2008), or L-talitol (Sasahara & Izumori, 2005). Studies in mice demonstrated the antiviral activity of L-psicose against herpes simplex virus 1, which causes keratitis (inflammation of the cornea) (Muniruzzaman et al., 2016).

[0018] There are essentially three different biotechnological routes known for producing L-psicose.

[0019] The aldol reaction of L-glyceraldehyde with dihydroxyacetone phosphate (DHAP), catalyzed by fructose-1,6-bisphosphate aldolase or tagatose-1,6-bisphosphate aldolase, followed by cleavage of the terminal phosphate group, yields a mixture of L-psicose and L-sorbose. The reaction cascade can be carried out in vitro and fermentatively in engineered Corynebacterium glutamicum strains (Yang et al., 2015). Yang et al. (2016) further developed the reaction cascade so that glycerol could also be used as a starting material for the fermentative production of L-psicose. The core component of the cascade is again the aldol reaction of L-glyceraldehyde with DHAP.

[0020] The Wen et al. (2015) pathway also begins with L-glyceraldehyde and dihydroxyacetone phosphate (DHAP), which are first converted to L-fructose-1-phosphate by rhamnulose bisphosphate aldolase and subsequently dephosphorylated to give L-fructose. This is isomerized to L-psicose by DTE, where the equilibrium is shifted to the product by phosphorylation using fructokinase, which requires adenosine triphosphate (ATP) as a cofactor. After removal of the phosphate group, L-psicose is obtained.

[0021] The second pathway involves the reaction of a donor bearing an α-hydroxycarbonyl group with C n - catalyzes the reaction between the aldehyde acceptor and C with CO2 elimination n+2It is based on the use of transketolase (EC 2.2.1.1), a thiamine-dependent enzyme that forms ketose. Thus, L-psicose can be produced by reacting L-erythrose with hydroxypyruvate using a thermostable transketolase mutant from Geobacillus stearothermophilus, but requires thiamine diphosphate as a cofactor (Lorilliere et al., 2019).

[0022] The third pathway is the microbial oxidation of allitol to L-psicose using Gluconobacter frateurii IFO3254, which can oxidize 100 g / L of allitol to 98% L-psicose (Takeshita et al., 1996). Oxidation by the same organism is also described in JP4761424B2 and JP3711296B2. Summary of the Invention

[0023] This is an object of the present invention. The present invention aims to provide an efficient and environmentally friendly method for preparing an aqueous solution containing L-psicose at a high conversion rate, which can be carried out in a one-pot process in particular. [Brief explanation of the drawings]

[0024] [Figure 1] FIG. 1 is a schematic diagram illustrating the method of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0025] According to the present invention, the object is to form a first D-psicose from D-fructose present in an aqueous solution by treatment with an epimerase in vitro, and then reduce the first D-psicose to allitol by treatment with the respective NAD(P)H-dependent oxidoreductase in vitro, and after inactivation of the epimerase and / or ultrafiltration, reduce the first D-psicose to allitol to form L-psicose. +This is achieved by adding the dependent oxidoreductase and then removing the inactivated epimerase and oxidoreductase.

[0026] The method of the present invention is illustrated diagrammatically in the accompanying drawing.

[0027] Surprisingly, it has been shown that the objectives set out in the present invention can be achieved when the reaction is not carried out fermentatively, but when the enzyme is contained intact in aqueous solution, i.e. when the reaction is carried out in vitro.

[0028] A preferred variant of the method of the invention is that the oxidoreductase for the formation of L-psicose from allitol is i) an amino acid sequence having at least 80% identity to SEQ ID NO: 2; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 1; and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 1 The amino acid sequence is selected from the group consisting of: SEQ ID NO:1:

number

number

[0029] A preferred variant of the method of the invention is the production of oxidized cofactor NAD(P) formed by the reduction of D-psicose to allitol. +The process involves the reduction of NAD(P)H by alcohol dehydrogenase (ADH) and a secondary alcohol, preferably D-glucose or 2-propanol (isopropanol), resulting in the formation of a ketone. 2-Propanol is a very inexpensive hydrogen donor for the regeneration of NAD(P)H, and the oxidation product, acetone, is easily separable due to its volatility (Xu et al., 2021). Acetone recovered from exhaust gas streams, present either in the gas phase or in solution as a 2-propanol / acetone / water or acetone / water mixture, can be heterogeneously catalytically rehydrogenated to 2-propanol (Al-Rabiah et al., 2022). In the future, hydrogen from sustainable sources ("green hydrogen") may be increasingly used. The use of such systems allows for the recycling of 2-propanol without emitting climate-harmful CO2 and also avoids large amounts of waste (such as unreacted sodium formate in the FDH regeneration system).

[0030] Cofactor regeneration using ADH is known, for example, from EP2812439B1 or described in Xu et al. (2021).

[0031] In a further preferred embodiment of the present invention, the oxidized cofactor NAD(P) formed by reduction + is reduced by glucose dehydrogenase and D-glucose with the formation of D-gluconic acid. The use of glucose dehydrogenase to regenerate NAD(P)H is +This is particularly advantageous because during the reduction of D-glucose, D-gluconic acid is formed from D-glucose, which can be obtained from the reaction mixture and used in several fields (e.g., in metal pickling detergents, pharmaceuticals, and as a stabilizer in foods). Furthermore, the use of glucose dehydrogenase allows the use of a mixture containing D-fructose and D-glucose as a substrate for the production of allitol without adding additional D-glucose to the reaction mixture and without prior isomerization of D-glucose to D-fructose. A mixture of D-fructose and D-glucose can be produced, for example, by hydrolysis of sucrose. Particularly preferably, a glucose dehydrogenase derived from Priestia megaterium and comprising the amino acid sequence available under NCBI accession number MDQ0804260.1 is used.

[0032] 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 the intermediate products.

[0033] In the method of the present invention, the enzymes are preferably used as lysates of the corresponding cells that produce them. In contrast to the method described by Wang et al. (2023), which is based on a whole-cell biocatalysis of E. coli with co-expressed recombinant enzymes, the enzymes are expressed individually in a suitable E. coli production strain.

[0034] This allows for optimization of enzyme ratios and is therefore independent of expression levels across the construct, compared to Wang et al. (2023).

[0035] Before carrying out the final step (oxidation), the enzymes (epimerase and reductase and / or dehydrogenase) of the first step (D-fructose → allitol) are inactivated by heat and / or removed by ultrafiltration to prevent the formation of enzymatic by-products. In particular, in the case of D-psicose, most of the D-psicose formed by oxidation can revert to D-fructose without appropriate treatment.

[0036] Regeneration of the nicotinamide-based cofactor (NAD or NADP) occurs in the reduction of D-psicose to allitol using NAD(P)-dependent alcohol dehydrogenase or glucose dehydrogenase, and in the oxidation reaction (second step) using HO-forming NAD(P)H oxidase.

[0037] A particularly preferred concentration of D-fructose is 50 to 250 g / l.

[0038] Particularly preferred temperature ranges for the first step (epimerization and reduction) are between 25 and 45°C, and for the second step (oxidation) between 20 and 30°C.

[0039] A particularly preferred pH range for both steps is between 7 and 8.5.

[0040] In a further preferred embodiment of the method of the invention, the enzymes are present in suspensions and / or homogenates and / or lysates of the respective cells forming them, with lysates being particularly preferred.

[0041] In this context, a suspension refers to a suspension of resting cells. These are harvested (separated from the growth medium) after cultivation and used as a paste or suspended in an appropriate buffer system. In contrast to fermentation methods, in which whole cells are also used, resting cells can no longer grow due to the lack of a carbon source and nutrients and are only useful for substrate conversion (Lin & Tao, 2017). In this context, a homogenate refers to a suspension that has been physically and / or chemically treated (e.g., with pressure, lysozyme, or ultrasound) so that cellular components are released from the cells. A lysate is obtained when the insoluble cellular components of the homogenate are removed, for example, by filtration or centrifugation (see Enzyme Production & Lysate Preparation for details).

[0042] In another variation, the enzyme may also be N-terminally modified with a water-soluble polymer such as polyethylene glycol, may be immobilized in or on a solid matrix, or may be part of a fusion protein.

[0043] In another variation, the enzyme may be present in powdered, freeze-dried or spray-dried form.

[0044] After separation of the enzyme, L-psicose is preferably present in an aqueous solution, from which solid L-psicose can be obtained, for example, by spray drying (for D-psicose, see US2019 / 0315790A1; Kawakami et al., 2013; Kawakami et al., 2014).

[0045] Because the obtained solution is highly pure, the filtrate can be concentrated to obtain L-psicose in the form of crystals or syrup.

[0046] In a further preferred variant, L-psicose is present in a syrup, which is prepared by concentrating the above-mentioned filtrate or by dissolving crystalline L-psicose, which can be prepared by the method according to the present invention, in water. The syrup according to the present invention preferably has a total solids content of approximately 50% to approximately 90% by weight. The L-psicose content in the syrup according to the present invention is approximately 80% to approximately 99% by weight based on dry matter.

[0047] Therefore, a further aspect of the present invention relates to an L-psicose-containing syrup that can be produced by the method according to the present invention.

[0048] In a particularly preferred embodiment of the method, only enzymes from the group of enzymes epimerases and oxidoreductases are used for the conversion of the starting material, one or more of these enzymes being selected from each of these groups.

[0049] The epimerase used in the present method may be 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.

[0050] The enzymes used for the reduction of D-psicose and the oxidation of allitol are from the group of oxidoreductases (see Table 1 for details).

[0051] The alcohol dehydrogenase (ADH) used for cofactor regeneration may belong to one of the groups EC 1.1.1.1 (NAD-dependent ADH) and EC 1.1.1.2 (NADP-dependent ADH).

[0052] The NAD(P)-dependent alcohol dehydrogenase for cofactor regeneration is preferably i) an amino acid sequence having at least 80% identity to SEQ ID NO: 6; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO:5, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 5 The amino acid sequence comprises or consists of an amino acid sequence selected from the group consisting of:

[0053] Generally, particularly suitable for cofactor regeneration are alcohol dehydrogenases whose amino acid sequence has at least 80% identity to SEQ ID NO:6, or that are encoded by a nucleic acid having at least 80% identity to SEQ ID NO:5, or that bind under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:5. SEQ ID NO:5:

number

number

[0054] The alcohol dehydrogenase for cofactor regeneration referred to herein preferably comprises an amino acid sequence which has at least 80%, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and especially 100% identity to SEQ ID NO: 6. Particularly preferably, the alcohol dehydrogenase according to the invention for cofactor regeneration comprises or consists of the amino acid sequence of SEQ ID NO: 6.

[0055] Alternatively, the alcohol dehydrogenase for cofactor regeneration preferably comprises an amino acid sequence encoded by a nucleic acid having at least 80%, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and especially 100% identity to SEQ ID NO: 5. Particularly preferably, the nucleic acid encoding the alcohol dehydrogenase for cofactor regeneration according to the invention comprises or consists of the nucleic acid sequence of SEQ ID NO: 5.

[0056] A further aspect of the invention relates to the use of an alcohol dehydrogenase for cofactor regeneration, the alcohol dehydrogenase comprising: i) an amino acid sequence having at least 80% identity to SEQ ID NO: 6; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO:5, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 5 The amino acid sequence comprises or consists of an amino acid sequence selected from the group consisting of:

[0057] The alcohol dehydrogenases disclosed herein are capable of converting NAD(P) into NAD(P) in various enzymatic reactions. + or regeneration of NAD(P)H, i.e., NAD(P) + or the oxidation of NAD(P)H. Particularly preferred is the reduction of NAD(P)H formed during the reduction of D-psicose to allitol by NAD(P)H-dependent oxidoreductases. + and the use of the alcohol dehydrogenase of the present invention in cofactor regeneration.

[0058] Glucose dehydrogenases (GDHs) used for cofactor regeneration are in the groups EC1.1.1.47 (glucose-1-dehydrogenase), EC1.1.1.118 (glucose-1-dehydrogenase (NAD + )), EC1.1.1.119 (glucose-1-dehydrogenase (NADP+ )) or EC 1.1.1.360 (glucose / galactose-1-dehydrogenase).

[0059] The NAD(P)-dependent glucose dehydrogenase for cofactor regeneration is preferably i) an amino acid sequence having at least 80% identity to SEQ ID NO: 10; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 9, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 9 The amino acid sequence comprises or consists of an amino acid sequence selected from the group consisting of:

[0060] Generally, particularly suitable for cofactor regeneration are glucose dehydrogenases whose amino acid sequence has at least 80% identity to SEQ ID NO: 10, or that are encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 9, or that bind under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 9. SEQ ID NO:9:

number

number

[0061] The glucose dehydrogenase for cofactor regeneration referred to herein preferably comprises an amino acid sequence having at least 80%, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and especially 100% identity to SEQ ID NO: 10. Particularly preferably, the glucose dehydrogenase of the invention for cofactor regeneration comprises or consists of the amino acid sequence of SEQ ID NO: 10.

[0062] Alternatively, the glucose dehydrogenase for cofactor regeneration preferably comprises an amino acid sequence encoded by a nucleic acid having at least 80%, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and especially 100% identity to SEQ ID NO: 9. Particularly preferably, the nucleic acid encoding the glucose dehydrogenase of the invention for cofactor regeneration comprises or consists of the nucleic acid sequence of SEQ ID NO: 9.

[0063] A further aspect of the present invention relates to the use of glucose dehydrogenase for cofactor regeneration, the glucose dehydrogenase comprising: i) an amino acid sequence having at least 80% identity to SEQ ID NO: 10; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 9, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 9 The amino acid sequence comprises or consists of an amino acid sequence selected from the group consisting of:

[0064] The NAD(P)H oxidase used for cofactor regeneration (see FIG. 1) may be from one of the groups EC 1.6.3.1 (NAD(P)H oxidase (HO forming)), EC 1.6.3.2 (NAD(P)H oxidase (HO forming)), EC 1.6.3.3 (NADH oxidase (HO forming)) and EC 1.6.3.4 (NADH oxidase (HO forming)), where the HO forming class is particularly preferred.

[0065] Particularly preferred HO-forming NAD(P)H oxidases preferably i) an amino acid sequence having at least 80% identity to SEQ ID NO: 8; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 7, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 7 The amino acid sequence comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NO:7:

number

number

[0066] Preferably used HO-forming NAD(P)H oxidases preferably comprise or consist of an amino acid sequence having at least 80%, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and especially 100% identity to SEQ ID NO: 8. Particularly preferably, the HO-forming NAD(P)H oxidase comprises or consists of the amino acid sequence of SEQ ID NO: 8.

[0067] Alternatively, the HO-forming NAD(P)H oxidase preferably has an amino acid sequence encoded by a nucleic acid having at least 80%, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and especially 100% identity to SEQ ID NO: 7. Particularly preferably, the nucleic acid encoding the HO-forming NAD(P)H oxidase comprises or consists of the nucleic acid sequence of SEQ ID NO: 7.

[0068] A further aspect of the present invention is i) an amino acid sequence having at least 80% identity to SEQ ID NO: 8; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 7, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 7 Cofactor regeneration (NAD(P)H to NAD(P)) comprising or consisting of an amino acid sequence selected from the group consisting of: + The present invention relates to the use of HO-forming NAD(P)H oxidase for the treatment of inflammatory bowel diseases.

[0069] In combination with cofactor regeneration, the enzymatic strategy presented here enables a biocatalytic, environmentally friendly, and highly efficient production method for L-psicose.

[0070] The oxidoreductase used according to the present invention to form L-psicose from allitol is preferably a mannitol dehydrogenase-type short-chain dehydrogenase / reductase. Surprisingly, the mannitol dehydrogenase-type short-chain dehydrogenase / reductase does not react with NAD(P) + It has been shown that allitol can be converted to L-psicose in the presence of the cofactor NAD(P). + The present invention relates to a method for producing L-psicose, which comprises a step of treating allitol with an oxidoreductase, preferably a mannitol dehydrogenase-type short-chain dehydrogenase / reductase, in the presence of a cofactor.

[0071] The oxidoreductase for forming L-psicose from allitol is i) an amino acid sequence having at least 80% identity to SEQ ID NO: 2; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 1; and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 1 The amino acid sequence comprises or consists of an amino acid sequence selected from the group consisting of:

[0072] The first NAD(P)H-dependent oxidoreductase for reducing D-psicose to allitol is preferably i) an amino acid sequence having at least 80% identity to SEQ ID NO: 4, SEQ ID NO: 12 or SEQ ID NO: 14; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO:3, SEQ ID NO:11 or SEQ ID NO:13, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 11, or SEQ ID NO: 13; The amino acid sequence comprises or consists of an amino acid sequence selected from the group consisting of:

[0073] Generally particularly suitable for reducing a first D-psicose to allitol or D-psicose to allitol are oxidoreductases whose amino acid sequence has at least 80% identity to SEQ ID NO:4, SEQ ID NO:12, or SEQ ID NO:14, or is encoded by a nucleic acid having at least 80% identity to SEQ ID NO:3, SEQ ID NO:11, or SEQ ID NO:13, or which bind under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:3, SEQ ID NO:11, or SEQ ID NO:13. Moreover, surprisingly, these oxidoreductases can be used to oxidize allitol to D-psicose. SEQ ID NO:3:

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[0074] The oxidoreductase referred to herein for the reduction of D-psicose to allitol and / or the formation of L-psicose from allitol preferably comprises an amino acid sequence having at least 80%, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and especially 100% identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 12, or SEQ ID NO: 14. Particularly preferably, the oxidoreductase of the invention for the reduction of D-psicose to allitol comprises or consists of the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 12, or SEQ ID NO: 14, and the oxidoreductase for the formation of L-psicose from allitol comprises or consists of the amino acid sequence of SEQ ID NO: 2.

[0075] Alternatively, the oxidoreductase for the reduction of D-psicose to allitol and / or the formation of L-psicose from allitol preferably comprises an amino acid sequence encoded by a nucleic acid having at least 80%, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and particularly 100% identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 11, or SEQ ID NO: 13. Particularly preferably, the nucleic acid encoding the oxidoreductase of the invention for the reduction of D-psicose to allitol comprises or consists of the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 11, or SEQ ID NO: 13, and the nucleic acid encoding the oxidoreductase for the formation of L-psicose from allitol comprises or consists of the nucleic acid sequence of SEQ ID NO: 1.

[0076] As used herein, the term "identity" refers to the percentage of identical nucleotides or amino acids between at least two nucleotide or amino acid sequences that have been aligned ("aligned") using a standardized algorithm. Such algorithms can insert gaps in the sequences being compared in a standardized and reproducible manner to optimize the alignment between the two sequences and thus achieve a more meaningful comparison of the two sequences.

[0077] The percentage identity between sequences can be determined using one or more computer algorithms or programs known in the state of 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 includes several programs, including a tool called "BLAST2 sequences," which is used for direct pairwise comparison of two nucleotide or amino acid sequences. "BLAST2 sequences" can also be searched and used interactively on the Internet via the NCBI World Wide Web page. The blastn program (for nucleotide sequences) uses the following default settings: word length (W) of 11, expectation (E) of 10, M=5, N=-4, and comparison of both strands. For amino acid sequences, the blastp program uses as defaults a word length of 3 and an expectation (E) of 10, and the BLOSUM62 scoring matrix (Henikoff & Henikoff, 1989), alignments (B) of 50, expectation (E) of 10, M=5, N=-4.

[0078] Alternatively, the oxidoreductase for reducing D-psicose to allitol and / or forming L-psicose from allitol preferably comprises an amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 11, or SEQ ID NO: 13 under stringent conditions. As used herein, "stringent conditions" refer to conditions under which so-called specific hybrids are formed but nonspecific hybrids are not. For example, stringent conditions include hybridization in 6xSSC (sodium chloride / sodium citrate) at 45°C, followed by washing with 0.2-1xSSC, 0.1% SDS at 50-65°C, or such conditions may include hybridization in 1xSSC at 65-70°C, followed by washing with 0.3xSSC at 65-70°C. Hybridization can be carried out by conventional methods such as those described in J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989).

[0079] One aspect of the present invention relates to the use of an oxidoreductase for the formation of L-psicose from allitol, the oxidoreductase comprising: i) an amino acid sequence having at least 80% identity to SEQ ID NO: 2; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 1; and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 1 The amino acid sequence comprises or consists of an amino acid sequence selected from the group consisting of:

[0080] The use of an oxidoreductase for reducing D-psicose to allitol is disclosed, the oxidoreductase comprising: i) an amino acid sequence having at least 80% identity to SEQ ID NO: 4, SEQ ID NO: 12 or SEQ ID NO: 14; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO:3, SEQ ID NO:11 or SEQ ID NO:13, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 11, or SEQ ID NO: 13; The amino acid sequence comprises an amino acid sequence selected from the group consisting of:

[0081] Depending on the reaction (reduction or oxidation), the oxidoreductase enzymes of the invention require the corresponding cofactor, as described above.

[0082] material D-psicose was purchased from TCI and Hunan Garden Naturals Inc. (China), allitol and L-psicose were purchased from TCI, D-fructose, NADPH tetrasodium salt, and methanol were purchased from PanReac AppliChem (ITW Reagents), D-glucose, sodium gluconate, and IPTG (isopropyl-β-D-thiogalactopyranoside) were purchased from Sigma-Aldrich, and potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and NAD + , NADH disodium salt, NADP + Disodium salt and sodium dodecyl sulfate (SDS) were purchased from Carl Roth, and triethanolamine (TEA) was purchased from Chem-Lab NV.

[0083] Enzyme production and lysate preparation General information regarding the expression of recombinant enzymes in E. coli For recombinant enzyme production in Escherichia coli strains, the gene to be expressed was first amplified in PCR using genomic DNA or its synthetic equivalent, adapted to E. coli codon usage, as a template, along with specific oligonucleotides containing additional restriction endonuclease recognition sequences, and isolated from the reaction mixture. After digestion of the nucleic acid with the restriction enzymes SphI and HindIII, the gene fragment encoding the target enzyme was ligated into the SphI- and HindIII-cleaved backbone of the expression vector pQE70-Kan. The ligation product was transformed into chemically competent Top10F E. coli cells, and the resulting colonies were used for plasmid isolation and restriction analysis.

[0084] The results of the cloning process were verified by restriction enzyme digestion and DNA sequencing. The resulting construct harbors the target gene under an IPTG-inducible T5 promoter.

[0085] For overexpression of the enzyme in E. coli, the resulting expression plasmid was transformed into competent expression cells RB791. After 24 hours of incubation at 37°C, the resulting colonies were inoculated into LB medium for expression assays.

[0086] The next day, grow the expression culture to an optical density OD of 0.02. 550 and inoculated at an OD of 0.3. 550 The mixture was shaken at 37°C until an OD of 0.5 was reached. The temperature was then lowered to 25°C. 550 Once the ATP concentration reached 0.1 mM, the cultures were induced with 0.1 mM IPTG. After 22 hours, the cultures were harvested (separated from the medium by centrifugation in the form of a cell pellet) and analyzed for expression of the recombinant enzyme using SDS gel electrophoresis and activity determination (using a test or optical enzyme assay).

[0087] Preparation of cell lysates using sonifier disruption To prepare the cell suspension, the cell pellet prepared according to the method described above was weighed into an appropriate container, mixed with buffer and lysozyme (final concentration 0.5 mg / ml) (e.g., triethanolamine (TEA)-HCl), and dissolved under stirring. The mass fraction of biomass was usually 20%, with the remainder being buffer.

[0088] A Branson Sonifier 450 was used for cell disruption. The suspension was treated three times with 15 ultrasonic pulses each (device settings: timer = 15; duty cycle = 50; output control = 3-5).

[0089] The resulting homogenate was centrifuged at 16,000 rpm for 10 minutes at 4°C (Eppendorf centrifuge 5417R) to separate insoluble cell fragments and obtain a lysate.

[0090] [Table 1]

[0091] Analysis method High-performance liquid chromatography D-psicose / L-psicose, D-fructose, D-glucose, and allitol were quantified 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). Measurements were performed using a Phenomenex Rezex RPM-Monosaccharide Pb+2 (8%) column with an appropriate precolumn, and eluted isocratically with ultrapure water.

[0092] High-Performance Anion-Exchange Chromatography D-gluconic acid / D-gluconate was quantified using HPAEC (high-performance anion exchange chromatography) on a Dionex ICS6000 system equipped with an AS-AP autosampler. Measurements were performed using conductivity detection (CD) coupled to a Dionex AERS500 electrolytically regenerated suppressor in external water mode. Analytes were separated using a Dionex lonPac AS11-HC-4 μm column with an appropriate precolumn and NaOH gradient. The mobile phase was further pretreated with a Dionex ATC anion trap column.

[0093] Determination of enzyme activity (optical-enzyme assay) The 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 via the change in absorbance at a wavelength of 340 nm. Measurements were performed in the presence of 0.2 mM of the cofactor (NAD(P) + or NAD(P)H). For this purpose, a cuvette (Greiner Bio-One Semi-Micro Cuvette made of polystyrene) was filled with 20 μl of a 10 mM stock solution of the cofactor and adjusted to the desired pH with 100 mM TEA-HCl buffer (870 μl). 10 μl of lysate (diluted or undiluted) and 100 μl of substrate solution were added to the cuvette and the measurement was started immediately. Measurements were carried out at 25°C by default. The enzyme activity of the lysate was determined by the extinction coefficient of NADH / NADPH at 340 nm (E = 6220 Lmol -1 cm -1 ) can be used to calculate the conversion rate in U / ml (based on the volume of lysate) or U / g (based on the biomass used for production), where 1 U represents 1 pmol of substrate converted per minute (1 U = 1 pmol / min = 1 μmol / min = 1.67 10 -8 kat).

[0094] The following examples explain in more detail preferred variants of the method according to the invention: The lysates used in these examples were prepared according to the procedure described above.

[0095] Example 1 Conversion of D-fructose to L-psicose (cofactor regeneration by ADH during the reduction step) The following components were mixed in a glass vial: 70.6 μl of deionized water, 250 μl of 200 mM TEA HCl buffer (pH 8), 50 μl of D-fructose solution (500 g / L), and 25 μl of D-psicose-3-epimerase lysate, followed by 35 μl of SDR I lysate, 8 U of alcohol dehydrogenase lysate, and 5 μl of 10 mM NAD. + solution, and 50 μl of 2-propanol were added. The mixture was incubated for a total of 20 h with continuous shaking (Eppendorf Thermomixer; 35° C., 800 rpm).

[0096] The mixture was heated to 70° C. for 60 minutes. After cooling, 25 μl of oxidoreductase lysate (SEQ ID NO: 2), 10 U of NAD(P)H oxidase lysate, and 5 μl of 5 mM NADP + The solution was added, and the mixture was incubated for a further 20 hours with continuous shaking (Eppendorf Thermomixer; 24°C, 800 rpm).

[0097] For analysis, 100 μl of the mixture was added to 200 μl of methanol and incubated for 15 min at 60 °C and 1200 rpm in an Eppendorf Thermomixer. The sample was briefly centrifuged in a centrifuge, 700 μl of deionized water was added, vortexed, and then centrifuged at maximum g for 5 min. 200 μl of the supernatant was transferred to an HPLC vial with an insert and measured by HPLC (RI detection).

[0098] In this way, 97.7% (50 g / l) of the D-fructose was converted into L-psicose (measured concentration: 45.8 g / l).

[0099] Because D-psicose and L-psicose are indistinguishable by the chromatographic method used (they elute at the same retention time), 25 μl of D-psicose-3-epimerase lysate was added to the vial, and the mixture was incubated at 35° C. for 2 hours and reanalyzed by HPLC.

[0100] No change in the amount of psicose was observed after addition of epimerase, confirming L-psicose as the product.

[0101] Example 2 Conversion of D-fructose to L-psicose (cofactor regeneration by GDH during the reduction step) The following components were mixed in a glass vial: 134 μl of deionized water, 50 μl of 500 mM TEA HCl buffer (pH 7.5), 250 μl of a solution containing D-fructose and D-glucose (300 g / L each), and 25 μl of D-psicose-3-epimerase lysate, followed by 35 μl of SDR I lysate, 2 U of glucose dehydrogenase lysate, and 5 μl of 10 mM NAD. + The mixture was incubated with continuous shaking (Eppendorf Thermomixer; 35°C, 800 rpm) for a total of 30 hours.

[0102] After the mixture was cooled to 24°C, 25 μl of oxidoreductase lysate (SEQ ID NO: 2), 10 U of NAD(P)H oxidase lysate, and 5 μl of 5 mM NADP + The solution was added, and the mixture was incubated for a further 20 hours with continuous shaking (Eppendorf Thermomixer; 24°C, 800 rpm).

[0103] For analysis, 100 μl of the mixture was added to 200 μl of methanol and incubated for 15 minutes at 60°C and 1200 rpm in an Eppendorf Thermomixer. The sample was briefly centrifuged in a centrifuge, 700 μl of deionized water was added, vortexed, and then centrifuged at maximum g for 5 minutes. 200 μl of the supernatant was transferred to an HPLC vial and measured by HPLC (RI detection). For HPAEC measurement (conductivity detection), the clear supernatant was diluted 1:250.

[0104] In this way, 25.6% of D-fructose (150 g / l) was converted to L-psicose (measured concentration: 29.9 g / l), and D-glucose was completely converted to D-gluconic acid (measured concentration: 158 g / l).

[0105] Example 3 Oxidation of allitol to L-psicose The following components were mixed in a glass vial: 30 μl of deionized water, 250 μl of 200 mM TEA HCl buffer (pH 8), 125 μl of allitol solution (200 g / l), 50 μl of oxidoreductase lysate (SEQ ID NO: 2), 40 μl of NAD(P)H oxidase lysate, and 5 μl of 10 mM NADP. + The mixture was incubated for a total of 20 hours with continuous shaking (Eppendorf Thermomixer; 35°C, 800 rpm).

[0106] For analysis, 100 μl of the mixture was added to 200 μl of methanol and incubated for 15 min at 60 °C and 1200 rpm in an Eppendorf Thermomixer. The sample was briefly centrifuged in a centrifuge, 700 μl of deionized water was added, vortexed, and then centrifuged at maximum g for 5 min. 200 μl of the supernatant was transferred to an HPLC vial and measured by HPLC (RI detection).

[0107] In this way, 98.2% of allitol (50 g / l) was oxidized to L-psicose (actual concentration: 50.1 g / l).

[0108] literature Table L1 Table L2

Table L3

Table L5

Claims

1. A first D-psicose is formed from D-fructose present in an aqueous solution by treatment with an epimerase in vitro, and then the first D-psicose is reduced to allitol by treatment with a respective NAD(P)H-dependent oxidoreductase in vitro, and after inactivation of the epimerase and / or ultrafiltration, the respective NAD(P)H-dependent oxidoreductase is reduced to form L-psicose. ＋ The method for preparing an aqueous solution containing L-psicose includes adding a deactivated epimerase and a deactivated oxidoreductase, and then removing the deactivated epimerase and the deactivated oxidoreductase.

2. the oxidoreductase for the formation of L-psicose from allitol is i) an amino acid sequence having at least 80% identity to SEQ ID NO:2; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 1; and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 1; 2. The method of claim 1, wherein the amino acid sequence comprises an amino acid sequence selected from the group consisting of:

3. The oxidized cofactor NAD(P) formed by the reduction ＋ 3. The method according to claim 1, wherein is reduced by an alcohol dehydrogenase and a secondary alcohol with the formation of a ketone.

4. 4. The method according to claim 3, wherein the secondary alcohol is D-glucose or 2-propanol.

5. 5. The process according to any one of claims 1 to 4, characterized in that it is carried out as a one-pot reaction without isolation of the intermediate products.

6. 6. The method according to claim 1, wherein the enzymes are present as lysates of the corresponding cells that produce them.

7. the NAD(P)H-dependent oxidoreductase for the reduction of the first D-psicose to allitol is i) an amino acid sequence having at least 80% identity to SEQ ID NO:4, SEQ ID NO:12 or SEQ ID NO:14; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO:3, SEQ ID NO:11, or SEQ ID NO:13; and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 11, or SEQ ID NO: 13; The method according to any one of claims 1 to 6, characterized in that the amino acid sequence is selected from the group consisting of:

8. The alcohol dehydrogenase i) an amino acid sequence having at least 80% identity to SEQ ID NO:6; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO:5, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 5 8. The method according to any one of claims 3 to 7, characterized in that it comprises or consists of an amino acid sequence selected from the group consisting of:

9. the oxidized cofactor NAD(P) formed by the reduction ＋ is reduced by glucose dehydrogenase and D-glucose with the formation of D-gluconic acid.

10. Glucose dehydrogenase is i) an amino acid sequence having at least 80% identity to SEQ ID NO: 10; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO:9, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 9 10. The method of claim 9, comprising or consisting of an amino acid sequence selected from the group consisting of:

11. Use of an aqueous solution preparable by the method according to any one of claims 1 to 10 for preparing a syrup containing L-psicose.

12. Use of an oxidoreductase for forming L-psicose from allitol, the oxidoreductase comprising: i) an amino acid sequence having at least 80% identity to SEQ ID NO:2; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 1; and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 1; Use of an oxidoreductase, characterized in that it comprises an amino acid sequence selected from the group consisting of:

13. Use of an oxidoreductase for reducing D-psicose to allitol, wherein the oxidoreductase comprises: i) an amino acid sequence having at least 80% identity to SEQ ID NO:4, SEQ ID NO:12 or SEQ ID NO:14; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO:3, SEQ ID NO:11, or SEQ ID NO:13; and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 11, or SEQ ID NO: 13; 10. Use of an oxidoreductase comprising an amino acid sequence selected from the group consisting of:

14. Use of an alcohol dehydrogenase for cofactor regeneration, the alcohol dehydrogenase comprising: i) an amino acid sequence having at least 80% identity to SEQ ID NO:6; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO:5, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 5 10. Use of an alcohol dehydrogenase comprising or consisting of an amino acid sequence selected from the group consisting of:

15. Use of glucose dehydrogenase for cofactor regeneration, said glucose dehydrogenase comprising: i) an amino acid sequence having at least 80% identity to SEQ ID NO: 10; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO:9, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 9 10. Use of a glucose dehydrogenase comprising or consisting of an amino acid sequence selected from the group consisting of:

16. i) an amino acid sequence having at least 80% identity to SEQ ID NO: 8; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 7; and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 7 H for cofactor regeneration comprising or consisting of an amino acid sequence selected from the group consisting of: ２ Use of O-forming NAD(P)H oxidase.

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