Method for preparing an aqueous solution containing allose

Abstract

Treatment with epimerase in vitro forms D-psicose from D-fructose dissolved in aqueous solution, and then D-psicose is converted into the oxidized cofactor NAD(P). + A method for preparing an aqueous solution containing allose by reducing allitol to allitol in vitro with NAD(P)H-dependent oxidoreductase, which is accompanied by the formation of a compound, and then enzymatically oxidizing allitol to allose. (Figure 1)

Description

[Technical Field]

[0001] The present invention relates to a method for preparing an aqueous solution of the rare monosaccharide allose. [Background technology]

[0002] D-Arrows D-allose, an aldohexose, is the C3 epimer of D-glucose and is a so-called rare sugar due to its limited natural occurrence. It is found, for example, as a component of glycosides in certain types of seagrass (Kannan et al., 2012) or in a South African plant called Protea rubropilosa (Transvaal sugarbush) (Perold et al., 1973).

[0003] D-allose is a sweet sugar (80% sweeter than sucrose), but unlike the latter, it contains almost no calories (Mooradian et al., 2017). Furthermore, D-allose possesses many other positive properties, including antitumor (e.g., bladder cancer (Tohi et al., 2022)), antioxidant (Ishihara et al., 2011), and anti-inflammatory (Gao et al., 2011) activity. Further examples and physiological effects of D-allose are described in review articles (Chen et al., 2018, Lim & Oh, 2011, Mijailovic et al., 2021).

[0004] Because of its diverse applications, the need for D-allose cannot be met solely by natural sources; therefore, synthetic methods (chemical or biocatalytic) for producing D-allose based on more common (and therefore less expensive) monosaccharides such as D-fructose or D-glucose have been developed.

[0005] US9109266B2 describes the conversion of D-fructose to D-allose using NaOH or a strongly basic ion exchange resin (by the Lobry-de-Bruyn-Alberda-van-Ekenstein rearrangement mechanism), which results in numerous byproducts that must be separated, such as D-glucose, D-mannose, D-altose, and D-psicose.

[0006] US5433793 discloses the ammonium molybdate-catalyzed isomerization of D-glucose to D-allose in an aqueous acetic acid medium at 130°C. This yields approximately 10 wt percent D-allose (based on dry mass), but isolation of the product requires activated carbon filtration, ion exchange, and pseudo-mobile bed chromatography.

[0007] Jumde et al. (2016) presented a chemosynthetic pathway based on D-glucose, which includes palladium-catalyzed regioselective oxidation at the C3 position, as well as stereoselective reduction to D-allose using borohydride reagents.

[0008] WO1997 / 042339A1 describes a method for converting sucrose to D-allose and the corresponding sugar alcohol, allitol. Fermentation by Agrobacterium tumefaciens oxidizes sucrose to 3-ketosucrose, which is converted to allosucrose by hydration (with Raney nickel). Cleavage of allosucrose can be catalyzed with an acid (cation exchanger) or enzymatically by invertase or β-fructosidase. Separation of the D-allose / D-fructose mixture can be achieved by either removal of D-fructose by yeast fermentation or by chromatography (ion exchanger). Allitol can be produced by hydrating D-allose (with Raney nickel).

[0009] The chemical methods described above are generally characterized by complex reaction steps with low conversion rates, poor atomic economics, and / or the formation of by-products, making them completely unsuitable for the industrial-scale production of D-allose.

[0010] These inefficient chemical synthesis routes have now been replaced by more efficient biocatalytic methods.

[0011] Based on D-glucose, three enzymatic steps result in D-allose. D-glucose is first isomerized to D-fructose (Nam, 2022) by glucose / xylose isomerase (GI / XI), which is then epimerized to D-psicose (also known as D-allulose) by 3-ketose epimerases (D-tagatose 3-epimerase (DTE), D-psicose / allulose 3-epimerase (DPE / DAE), or L-ribulose 3-epimerase (LRE)) (Zhang et al., 2016; Jiang et al., 2020). The final step (D-psicose → D-allose) is achieved through the effects of various isomerases: L-rhamnose isomerase (L-RI, EC5.3.1.14), D-ribose-5-phosphate isomerase (RPI, EC5.3.1.6), D-galactose-6-phosphate isomerase (GaPI, EC5.3.1.26), and D-glucose-6-phosphate isomerase (GlPI, EC5.3.1.9) (Chen et al., 2018; Lim & Oh, 2011).

[0012] L-rhamnose isomerase is by far the most important enzyme class for the isomerization of D-psicose and D-allose (Chen et al., 2018), and is described, for example, in EP1589102B1, EP1788089B1, EP1860195B1, US7205141B2, US7501267B2, US7691619B2, and US8748589B2.

[0013] The conversion of D-fructose to D-psicose by ketose-3-epimerase does not proceed completely, and an equilibrium relationship is formed between the two epimers. Depending on the reaction conditions (temperature 40-70°C, pH 6-11), the ratio ranges from 80:20 to 62.5:37.5 (D-fructose:D-psicose) (Zhang et al., 2016; Jiang et al., 2020).

[0014] The subsequent isomerization is also an equilibrium reaction, with the equilibrium on the D-psicose side, and depending on the conditions, the mass ratio is between 77:23 and 62.5:37.5 (D-psicose:D-allose) (Chen et al., 2018; Lim & Oh, 2011).

[0015] Lee et al. (2018) described a method for converting D-fructose to D-allose using DPE derived from Flavonifractor plautii and RPI (R132E mutant) derived from Clostridium thermocellum. It was possible to convert 600 g / l of D-fructose to 79 g / l of D-allose within 2 hours, which corresponds to a 13% conversion. Furthermore, 1 mM Co 2+ Ions are required as cofactors for DPE.

[0016] Li et al. (2020) presented a similar method: DPE derived from Ruminococcus sp. and L-RI derived from Bacillus subtilis were expressed in Escherichia coli and immobilized on resin. After a reaction time of 5 hours, equilibrium was achieved with a mass ratio of 66:24:10 (D-fructose:D-psicose:D-allose) (using 500 g / l D-fructose as the substrate).

[0017] US10689668B2 describes a fusion enzyme consisting of DPE from Ensifer adhaerens or Clostridium scindens and RPI from Persephonella marina EX-H1 (EDPE_RPI or CDPE_RPI), which also enables up to 13% conversion.

[0018] An alternative to the above isomerase is a commercially available glucose isomerase from Sweetzyme IT, which was used by Choi et al. to isomerize D-psicose to D-allose. Immobilizing the enzyme in a fixed-bed reactor enables easy separation of the product solution (Choi et al., 2021).

[0019] Since the combination of epimerization and isomerization does not allow for complete conversion, D-allose must be separated from the resulting product mixture.

[0020] Depending on the isomerase used, the process can sometimes be further complicated by the fact that D-psicose can also result in D-altrose, the C2 epimer of D-allose (Chen et al., 2018, Lim & Oh, 2011). For example, by using recombinant L-RI from Pseudomonas stutzeri LL172, a product mixture consisting of 25% D-allose, 8% D-altrose, and 67% D-psicose was obtained based on D-psicose (Menavuvu et al., 2006).

[0021] Two methods for isolating D-allose from enzyme conversions are available: 1) crystallization from concentrated sugar solutions by adding ethanol (Menavuvu et al., 2006), or 2) SMB chromatography with subsequent crystallization from syrup (Morimoto et al., 2006).

[0022] Furthermore, fermentation methods for synthesizing D-allose are also known. Zheng et al. (2022) designed a new metabolic pathway in Escherichia coli (E. coli) based on GI, DAE, and RPI to convert D-glucose to D-allose - in this way, they obtained 0.127 g / l of D-allose after 84 hours, which corresponds to a yield of 0.045 g of D-allose per 1 g of D-glucose.

[0023] One option to avoid thermodynamically unfavorable epimerization and isomerization reactions is an enzyme cascade involving phosphorylated intermediates. The final step, dephosphorylation, is irreversible and thus drives the cascade (Li et al., 2021).

[0024] The cascades described in US10745683B2 and US11236320B2 feature D-glucose-1-phosphate (G1P) as the central intermediate. G1P is first converted to D-glucose-6-phosphate (G6P) by phosphoglucomutase and then further converted to D-fructose-6-phosphate (F6P) by phosphoglucose isomerase. Next, F6P is epimerized to D-psicose-6-phosphate by D-psicose-6-phosphate 3-epimerase and then reacts with D-allose-6-phosphate isomerase to become D-allose-6-phosphate. The final step is the dephosphorylation of D-allose-6-phosphate to D-allose by D-allose-6-phosphate phosphatase.

[0025] G1P can be directly produced, for example, from amylodextrin (obtained by hydrolysis of starch) or sucrose, by the action of phosphorylase with the consumption of phosphate. Since the terminal sugar monomers of oligosaccharides and polysaccharides cannot be phosphorylated by the corresponding phosphorylases, polyphosphate glucokinase (D-glucose → G6P) or polyphosphate fructokinase (D-fructose → F6P) must be used, and polyphosphate must be further added as a phosphate source to increase the yield (US10745683B2, US11236320B2).

[0026] The main drawback of pathways proceeding via phosphorylated sugar derivatives is the use of expensive, energy-rich phosphate compounds, such as polyphosphates, in stoichiometric amounts to introduce phosphate groups. While this problem can be partially circumvented by using phosphorylase, terminal monosaccharides cannot be phosphorylated without the help of energy-rich phosphate compounds. Furthermore, any remaining phosphate compounds and phosphate ions must be removed after the reaction is complete.

[0027] L-Arrows Unlike its enantiomer, D-allose, L-allose does not exist in nature. Only a few methods for producing L-allose are known. A biocatalytic pathway was proposed by Terami et al., which is also based on the rare monosaccharide L-psicose. This is isomerized to L-allose by immobilized L-ribose isomerase from Cellulomonas parahominis MB426. At equilibrium, it is a mixture of 33:77 (L-allose:L-psicose), which can be separated by chromatography (Terami et al., 2015).

[0028] Alitol as an intermediate Reduction of D-allose at the C1 position yields the achiral sugar alcohol allitol (see, e.g., WO1997 / 042339A1), which plays a central role in the so-called Izumoring strategy for the bioproduction of rare sugars as an interface between D-hexoses and L-hexoses (Izumori, 2006; Hassanin et al., 2017).

[0029] On the other hand, allitol can be produced based on a combination of D-fructose, as well as DTE, ribitol dehydrogenase (RDH), and formate dehydrogenase (FDH, as the regenerating enzyme for the cofactor nicotinamide adenine dinucleotide (NADH)). In this way, the equilibrium can be completely shifted to the product side (D-psicose, followed by allitol), and allitol can be obtained in high purity (Takeshita et al., 2000).

[0030] Wang et al. (2023) described a whole-cell biocatalyst in Escherichia coli (E. coli) for the conversion of D-fructose to alitol. The E. coli cells used contained DPE from Clostridiales, RDH from Providencia alcalifaciens, FDH from Starkeya, and another DPE from Rhizobium straminoryzae. In this way, D-fructose (500 mM = 90 g / l) was converted to 452 mM alitol within 12 hours at 37°C and pH 6 using 1000 mM sodium formate (conversion rate 90.4%). Approximately 30 mM D-sorbitol (the reduction product of D-fructose) was obtained as a byproduct.

[0031] Subsequent oxidation of the hydroxyl group at the C1 position of allitol yields the C6 position of D-allose and L-allose. Currently, no biocatalytic methods are known for either of these oxidation processes.

[0032] Based on these findings, the present invention aims to provide a method for efficiently preparing an aqueous solution containing allose enzymatically. [Overview of the project]

[0033] The purpose is to form D-psicose in vitro from D-fructose dissolved in aqueous solution by treatment with epimerase, and then to convert D-psicose into the oxidized cofactor NAD(P). + This is achieved by reducing allitol to alitol by in vitro treatment with NAD(P)H-dependent oxidoreductase, which is accompanied by the formation of a compound, and then enzymatically oxidizing allitol to allose, preferably in vitro.

[0034] A further preferred variation of the method of the present invention is characterized in that, before allitol is enzymatically oxidized to allose, preferably in vitro, epimerase and NAD(P)H-dependent oxidoreductase are inactivated or removed from the aqueous solution by ultrafiltration. [Brief explanation of the drawing]

[0035] [Figure 1] The reaction steps of the method of the present invention are schematically shown in attached Figure 1.

[0036] Reference A refers to D-fructose, B to D-psicose, C to alitol, D to allose, E to 2-propanol, F to acetone, 1 to epimerization, 2 to reduction, 3 to alcohol dehydrogenase, 4 to oxidation, and 5 to NAD(P)H oxidase. [Modes for carrying out the invention]

[0037] A preferred variation of the method of the present invention involves the formation of the oxidized cofactor NAD(P) by reduction. + It is reduced by alcohol dehydrogenase and secondary alcohols, resulting in ketone formation.

[0038] 2-propanol has been shown to be particularly well-suited as a secondary alcohol.

[0039] Furthermore, the enzymatic oxidation of alitol to allose is equivalent to the corresponding NAD(P) +It is preferable to use a dependent oxidoreductase.

[0040] The present invention has been shown to be a one-pot reaction that does not require the isolation of intermediate products.

[0041] Furthermore, the present invention relates to a method for preparing an aqueous solution containing allose by oxidizing allitol, characterized in that the oxidation is performed enzymatically, preferably in vitro.

[0042] The enzymatic oxidation of alitol to allose is equivalent to the corresponding NAD(P) + This can be easily done using dependent oxidoreductase, which leads to the formation of the reduced cofactor NAD(P)H.

[0043] Furthermore, it has been shown that the enzymes in the method of the present invention are present in a suspension, homogenate, and / or lysate of the corresponding cells that produce them, with the lysate being particularly preferred.

[0044] In this context, suspension refers to a suspension of quiescent cells. These are collected after culturing (separated from the culture medium) and suspended in a suitable buffer system. In contrast to fermentation methods, which can also use whole cells, quiescent cells are no longer able to grow due to the removal of carbon sources and nutrients and are only useful for reacting substrates (Lin & Tao, 2017). In this context, homogenate refers to a suspension that has been physically and / or chemically (e.g., using pressure, lysozyme, or sonication) processed from which cellular components are released from the cells. 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).

[0045] In further variations, the enzyme may also be modified at its N-terminus with a water-soluble polymer such as polyethylene glycol, immobilized in or on a solid matrix, or be part of a fusion protein.

[0046] In further modifications, the enzyme can exist in powder, freeze-dried, or spray-dried form.

[0047] The reduced cofactor NAD(P)H, formed by oxidation, is converted using oxidase and oxygen. + It can be oxidized favorably.

[0048] In a more preferred variation of the method of the present invention, the epimerase and NAD(P)H-dependent oxidoreductase are inactivated by heat or removed from the aqueous solution by ultrafiltration.

[0049] The preferred concentration of D-fructose is 50-250 g / l.

[0050] The preferred concentration of allitol is 50-250 g / l.

[0051] The preferred temperature range for the method of the present invention is 25 to 45°C.

[0052] The preferred pH range for the method of the present invention is 7 to 8.5.

[0053] In a more particularly preferred embodiment of the method, only enzymes from the group of enzymes epimerases, dehydrogenases, reductases, and oxidases are used to convert the starting material, and one or more of these enzymes are selected from each of these groups.

[0054] The epimerase used in this method may be one of the groups called EC5.1.3.30 (D-psicose 3-epimerase) or EC5.1.3.31 (D-tagatose 3-epimerase / L-ribulose 3-epimerase), with the first being particularly preferred.

[0055] The enzyme used for the reduction of D-psicose belongs to the group of oxidoreductases, with ribitol dehydrogenase (EC 1.1.1.56) being particularly preferred.

[0056] The enzymes used for the oxidation of alitol to allose belong to the group of oxidoreductases, with xylose reductases (EC1.1.1.307, EC1.1.1.430, EC1.1.1.431) or aldo / ketoreductases being particularly preferred.

[0057] For the oxidation of alitol to allose, the oxidoreductase is preferably i) An amino acid sequence having at least 75% identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 23, ii) Amino acid sequences encoded by nucleic acids having at least 75% identity with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 24, and iii) An amino acid sequence encoded by a nucleic acid bound to the complementary strand of a nucleic acid molecule containing or consisting of the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 24, under stringent conditions (where stringent conditions include one or more washing steps at 65°C and a salt concentration of 0.1x~2xSSC). It contains an amino acid sequence selected from the group consisting of the following.

[0058] Sequence ID 1: [ka]

[0059] Sequence ID 2: [ka]

[0060] Sequence ID 3: [ka]

[0061] Sequence ID 4: [ka]

[0062] Sequence ID 5: [ka]

[0063] Sequence ID 6: [ka]

[0064] Sequence ID 7: [ka]

[0065] Sequence ID 8: [ka]

[0066] Sequence ID 9: [ka]

[0067] Sequence ID 10: [ka]

[0068] Sequence ID 11: [ka]

[0069] Sequence ID 12: [ka]

[0070] Sequence ID 13: [ka]

[0071] Sequence ID 14: [ka]

[0072] Sequence ID 15: [ka]

[0073] Sequence ID 16: [ka]

[0074] Sequence ID 17: [ka]

[0075] Sequence ID 18: [ka]

[0076] Sequence ID 23: [ka]

[0077] Sequence ID 24: [ka]

[0078] The oxidoreductase for the oxidation of allitol to allose preferably comprises or consists of an amino acid sequence having at least 75%, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and most preferably 100% identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 23. Particularly preferably, the oxidoreductase of the present invention for the oxidation of allitol to allose comprises or consists of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 23.

[0079] Alternatively, the oxidoreductase for the oxidation of alitol to allose preferably comprises or consists of an amino acid sequence encoded by a nucleic acid having at least 75% identity, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and most preferably 100% identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 24. Particularly preferably, the nucleic acid encoding the oxidoreductase of the present invention for the oxidation of alitol to allose comprises or consists of the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 24.

[0080] 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 using a standardized algorithm. Such algorithms can optimize the alignment between two sequences by inserting gaps into the sequences being compared in a standardized and reproducible manner, thereby achieving a more meaningful comparison of the two sequences.

[0081] The percentage of identity between sequences is known in the latest technology or can be determined using one or more computer algorithms or programs 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" used for direct pairwise comparison of two nucleotide or amino acid sequences. "BLAST2 Sequences" can also be interactively searched and used on the internet via the NCBI's Worldwide Webpage. The blastn program (for nucleotide sequences) uses, by default, a word size (W) of 28, an expected value (E) of 0.05, M=1, N=-2, and a comparison of both strands. For amino acid sequences, the blastp program defaults to a word size of 3 and an expected value of 0.05 (E), as well as a BLOSUM62 score matrix (Henikoff & Henikoff, 1989), alignment of 50 (B), expected value of 0.05 (E), M=1, and N=-2.

[0082] Alternatively, the oxidoreductase for the oxidation of alitol to allose preferably comprises or consists of an amino acid sequence encoded by a nucleic acid that binds to the complementary strand of a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 24, under stringent conditions. As used herein, stringent conditions mean conditions under which so-called specific hybrids are formed, but nonspecific hybrids are not.

[0083] Hybridization can be carried out by conventionally known methods, such as those described by J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989). Stringent conditions refer to washing at 65°C with a salt concentration of 0.1x to 2x SSC (1x SSC refers to a mixture of 0.15 M sodium chloride and 0.015 M sodium citrate).

[0084] The alcohol dehydrogenase (ADH) used for cofactor regeneration may come from one of two groups called EC1.1.1.1 (NAD-dependent ADH) and EC1.1.1.2 (NADP-independent ADH).

[0085] Cofactor regeneration (NAD(P)) + For the reduction of NAD(P) to NAD(P)H, an NAD(P)-dependent alcohol dehydrogenase is preferably, i) An amino acid sequence having at least 80% identity with SEQ ID NO: 19, ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO: 20, and iii) Under stringent conditions, the amino acid sequence encoded by the nucleic acid that binds to the complementary strand of the nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO: 20. It contains or consists of an amino acid sequence selected from the group comprising the following.

[0086] In general, an alcohol dehydrogenase having an amino acid sequence encoded by a nucleic acid that has at least 80% identity to SEQ ID NO: 19, or at least 80% identity to SEQ ID NO: 20, or that binds to the complementary strand of a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO: 20 under stringent conditions, is particularly suitable for cofactor regeneration.

[0087] Sequence ID 19: [ka]

[0088] Sequence ID 20: [ka]

[0089] The alcohol dehydrogenases referred to herein for cofactor regeneration preferably contain 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 most preferably 100% identity with SEQ ID NO: 19. Particularly preferably, the alcohol dehydrogenases of the present invention for cofactor regeneration contain or consist of the amino acid sequence of SEQ ID NO: 19.

[0090] 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 with SEQ ID NO: 20. Particularly preferably, the nucleic acid encoded by the alcohol dehydrogenase of the present invention for cofactor regeneration comprises or consists of the nucleic acid sequence of SEQ ID NO: 20.

[0091] A further aspect of the present invention relates to the use of alcohol dehydrogenase for cofactor regeneration (reduction of NAD(P) + to NAD(P)H), wherein the alcohol dehydrogenase is i) an amino acid sequence having at least 80% identity to SEQ ID NO: 19, ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 20, and iii) an amino acid sequence encoded by a nucleic acid that binds to the complementary strand of a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO: 20 under stringent conditions and comprises or consists of an amino acid sequence selected from the group consisting of

[0092] The NAD(P)H oxidase used for cofactor regeneration may be from one of the group called EC 1.6.3.1 (NAD(P)H oxidase (H2O2 formation)), EC 1.6.3.2 (NAD(P)H oxidase (H2O formation)), EC 1.6.3.3 (NADH oxidase (H2O2 formation)), and EC 1.6.3.4 (NADH oxidase (H2O formation)), and the class of H2O formation is particularly preferred.

[0093] For cofactor regeneration (oxidation of NAD(P)H to NAD(P) + ), a particularly preferred H2O-forming NAD(P)H oxidase is preferably i) an amino acid sequence having at least 80% identity to SEQ ID NO: 21, ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 22, and iii) an amino acid sequence encoded by a nucleic acid that binds to the complementary strand of a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO: 22 under stringent conditions and comprises or consists of an amino acid sequence selected from the group consisting of

[0094] SEQ ID NO: 21:

Chemical formula

[0095] Sequence ID 22: [ka]

[0096] A preferred H2O-forming NAD(P)H oxidase preferably contains or 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 with SEQ ID NO: 21. Particularly preferred is that the H2O-forming NAD(P)H oxidase contains or comprises the amino acid sequence of SEQ ID NO: 21.

[0097] Alternatively, the H2O-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 with SEQ ID NO: 22. Particularly preferably, the nucleic acid encoding the H2O-forming NAD(P)H oxidase contains or consists of the nucleic acid sequence of SEQ ID NO: 22.

[0098] Further aspects of the present invention include: i) An amino acid sequence having at least 80% identity with SEQ ID NO: 21, ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO: 22, and iii) Under stringent conditions, the amino acid sequence encoded by the nucleic acid that binds to the complementary strand of the nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO: 22. Cofactor regeneration (NAD(P)H of NAD(P)) includes or consists of an amino acid sequence selected from the group comprising + Regarding the use of H2O-forming NAD(P)H oxidase for oxidation to H2O. [Examples]

[0099] The enzymatic strategies presented herein, in combination with cofactor regeneration, enable a biocatalytic, environmentally friendly, and highly efficient production process for allose preparation.

[0100] material D-psicose is purchased from TCI and Hunan Garden Naturals Inc. (China), D-allose and alitol are purchased from TCI, and D-fructose, lysozyme, and NAD are also used. + NADH disodium salt, NADP + Disodium salt, tetrasodium NADPH, and methanol were purchased from PanReac AppliChem (ITW Reagents); zinc chloride and IPTG (isopropyl-β-D-thiogalactopyranoside) were purchased from Sigma-Aldrich; magnesium chloride hexahydrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and sodium dodecyl sulfate (SDS) were purchased from Carl Roth; and triethanolamine (TEA) was purchased from Chem-Lab NV.

[0101] Enzyme production and preparation of lysates General information on recombinant enzyme expression in Escherichia coli (E. coli) To produce recombinant enzymes in Escherichia coli strains, the gene to be expressed was first amplified by PCR using genomic DNA or a synthetic equivalent adapted to the codon usage frequency of E. coli as a template, along with a specific oligonucleotide further containing the recognition sequence of the restriction endonuclease, and isolated from the reaction mixture. After digestion of the nucleic acid with restriction enzymes SphI and HindIII, the gene fragment encoding the target enzyme was ligated to the SphI and HindIII-cleaved backbone of the expression vector pQE70-Kan. The ligation product was transformed into chemically competent E. coli Top10F cells, and the resulting colonies were used for plasmid isolation and restriction analysis.

[0102] The results of the cloning process were validated by restriction enzyme digestion and DNA sequencing. The resulting constructs possess the target gene under an IPTG-inducible T5 promoter.

[0103] To overexpress the enzyme in Escherichia coli (E. coli), the obtained 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 testing.

[0104] The following day, the expression culture was subjected to an optical density (OD) of 0.02. 550 Inoculated with 0.3 OD 550 It was shaken at 37°C until it reached 0.5 OD. Then the temperature was lowered to 25°C and the OD was reduced to 0.5. 550 Once the target was reached, the culture was induced with 0.1 mM IPTG. After 22 hours, the culture was collected (separated from the medium in the form of a cell pellet by centrifugation) and recombinant enzyme expression was analyzed by SDS gel electrophoresis and activity determination (application of a test or optical enzyme assay).

[0105] Preparation of cell lysates by sonifier disruption To prepare the cell suspension, the cell pellet prepared according to the method described above was weighed into a suitable 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 is typically 20%, with the remainder being buffer.

[0106] 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, power control=3-5).

[0107] The resulting homogenate was centrifuged at 4°C and 16,000 rpm for 10 minutes (Eppendorf 5417R) to separate insoluble cell fragments and obtain a lysate. [Table 1]

[0108] Analysis method High-performance liquid chromatography D-psicose, D-fructose, and the intermediate alitol 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). For the measurements, a Phenomenex Rezex RPM-monosaccharide Pb+2 (8%) column was used with an appropriate pre-column, and elution was performed isocratically with ultrapure water.

[0109] High-speed anion exchange chromatography HPAEC was used to determine substrate conversion and product concentration during the oxidation of alitol to allose. The analytes were detected by pulsed amperometric detection (PAD). Here, a Dionex® CarboPac® PA210-fast-4μm column was used with a suitable pre-column, and elution was performed with a NaOH gradient.

[0110] Determination of enzyme activity (optical-enzyme assay) Enzyme activity in the lysate was determined using a Shimadzu UV-1900 spectrophotometer. For this purpose, the formation or consumption of NAD(P)H was monitored at a wavelength of 340 nm by changes in absorption. The measurement was performed using a 0.2 mM cofactor (NAD(P) + Alternatively, the procedure was performed using NAD(P)H. For this purpose, 20 μl of 10 mM stock solution of the cofactor was placed in a cuvette (Greiner bio-one Semi-Micro Cuvette made of polystyrene), and the pH was adjusted to the desired level using 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 immediately started. The measurement was performed at 25°C according to standards. The enzyme activity of the lysate was expressed as the extinction coefficient of NADH / NADPH at 340 nm (ε = 6220 l mol). -1 cm -1 This can be determined in U / ml (based on the volume of dissolved material) or U / g (based on the biomass used in production) using the following formula: where 1 U is the substrate conversion rate of 1 μmol per minute (1 U = 1 μmol / min = 1.67·10⁻¹⁰). -8 It refers to Kat.

[0111] The following examples illustrate in more detail preferred variations of the method according to the present invention. The solutions used in these examples were prepared according to the procedure described above.

[0112] Example 1 Oxidation of allitol to allose (accompanied by cofactor regeneration) The oxidation reaction was carried out in a 2 ml glass vial using allitol (final concentration 50 g / l) and NADP. +The final concentration of 0.05 mM was dissolved in 100 mM TEA-HCl buffer (pH 8). Furthermore, each solution contained 10 U of NAD(P)H oxidase lysate for cofactor regeneration. To initiate the reaction, 50 μl of oxidoreductase lysate (see Table 2 below) was added to each solution. The vials were incubated in an Eppendorf thermomixer with continuous shaking (30°C, 800 rpm) for 20 hours.

[0113] For analysis, 40 μl of the preparation was treated 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, treated with 760 μl of ultrapure water, vortexed, and then centrifuged at maximum g for 5 minutes. The supernatant was diluted 1:400 with ultrapure water and measured by HPAEC (PAD). The results are shown in Table 2 below. [Table 2]

[0114] Example 2 Oxidation of allitol to allose (without cofactor regeneration) The following components were mixed in two 2 ml glass vials (Preparations 1 and 2): 225 μl deionized water, 50 μl 500 mM TEA-HCl buffer (pH 8), 25 μl 100 g / l alitol solution (final concentration 5 g / l), and 275 μl 10 mM NADP. + Solution (final concentration 5.5 mM). To initiate the reaction, 25 μl of oxidoreductase lysate (see Table 3 below) was added to the preparation. The vial was incubated under continuous shaking (30°C, 800 rpm) for a total of 20 hours.

[0115] For analysis, 100 μl of the preparation was treated 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, treated with 700 μl of deionized water, vortexed, and then centrifuged at maximum g for 5 minutes. The supernatant was diluted 1:100 with ultrapure water and measured by HPAEC (PAD). The results are shown in Table 3 below. [Table 3]

[0116] The results in Table 3 show that cofactor regeneration during the conversion of alitol to allose (see Example 1) was advantageous in terms of yield, but not absolutely necessary.

[0117] Example 3 Preparation of allose from D-fructose The following components were mixed in three 2 ml glass vials (Preparations 1-3): 210.4 μl deionized water, 100 μl 500 mM TEA HCl buffer (pH 8), 50 μl D-fructose solution (500 g / l), and 25 μl D-psicose-3-epimerase lysate. Then, 35 μl ribitol dehydrogenase lysate, 12 U alcohol dehydrogenase lysate, and 5 μl 10 mM NAD + The solution and 50 μl of 2-propanol were added to the preparation. The preparation was incubated under continuous shaking (35°C, 800 rpm) for a total of 20 hours.

[0118] Next, preparations 1-3 were heated at 70°C for 60 minutes. After cooling, 25 μl of oxidoreductase lysate (see Table 4 below), 10 U of NAD(P)H oxidase lysate, and 5 μl of 5 mM NADP were added. + The solution was added. The preparation was incubated in an Eppendorf Thermomixer with continuous shaking (24°C, 800 rpm) for 24 hours.

[0119] The following components were mixed in three additional 2 ml glass vials (Preparations 4-6): 142.7 μl deionized water, 100 μl 500 mM TEA HCl buffer (pH 8), 50 μl D-fructose solution (500 g / l), and 25 μl D-psicose-3-epimerase lysate. Then, 35 μl ribitol dehydrogenase lysate, 12 U alcohol dehydrogenase lysate, and 5 μl 10 mM NAD + Solution, 50 μl of 2-propanol, 25 μl of oxidoreductase lysate (see Table 4 below), 10 U of NAD(P)H oxidase lysate, and 5 μl of 5 mM NADP + The solution was added to the prepared material. The prepared material was incubated under continuous shaking (30°C, 800 rpm) for a total of 44 hours.

[0120] For analysis, 50 μl of the preparation was treated 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, treated with 350 μl of deionized water, vortexed, and then centrifuged at maximum g for 5 minutes. 200 μl of the supernatant was transferred to an HPLC vial with an insert and measured by HPLC (RI detection). The supernatant was diluted 1:1000 with ultrapure water and measured by HPAEC (PAD). [Table 4]

[0121] Table 4 shows that the one-pot procedure for D-fructose conversion can be performed with or without thermal inactivation of the enzymes (epimerase and ribitol dehydrogenase) in the first step, and that conversion without inactivation provides better results. References

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Claims

1. Treatment with epimerase forms D-psicose in vitro from D-fructose dissolved in aqueous solution, and then the D-psicose is converted into the oxidized cofactor NAD(P). ＋ A method for preparing an aqueous solution containing allose by in vitro reduction of allitol to allitol by treatment with NAD(P)H-dependent oxidoreductase, which is accompanied by the formation of a compound, and then enzymatically oxidizing the allitol to allose.

2. The method according to claim 1, characterized in that the epimerase and the NAD(P)H-dependent oxidoreductase are inactivated or removed from the aqueous solution by ultrafiltration before the allitol is enzymatically oxidized to allose.

3. The oxidized cofactor NAD(P) formed by the reduction ＋ The method according to claim 1 or 2, characterized in that it is reduced by an alcohol dehydrogenase and a secondary alcohol, which results in the formation of a ketone.

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

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

6. A method for preparing an aqueous solution containing allose by oxidizing allitol, characterized in that the oxidation is carried out enzymatically.

7. The enzymatic oxidation of allitol to allose is accompanied by the formation of the corresponding NAD(P) H, which is a reduced cofactor. ＋ The method according to any one of claims 1 to 6, characterized in that it is carried out using a dependent oxidoreductase.

8. The method according to any one of claims 1 to 7, characterized in that the enzymes exist as lysates of the cells that produce them.

9. The reduced cofactor NAD(P)H formed by the aforementioned oxidation uses NAD(P)H oxidase and oxygen to produce NAD(P) ＋ The method according to claim 7, characterized by being oxidized to

10. The enzymatic oxidation of allitol to allose, i) An amino acid sequence having at least 75% identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 23, ii) Amino acid sequences encoded by nucleic acids having at least 75% identity with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 24, and iii) Amino acid sequences encoded by nucleic acids that bind to the complementary strand of a nucleic acid molecule containing or consisting of the nucleic acid sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 24 under stringent conditions. NAD(P) containing an amino acid sequence selected from the group consisting of ＋ The method according to any one of claims 1 to 9, characterized in that it is carried out using a dependent oxidoreductase.

11. The aforementioned cofactor NAD(P) ＋ The alcohol dehydrogenase for the reduction of the above is i) An amino acid sequence having at least 80% identity with SEQ ID NO: 19, ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO: 20, and iii) Amino acid sequence encoded by nucleic acid that binds to the complementary strand of a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO: 20 under stringent conditions. The method according to claim 3 or 4, characterized by comprising or consisting of an amino acid sequence selected from the group comprising the following.

12. NAD(P)H's NAD(P) ＋ The NAD(P)H oxidase for the oxidation of the above, i) An amino acid sequence having at least 80% identity with SEQ ID NO: 21, ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO: 22, and iii) Amino acid sequence encoded by nucleic acid that binds to the complementary strand of a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO: 22 under stringent conditions. The method according to claim 9, characterized in that it includes or consists of an amino acid sequence selected from the group consisting of the following.

13. To oxidize allitol to allose, i) An amino acid sequence having at least 75% identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 23, ii) Amino acid sequences encoded by nucleic acids having at least 75% identity with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 24, and iii) Amino acid sequences encoded by nucleic acids that bind to the complementary strand of a nucleic acid molecule containing or consisting of the nucleic acid sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 24 under stringent conditions. NAD(P) containing an amino acid sequence selected from the group consisting of ＋ Use of dependent oxidoreductase.

14. i) An amino acid sequence having at least 80% identity with SEQ ID NO: 19, ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO: 20, and iii) Amino acid sequence encoded by nucleic acid that binds to the complementary strand of a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO: 20 under stringent conditions. The use of an alcohol dehydrogenase for the reduction of NAD(P) to NAD(P)H, comprising or consisting of an amino acid sequence selected from the group consisting of ＋ .

15. i) An amino acid sequence having at least 80% identity with SEQ ID NO: 21, ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID NO: 22, and iii) Amino acid sequence encoded by nucleic acid that binds to the complementary strand of a nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO: 22 under stringent conditions. NAD(P)H contains or consists of an amino acid sequence selected from the group comprising the following: ＋ H for oxidation ２ Use of O-forming NAD(P)H oxidase.

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