Enzymatic preparation of dulcitol

Abstract

The invention relates to a preparation method of dulcitol, in particular to a method for preparing dulcitol through in-vitro enzyme catalysis, and belongs to the field of enzyme catalysis preparation of dulcitol. The method comprises the following steps: catalyzing by adopting galactiol-1-phosphate-5-dehydrogenase (EC 1.1. 1.251, GatD), converting tagatose-6-phosphoric acid (T6P) and NADH (Nicotinamide Adenine Dinucleotide) into galactiol-6-phosphoric acid (D6P) and NAD + (Nicotinamide Adenine Dinucleotide +), and carrying out a reaction on the galactiol-1-phosphate-5-dehydrogenase (EC 1.1. 1.251, GatD) to obtain the galactiol-6-phosphoric acid (T6P) and the NADH + (Nicotinamide Adenine Dinucleotide +). And catalyzing the D6P dephosphorylation group to generate dulcitol by adopting an enzyme capable of dephosphorylation group.

Description

Technical Field

[0001] The invention relates to a method for preparing dulcitol, in particular to a method for preparing dulcitol through in vitro enzyme catalysis, and belongs to the field of enzyme catalysis preparation of dulcitol. Background Art

[0002] Dulcitol, also known as galactitol, sweet alcohol, and hexane hexol, occurs naturally in some plants of the Celastraceae and Rosaceae families. It is one of the main active ingredients in Euonymus fortunei and has broad application prospects in medicine, food, and derivative synthesis. In the pharmaceutical industry, dulcitol has inhibitory effects on hepatocellular carcinoma and gliomas, is used to alleviate acute lung injury, and prevent and treat rheumatoid arthritis. In the food sector, dulcitol has prebiotic effects by regulating human intestinal flora and promoting bowel movements. It can be used as a substrate to produce healthy sugars such as D-tagatose and L-tagatose. Furthermore, dulcitol has applications in improving plant drought resistance and synthesizing dehydrated dulcitol and galactitol-based cross-linked polyesters.

[0003] The main method for producing dulcitol is to ferment galactose using yeast. For example, in 2019, Christopher V. Rao's group at the University of Illinois at Urbana-Champaign optimized the fermentation conditions of Rhodosporidium toruloides and converted 40 g / L galactose into 8.4 g / L dulcitol within 120 hours (Jagtap SS, Bedekar AA, Liu JJ, et al. Production of galactitol from galactose by the oleaginous yeast Rhodosporidium toruloides IFO0880. Biotechnology Biofuels, 2019, 12: 250). In 2022, Qi Xianghui's research group at Jiangsu University used Maggi yeast to produce D-tagatose, D-arabitol, and galactitol from dairy waste. They achieved 28.46 g / L of galactitol after 12 days of fermentation (Zhang G., Zabed H. M., An Y., et al. Biocatalytic conversion of a lactose-rich dairy waste into D-tagatose, D-arabitol and galactitol using sequential whole cell and fermentation technologies. Bioresource Technol, 2022, 358: 127422). This is the highest reported yield of galactitol to date. In summary, existing galactitol production technologies suffer from low product yields and low production intensity.

[0004] Therefore, there is an urgent need to develop cost-effective and feasible biomanufacturing methods for dulcitol. Summary of the Invention

[0005] The invention relates to a method for preparing dulcitol by an in vitro enzymatic method. The method produces dulcitol by catalysis of an in vitro enzyme reaction system. The method has the advantages of cheap raw materials, abundant sources, low production cost, and environmental friendliness.

[0006] The present invention is achieved through the following technical solutions:

[0007] The present invention provides a method for preparing dulcitol by using an in vitro enzyme catalytic reaction, comprising: using galactitol-1-phosphate 5-dehydrogenase (EC 1.1.1.251, GatD) to catalyze tagatose-6-phosphate (T6P) and NADH to convert dulcitol-6-phosphate (D6P) and NAD + and using an enzyme capable of dephosphorylating the D6P to dephosphorylate the D6P to generate dulcitol.

[0008] According to the present invention, preferably, the method further comprises a reaction step of converting fructose-6-phosphate (F6P) into T6P, which is catalyzed by tagatose 6-phosphate 4-epimerase (EC5.1.3.40, TPE).

[0009] According to the present invention, preferably, the method further comprises a reaction step of converting glucose-6-phosphate (G6P) into F6P, which is catalyzed by phosphoglucose isomerase (EC 5.3.1.9, PGI).

[0010] According to the present invention, preferably, the method further comprises a reaction step of converting glucose-1-phosphate (G1P) into G6P, which is catalyzed by phosphoglucomutase (EC 5.4.2.2, PGM).

[0011] According to the present invention, preferably, the method further comprises a reaction step of converting a substrate and phosphate into G1P, wherein the substrate is a disaccharide, a polysaccharide or any mixture thereof containing a D-glucose unit, and this step is catalyzed by an enzyme capable of converting the substrate and phosphate into G1P.

[0012] According to the present invention, various phosphates can be used in the present invention. Preferably, the phosphate is selected from one or a mixture of two or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, and disodium hydrogen phosphate. More preferably, the phosphate is derived from potassium dihydrogen phosphate and / or dipotassium hydrogen phosphate.

[0013] According to the present invention, preferably, the disaccharide comprising a D-glucose unit is sucrose, and sucrose and phosphate are converted into G1P using sucrose phosphorylase (EC 2.4.1.7, SP).

[0014] According to the present invention, preferably, the polysaccharide containing D-glucose units is selected from starch, starch derivatives, or any mixture thereof, and is converted into G1P by catalysis with phosphate using starch phosphorylase (α-glucan phosphorylase, EC 2.4.1.1, αGP). Preferably, the starch or starch derivative is selected from soluble starch, soluble amylose, soluble amylopectin, amylopectin, maltodextrin, and maltopolysaccharide. Preferably, the starch is soluble starch.

[0015] According to the present invention, preferably, the polysaccharide containing D-glucose units is further selected from cellulose, cellulose derivatives, or any mixture thereof. Preferably, the cellulose derivative is selected from cellopolysaccharide, cellobiose, or any mixture thereof. Preferably, the cellulose derivative is the product of acid or enzyme pretreatment of cellulose, and the product is cellopolysaccharide, cellobiose, or any mixture thereof. Preferably, the cellulose derivative is cellopolysaccharide. When the polysaccharide containing D-glucose units contains cellulose and / or cellopolysaccharide, for example, cellulose and / or cellopolysaccharide, cellopolysaccharide phosphorylase (EC 2.4.1.49, CDP) is used to catalyze the conversion of the cellobiose and phosphate produced by the degradation of cellulose and / or cellopolysaccharide into G1P. Preferably, cellobiose phosphorylase (EC 2.4.1.20, CBP) is further used to catalyze the conversion of the cellobiose and phosphate produced by the degradation of cellulose and / or cellopolysaccharide into G1P. When the polysaccharide comprising D-glucose units contains cellobiose, for example, cellobiose, cellobiose and phosphate are converted into G1P catalyzed by cellobiose phosphorylase (EC 2.4.1.20, CBP).

[0016] Those skilled in the art will appreciate that the above steps included in the method of the present invention can be performed step by step, for example, in one bioreactor or reaction vessel or in multiple bioreactors or reaction vessels arranged in series.

[0017] Those skilled in the art will appreciate that the above steps included in the method of the present invention may also be performed simultaneously, for example, in one bioreactor or reaction vessel. Preferably, the above steps included in the method of the present invention are performed simultaneously.

[0018] Preferably, the present invention provides a method for preparing dulcitol using an in vitro enzyme-catalyzed reaction, comprising: using a disaccharide, polysaccharide or any mixture thereof containing a D-glucose unit as a substrate, adding phosphate, NADH, and an enzyme capable of converting the disaccharide, polysaccharide or any mixture thereof containing a D-glucose unit and phosphate into G1P, phosphoglucomutase, phosphoglucose isomerase, tagatose-6-phosphate-4-epimerase, dulcitol-1-phosphate-5-dehydrogenase and an enzyme capable of dephosphorylating the phosphate group, and carrying out a catalytic reaction to obtain dulcitol.

[0019] Those skilled in the art will appreciate that dulcitol-1-phosphate-5-dehydrogenase from various sources can be used in the present invention. Preferably, dulcitol-1-phosphate-5-dehydrogenase can be derived from Escherichia coli (UniProt No. P0A9S3), Parageobacillus caldoxylosilyticus (UniProt nos. A0A150KWR3, A0A150KXC6), Moraxella thermoaceticum Moorella thermoacetica (UniProt number A0A1J5NLK4), Thermoleophilia bacterium (UniProt no. A0A6J4TB39) or Clostridium thermopalmarium (UniProt no. A0A2T0APR6).

[0020] It will be understood by those skilled in the art that enzymes capable of dephosphorylating groups from various sources can be used in the present invention. According to one aspect of the present invention, the enzyme capable of dephosphorylating groups is galactitol-6-phosphate phosphatase (D6PP) with substrate specificity. Preferably, galactitol-6-phosphatase can be derived from Haemophilus influenzae (UniProt No. P46452), Thermospora maritima ( Thermotoga maritima , UniProt number Q9X0Y1), Thermoplasma acidophilum (UniProt number Q9X0Y1), Streptomyces avermitilis (UniProt number Q82E90, Q82B76, Q82B95, etc.), Salmonella typhi ( Salmonella typhi , UniProt number Q8Z989) etc.

[0021] It will be understood by those skilled in the art that enzymes capable of converting substrates into G1P, phosphoglucomutase, phosphoglucose isomerase, and tagatose-6-phosphate-4-epimerase from various sources can be used in the present invention. Preferably, starch phosphorylase can be derived from Escherichia coli (Uniprot No. A0A0A0HB49), Thermotoga maritima ( Thermotoga maritima , Uniprot number G4FEH8), Clostridium thermocellum ( Clostridium thermocellum , Uniprot number A3DCB6) etc.; sucrose phosphorylase can be derived from Bifidobacterium adolescentis ( Bifidobacterium adolescentis , UniProt number A0ZZH6), Thermoanaerobacterium thermosaccharolyticum (UniProt number D9TT09), etc.; Cellulose polysaccharide phosphorylase can be derived from Clostridium thermocellum ( Clostridium thermocellum , UniProt number A3DJQ6), Clostridium stercorarium (UniProt No. P77846), etc.; Cellobiose phosphorylase can be derived from Clostridium thermocellum ( Clostridium thermocellum , UniProt number A3DC35), Thermotoga neapolitana (UniProt number B9K7M6), etc. Glucose phosphotyrosine mutase can be derived from Clostridium thermocellum ( Clostridium thermocellum , Uniprot number A3DEW8), Thermococcus kodakarensis (UniProt number Q68BJ6), etc.; phosphoglucose isomerase can be derived from Clostridium thermocellum ( Clostridium thermocellum , Uniprot number A3DBX9), Thermus thermophilus ( Thermus thermophilus , Uniprot No. Q5SLL6) etc. Tagatose-6-phosphate-4-epimerase can be derived from Ruminiclostridium herbifermentans (Uniprot number A0A4U7JK39), Dictyoglomus thermophilum (Uniprot No. B5YBD7), Agrobacterium tumefaciens ( Agrobacterium fabrum , Uniprot number A9CES6) etc.

[0022] According to the present invention, preferably, the temperature of the catalytic reaction is 30-80°C, more preferably 30-60°C, and most preferably 37°C.

[0023] According to the present invention, preferably, the pH of the catalytic reaction is 5.0-8.0, more preferably 6.0-7.5, most preferably 7.0.

[0024] According to the present invention, when the above steps are performed simultaneously, preferably, the catalytic reaction time is 1-48 hours, more preferably 8-36 hours, more preferably 10-24 hours, and most preferably 15 hours.

[0025] According to the present invention, when the above steps are performed step by step, preferably, the catalytic reaction time of each step is independently 0.5-10 hours, more preferably 1-3 hours, and most preferably 2 hours.

[0026] According to the present invention, preferably, the concentration of the substrate in the reaction system is 1-200 g / L, more preferably 5-50 g / L, more preferably 8-20 g / L, and most preferably 10 g / L.

[0027] According to the present invention, preferably, the concentration of the enzyme that converts the substrate into G1P in the reaction system is 0.1-10 U / mL, more preferably 0.2-5 U / mL, more preferably 1-3 U / mL, and most preferably 2 U / mL; preferably, the concentration of the phosphoglucomutase is 0.1-10 U / mL, more preferably 0.2-5 U / mL, more preferably 1-3 U / mL, and most preferably 2 U / mL; preferably, the concentration of the phosphoglucoisomerase is 0.1-10 U / mL, more preferably 1-5 U / mL, and most preferably 3 U / mL; preferably, the concentration of the tagatose-6-phosphate-4-epimerase is 0.1-10 U / mL, more preferably 1-5 U / mL, and most preferably 2 U / mL; preferably, the concentration of the dulcitol-1-phosphate-5-dehydrogenase is 0.1-10 U / mL, more preferably 0.2-5 U / mL, more preferably 2-4 U / mL, and most preferably 3 U / mL; preferably, the concentration of the enzyme capable of dephosphorylating the phosphoryl group is 0.1-10 U / mL, more preferably 0.2-5 U / mL, more preferably 1-3 U / mL, and most preferably 2 U / mL.

[0028] According to the present invention, preferably, the concentration of NADH in the reaction system is 0.1-50 mM, more preferably 0.5-10 mM, and most preferably 1 mM.

[0029] According to the present invention, preferably, the concentration of phosphate in the reaction system is 1-150 mM, more preferably 2-50 mM, more preferably 5-20 mM, and most preferably 10 mM. It will be appreciated by those skilled in the art that the phosphate produced during the dephosphorylation of D6P to produce dulcitol can be used as a phosphorus source in the step of converting a disaccharide, polysaccharide, or any mixture thereof containing D-glucose units into G1P.

[0030] According to the present invention, preferably, the above reaction system further contains a magnesium salt. Those skilled in the art will appreciate that various magnesium salts can be used in the present invention, such as magnesium chloride, magnesium sulfate, etc. Preferably, the magnesium salt is magnesium chloride. Preferably, the concentration of the magnesium salt in the reaction system is 1-20 mM, more preferably 2-15 mM, and most preferably 10 mM.

[0031] According to the present invention, preferably, the reaction system further contains a buffer. Those skilled in the art will appreciate that various buffers can be used in the present invention, such as HEPES buffer, Tris-HCl buffer, MOPS buffer, and citrate buffers, such as sodium citrate buffer. Preferably, the buffer is HEPES buffer. Preferably, the concentration of the buffer in the reaction system is 20 to 300 mM, preferably 50 to 200 mM, and most preferably 100 mM.

[0032] In order to achieve the regeneration of NADH in the reaction system, the method of the present invention further comprises using an enzyme that catalyzes the regeneration of NADH to achieve the regeneration of NAD + The enzymes that can regenerate NADH include formate dehydrogenase (EC1.17.1.9, FDH) and glucose dehydrogenase (EC 1.1.1.118, GDH).

[0033] It will be appreciated by those skilled in the art that enzymes capable of regenerating NADH from various sources can be used in the present invention. Preferably, formate dehydrogenase can be derived from Thiobacillus sp. (UniProt number Q76EB7), Candida boidinii ( Candida boidinii , UniProt No. O13437). Glucose dehydrogenase can be derived from Sulfolobus solfataricus (UniProt number Q97U21).

[0034] According to the present invention, preferably, the concentration of the enzyme for achieving NADH regeneration in the reaction system is 0.1-10 U / mL, more preferably 1-3 U / mL, and most preferably 2 U / mL.

[0035] As a preferred embodiment, the enzyme-catalyzed reaction of the present invention adopts the following method:

[0036] In a reaction system, starch was used as substrate, magnesium chloride, phosphate, NAD + , sodium formate, HEPES buffer (pH 7.0), starch phosphorylase, glucose phosphomutase, phosphoglucose isomerase, tagatose-6-phosphate-4-epimerase, dulcitol-1-phosphate-5-dehydrogenase, an enzyme capable of dephosphorylating the phosphate group (preferably dulcitol-6-phosphatase), and formate dehydrogenase to carry out a catalytic reaction to obtain dulcitol.

[0037] In a further preferred embodiment, in a reaction system, 10 g / L starch was used as substrate, 10 mM magnesium chloride, 10 mM potassium dihydrogen phosphate, 55 mM sodium formate, 1 mM NAD + , 100 mM HEPES buffer (pH 7.0), 2 U / mL starch phosphorylase, 2 U / mL phosphoglucomutase, 3 U / mL phosphoglucose isomerase, 2 U / mL tagatose-6-phosphate-4-epimerase, 3 U / mL dulcitol-1-phosphate-5-dehydrogenase, 2 U / mL of an enzyme capable of dephosphorylating a phosphate group (preferably dulcitol-6-phosphatase), and 2 U / mL formate dehydrogenase, and the reaction mixture is catalyzed at 37°C for 20 h to obtain dulcitol.

[0038] The present invention also provides the use of dulcitol-1-phosphate-5-dehydrogenase and an enzyme capable of dephosphorylating a phosphate group (preferably dulcitol-6-phosphatase) in preparing dulcitol, preferably in catalyzing T6P to generate dulcitol.

[0039] The present invention also provides dulcitol prepared by the above method.

[0040] The present invention is the first to utilize an in vitro enzymatic reaction to prepare dulcitol. Specifically, it is the first to utilize dulcitol-1-phosphate-5-dehydrogenase and an enzyme capable of dephosphorylating T6P to catalyze the production of dulcitol. The method of the present invention can yield the target product with a good conversion rate. Furthermore, the method of the present invention can utilize a variety of raw materials, such as disaccharides, polysaccharides, or any mixture thereof containing D-glucose units. Therefore, the method of the present invention offers advantages such as inexpensive raw materials, abundant sources, low production costs, and environmental friendliness, making it suitable for widespread application. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Schematic diagram of the in vitro enzymatic pathway for producing dulcitol using starch as a substrate. αGP represents starch phosphorylase, PGM represents phosphoglucomutase, PGI represents phosphoglucose isomerase, TPE represents tagatose-6-phosphate-4-epimerase, GatD represents dulcitol-1-phosphate-5-dehydrogenase, D6PP represents dulcitol-6-phosphatase, and FDH represents formate dehydrogenase.

[0042] Figure 2 Gibbs energy changes of reactions between intermediates in the in vitro enzymatic pathway for the preparation of dulcitol from starch.

[0043] Figure 3 3A is the HPLC peak diagram of a standard dulcitol; 3B is the quantitative analysis of dulcitol concentration by HPLC. The concentration of dulcitol can be quantified by the intensity of the dulcitol peak.

[0044] Figure 4 This is the reaction progress curve of fructose-6-phosphate to dulcitol catalyzed by enzyme in vitro.

[0045] Figure 5 This is the reaction process curve of in vitro enzyme-catalyzed starch synthesis of dulcitol.

[0046] Figure 6 This is the reaction progress curve of in vitro enzymatic conversion of sucrose to dulcitol.

[0047] Figure 7 This is the reaction progress curve of the in vitro enzymatic conversion of cellobiose to dulcitol. DETAILED DESCRIPTION

[0048] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0049] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0050] Some of the material information used in the examples of the present invention is as follows:

[0051] Starch, product of ACROS, product number: 424490020;

[0052] pET28a vector, Novagen, Madison, WI;

[0053] Escherichia coli expression strain BL21 (DE3), Invitrogen, Carlsbad, CA;

[0054] Example 1 Determination of enzyme activity in the enzymatic synthesis pathway of dulcitol

[0055] The catalytic pathway for the conversion of starch to dulcitol by an in vitro enzymatic system is shown in Figure 1 Among them, dulcitol-6-phosphatase is an example of an enzyme that can dephosphorylate groups. Figure 2 The Gibbs energy changes of reactions between intermediates in the enzyme-catalyzed pathway for the conversion of starch to dulcitol are shown. In this example, (1) starch phosphorylase was derived from Escherichia coli (Uniprot No. A0A0A0HB49); (2) glucose phosphomutase was derived from Clostridium thermocellum ( Clostridium thermocellum , Uniprot number A3DEW8); (3) phosphoglucose isomerase from Clostridium thermocellum ( Clostridium thermocellum, Uniprot number A3DBX9); (4) Tagatose-6-phosphate-4-epimerase from Agrobacterium falciparum ( Agrobacterium fabrum , Uniprot number A9CES6); (5) dulcetol-1-phosphate-5-dehydrogenase from Escherichia coli (UniProt number P0A9S3); (6) enzyme capable of dephosphorylating the phosphate group from Streptomyces avermitilis (UniProt number Q82E90); (7) formate dehydrogenase from Thiobacillus sp. (UniProt No. Q76EB7). The above genomic DNA can be obtained from the official website of ATCC (www.atcc.org). Simple cloning (You C, Zhang XZ, Zhang Y-HP. 2012. Simple cloning via direct transformation of PCR product (DNA Multimer) to Escherichia coli and Bacillus subtilis The above genes were cloned into the pET28a vector (Novagen, Madison, WI) using the method described in [Appl. Environ. Microbiol. 78(5):1593-5], resulting in the corresponding expression vectors pET28a-EcαGP, pET28a-CtPGM, pET28a-CtPGI, pET28a-AfTPE, pET28a-EcGatD, and pET28a-SaD6PP. The recombinant proteins were expressed in Escherichia coli BL21(DE3) and purified.

[0056] According to the method reported in the literature (Meng D, Wei X, Bai X, et al. Artificial in vitro synthetic enzymatic biosystem for the one-pot sustainable biomanufacturing ofglucosamine from starch and inorganic ammonia. ACS Catalysis, 2020, 10, 13809−13819), the glucosamine from Escherichia coli ( E. coli ) starch phosphorylase, derived from Clostridium thermocellum ( Clostridium thermocellum ) of glucose phosphotyrosinase, derived from Clostridium thermocellum ( Clostridium thermocellum ) were 5.6 U / mg, 20 U / mg and 396 U / mg at 37°C, respectively.

[0057] Derived from Agrobacterium facilis ( Agrobacterium fabrum The enzymatic activity of tagatose-6-phosphate-4-epimerase from Agrobacterium faucalis was determined in 100 mM HEPES buffer (pH 7.0). The reaction was performed at 37°C for 10 minutes using 10 mM fructose-6-phosphate as the substrate, and the amount of tagatose-6-phosphate (T6P) produced was determined. The T6P amount was determined as follows: 100 μl of the T6P-containing sample was added to 100 μl of 100 mM HEPES buffer (pH 7.0) containing 1.5 mM NADH and 1 U / mL dulcitol-1-phosphate-5-dehydrogenase (GatD). The reaction was incubated at 37°C for 30 minutes. The absorbance at 340 nm was measured, and the amount of NADH consumed was calculated. The results showed that the specific enzyme activity of tagatose-6-phosphate-4-epimerase from Agrobacterium faucalis at 37°C was 10 U / mg.

[0058] The enzymatic activity of dulcitol-1-phosphate-5-dehydrogenase from Escherichia coli was determined in 100 mM HEPES buffer (pH 7.0) containing 1 U / mL tagatose-6-phosphate-4-epimerase. The reaction was performed at 37°C for 10 minutes using 20 mM fructose-6-phosphate and 1.5 mM NADH as substrates, and NADH consumption was measured. The results showed that the specific enzyme activity of E. coli dulcitol-1-phosphate-5-dehydrogenase at 37°C was 3.8 U / mg.

[0059] Originated from Thiobacillus The activity of formate dehydrogenase from sp. was determined in the presence of 1.5 mM NAD + The reaction was carried out in 100 mM HEPES buffer (pH 7.0). 10 mM sodium formate was used as substrate and the reaction was carried out at 37°C for 10 min. The amount of NADH produced was determined. Thiobacillus The specific enzyme activity of formate dehydrogenase from sp. at 37°C was 6 U / mg.

[0060] In this reaction pathway, a key feature of the present invention is the presence of an enzyme capable of dephosphorylating D6P with specific dephosphorylation activity. The dephosphorylation activity of these enzymes on G1P, G6P, F6P, T6P, and D6P was measured in 100 mM HEPES buffer (pH 7.0) containing 10 mM magnesium chloride at 37°C. The results showed that the dulcitol-6-phosphatase from Streptomyces avermitilis (UniProt number Q82B76) had specific enzyme activities of 0 U / mg, 0.06 U / mg, 0.002 U / mg, 0.55 U / mg, and 2.3 U / mg for G1P, G6P, F6P, T6P, and D6P, respectively. This enzyme exhibited high specific activity for D6P and specific dephosphorylation activity.

[0061] Example 2 In vitro enzyme catalysis to convert fructose-6-phosphate into dulcitol

[0062] High performance liquid chromatography (HPLC) was used to quantitatively analyze dalucitol. The column used was a Bio-Rad Aminex HPX-87H, the mobile phase was a 5 mM aqueous sulfuric acid solution, the flow rate was 0.6 mL / min, the column temperature was 55°C, and the detector used was a differential refractive index detector. Figure 3 As shown in A, the retention time of dulcitol is about 11.906 minutes. The concentration of dulcitol is proportional to the response intensity of the HPLC characteristic peak of dulcitol. The standard curve is as follows Figure 3 As shown in B.

[0063] The mixture contained 50 mM fructose-6-phosphate, 10 mM magnesium chloride, 1 mM NAD + A 5 mL reaction mixture containing 50 mM sodium formate, 100 mM HEPES buffer (pH 7.0), 2 U / mL TPE, 3 U / mL GatD, 2 U / mL D6PP, and 2 U / mL FDH was incubated at 37°C for 10 h. Samples were taken at different times, and the concentration of dulcitol in the reaction solution was determined by high-performance liquid chromatography. After 6 h of reaction, the concentration of dulcitol was 41.5 mM, and the conversion rate relative to the initial amount of fructose-6-phosphate added was 83% ( Figure 4 ).

[0064] Example 3 In vitro enzyme-catalyzed synthesis of dulcitol from starch

[0065] The mixture contained 10 g / L starch, 10 mM magnesium chloride, 10 mM potassium dihydrogen phosphate, 55 mM sodium formate, 1 mM NAD +A 5 mL reaction mixture containing 100 mM HEPES buffer (pH 7.0), 2 U / mL αGP, 2 U / mL PGM, 3 U / mL PGI, 2 U / mL TPE, 3 U / mL GatD, 2 U / mL D6PP, and 2 U / mL FDH was incubated at 37°C for 20 h. Samples were taken at different times, and the concentration of dulcitol was determined by high-performance liquid chromatography. After 15 h of reaction, the concentration of dulcitol was 6.2 g / L, and the conversion rate relative to the initial amount of starch added was 62% ( Figure 5 ).

[0066] Example 4 In vitro enzymatic conversion of sucrose to dulcitol

[0067] The mixture contained 10 g / L sucrose, 10 mM magnesium chloride, 10 mM potassium dihydrogen phosphate, 28 mM sodium formate, 1 mM NAD + A 5 mL reaction mixture containing 100 mM HEPES buffer (pH 7.0), 2 U / mL SP, 2 U / mL PGM, 3 U / mL PGI, 2 U / mL TPE, 3 U / mL GatD, 2 U / mL D6PP, and 2 U / mL FDH was incubated at 37°C for 12 h. Samples were taken at different times, and the concentration of dulcitol in the reaction solution was determined by high-performance liquid chromatography. After 8 h of reaction, the concentration of dulcitol was 4.5 g / L, and the conversion rate relative to the initial amount of sucrose added was 45% ( Figure 6 ).

[0068] Example 5 In vitro enzymatic conversion of cellobiose to dulcitol

[0069] The mixture contained 10 g / L cellobiose, 10 mM magnesium chloride, 10 mM potassium dihydrogen phosphate, 28 mM sodium formate, 1 mM NAD + A 5 mL reaction mixture containing 100 mM HEPES buffer (pH 7.0), 2 U / mL CBP, 2 U / mL PGM, 3 U / mL PGI, 2 U / mL TPE, 3 U / mL GatD, 2 U / mL D6PP, and 2 U / mL FDH was incubated at 37°C for 24 h. Samples were taken at different times, and the concentration of dulcitol in the reaction solution was determined by high-performance liquid chromatography. After 18 h of reaction, the concentration of dulcitol was 3.6 g / L, and the conversion rate was 36% ( Figure 7 ).

[0070] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A method for preparing dulcitol using an in vitro enzyme-catalyzed reaction, characterized in that: include: Galactitol-1-phosphate 5-dehydrogenase (EC 1.1.1.251, GatD) catalyzes the conversion of tagatose-6-phosphate (T6P) and NADH into galactitol-6-phosphate (D6P) and NAD + and using an enzyme capable of dephosphorylating the D6P to dephosphorylate the D6P to generate dulcitol.

2. The method according to claim 1, characterized in that The method further comprises a reaction step of converting fructose-6-phosphate (F6P) into T6P, which is catalyzed by tagatose 6-phosphate 4-epimerase (EC 5.1.3.40, TPE); Preferably, the method further comprises a reaction step of converting glucose-6-phosphate (G6P) into F6P, which is catalyzed by phosphoglucose isomerase (EC 5.3.1.9, PGI); Preferably, the method further comprises a reaction step of converting glucose-1-phosphate (G1P) into G6P, which is catalyzed by phosphoglucomutase (EC 5.4.2.2, PGM); Preferably, the method further comprises a reaction step of converting a substrate and phosphate into G1P, wherein the substrate is a disaccharide, a polysaccharide or any mixture thereof containing D-glucose units, and the step is catalyzed by an enzyme capable of converting the substrate and phosphate into G1P; Preferably, the phosphate is selected from one or a mixture of two or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, and disodium hydrogen phosphate; Preferably, the disaccharide containing D-glucose units is sucrose, which is converted into G1P with phosphate using sucrose phosphorylase (EC 2.4.1.7, SP); Preferably, the polysaccharide containing D-glucose units is selected from starch, starch derivatives or any mixture thereof, and is converted into G1P by starch phosphorylase (α-glucan phosphorylase, EC 2.4.1.1, αGP) and phosphate; Preferably, the polysaccharide containing D-glucose units is further selected from cellulose, cellulose derivatives or any mixture thereof; preferably, the cellulose derivative is a product of cellulose pretreated with acid or enzyme; Preferably, when the polysaccharide containing D-glucose units contains cellulose and / or cellulosic polysaccharide, cellodextrin phosphorylase (EC 2.4.1.49, CDP) is used to catalyze the conversion of cellulosic polysaccharide and phosphate into G1P; preferably, cellobiose phosphorylase (EC 2.4.1.20, CBP) is further used to catalyze the conversion of cellobiose and phosphate produced by the degradation of cellulose and / or cellulosic polysaccharide into G1P; Preferably, when the polysaccharide comprising D-glucose units contains cellobiose, cellobiose and phosphate are converted into G1P catalyzed by cellobiose phosphorylase (EC 2.4.1.20, CBP).

3. The method according to claim 1 or 2, characterized in that The dulcitol-1-phosphate-5-dehydrogenase is derived from Escherichia coli (UniProt number P0A9S3), Parageobacillus caldoxylosilyticus (UniProt nos. A0A150KWR3, A0A150KXC6), Moraxella thermoaceticum Moorella thermoacetica (UniProt number A0A1J5NLK4), Thermoleophilia bacterium (UniProt no. A0A6J4TB39) or Clostridium thermopalmarium (UniProt no. A0A2T0APR6); Preferably, the enzyme capable of dephosphorylating the phosphatase is galactitol-6-phosphatephosphatase (D6PP); preferably, the galactitol-6-phosphatase is derived from Haemophilus influenzae (UniProt No. P46452), Thermospora maritima ( Thermotoga maritima , UniProt number Q9X0Y1), Thermoplasma acidophilum (UniProt number Q9X0Y1), Streptomyces avermitilis (UniProt number Q82E90, Q82B76, Q82B95, etc.), Salmonella typhi ( Salmonella typhi , UniProt number Q8Z989); Preferably, the starch phosphorylase is derived from Escherichia coli (Uniprot No. A0A0A0HB49), Thermotoga maritima ( Thermotoga maritima , Uniprot number G4FEH8), Clostridium thermocellum ( Clostridium thermocellum , Uniprot number A3DCB6); Preferably; the sucrose phosphorylase is derived from Bifidobacterium adolescentis ( Bifidobacterium adolescentis , UniProt number A0ZZH6), Thermoanaerobacterium thermosaccharolyticum (UniProt accession number D9TT09); Preferably, the cellulosic polysaccharide phosphorylase is derived from Clostridium thermocellum ( Clostridium thermocellum , UniProt number A3DJQ6), Clostridium stercorarium (UniProt no. P77846); Preferably, the cellobiose phosphorylase is derived from Clostridium thermocellum ( Clostridium thermocellum , UniProt number A3DC35), Thermotoga neapolitana (UniProt number B9K7M6); Preferably, the phosphoglucomutase is derived from Clostridium thermocellum ( Clostridium thermocellum , Uniprot number A3DEW8), Thermococcus kodakarensis (UniProt number Q68BJ6); Preferably, the phosphoglucose isomerase is derived from Clostridium thermocellum ( Clostridium thermocellum , Uniprot number A3DBX9), Thermus thermophilus ( Thermus thermophilus , Uniprot number Q5SLL6); Preferably, the tagatose-6-phosphate-4-epimerase is derived from Ruminiclostridium herbifermentans (Uniprot number A0A4U7JK39), Dictyoglomus thermophilum (Uniprot No. B5YBD7), Agrobacterium tumefaciens ( Agrobacterium fabrum , Uniprot number A9CES6).

4. The method according to any one of claims 1 to 3, characterized in that The temperature of the catalytic reaction is 30-80°C, more preferably 30-60°C, and most preferably 37°C; Preferably, the pH of the catalytic reaction is 5.0-8.0, more preferably 6.0-7.5, most preferably 7.0; Preferably, each step is carried out simultaneously, and the catalytic reaction time is 1-48 hours, more preferably 8-36 hours, more preferably 10-24 hours, and most preferably 15 hours; Preferably, each step is carried out step by step, and the catalytic reaction time of each step is independently 0.5-10 hours, more preferably 1-3 hours, and most preferably 2 hours; Preferably, the concentration of the substrate is 1-200 g / L, more preferably 5-50 g / L, more preferably 8-20 g / L, and most preferably 10 g / L; Preferably, the concentration of the enzyme that converts the substrate into G1P is 0.1-10 U / mL, more preferably 0.2-5 U / mL, more preferably 1-3 U / mL, and most preferably 2 U / mL; Preferably, the concentration of phosphoglucomutase is 0.1-10 U / mL, more preferably 0.2-5 U / mL, more preferably 1-3 U / mL, and most preferably 2 U / mL; Preferably, the concentration of phosphoglucose isomerase is 0.1-10 U / mL, more preferably 1-5 U / mL, most preferably 3 U / mL; Preferably, the concentration of tagatose-6-phosphate-4-epimerase is 0.1-10 U / mL, more preferably 1-5 U / mL, and most preferably 2 U / mL; Preferably, the concentration of dulcitol-1-phosphate-5-dehydrogenase is 0.1-10 U / mL, more preferably 0.2-5 U / mL, more preferably 2-4 U / mL, and most preferably 3 U / mL; Preferably, the concentration of the enzyme capable of dephosphorylating the phosphoryl group is 0.1-10 U / mL, more preferably 0.2-5 U / mL, more preferably 1-3 U / mL, and most preferably 2 U / mL; Preferably, the concentration of NADH is 0.1-50 mM, more preferably 0.5-10 mM, most preferably 1 mM; Preferably, the concentration of phosphate is 1-150 mM, more preferably 2-50 mM, more preferably 5-20 mM, and most preferably 10 mM; Preferably, the reaction system further contains a magnesium salt; preferably, the magnesium salt is magnesium chloride and / or magnesium sulfate; preferably, the concentration of the magnesium salt in the reaction system is 1-20 mM, more preferably 2-15 mM, and most preferably 10 mM; Preferably, the reaction system further comprises a buffer; preferably, the buffer is selected from HEPES buffer, Tris-HCl buffer, MOPS buffer, and citrate buffer; preferably, the buffer is HEPES buffer. Preferably, the concentration of the buffer is 20-300 mM, preferably 50-200 mM, and most preferably 100 mM.

5. The method according to any one of claims 1 to 4, characterized in that In order to achieve the regeneration of NADH in the reaction system, the method of the present invention further comprises using an enzyme that catalyzes the regeneration of NADH to achieve the regeneration of NAD + The enzymes that can regenerate NADH are formate dehydrogenase (EC 1.17.1.9, FDH) and glucose dehydrogenase (EC 1.1.1.118, GDH). Preferably, the formate dehydrogenase is derived from Thiobacillus sp. (UniProt number Q76EB7), Candida boidinii ( Candida boidinii , UniProt number O13437); glucose dehydrogenase is derived from Sulfolobus solfataricus (UniProt number Q97U21); Preferably, the concentration of the enzyme for achieving NADH regeneration in the reaction system is 0.1-10 U / mL, more preferably 1-3 U / mL, and most preferably 2 U / mL.

6. The method according to any one of claims 1 to 5, characterized in that 10 g / L starch was used as substrate, and 10 mM magnesium chloride, 10 mM potassium dihydrogen phosphate, 55 mM sodium formate, 1 mM NAD + , 100 mM HEPES buffer (pH 7.0), 2 U / mL starch phosphorylase, 2 U / mL phosphoglucomutase, 3 U / mL phosphoglucose isomerase, 2 U / mL tagatose-6-phosphate-4-epimerase, 3 U / mL dulcitol-1-phosphate-5-dehydrogenase, 2 U / mL of an enzyme capable of dephosphorylating a phosphate group (preferably dulcitol-6-phosphatase), and 2 U / mL formate dehydrogenase, and the reaction mixture is catalyzed at 37°C for 20 h to obtain dulcitol.

7. Use of dulcitol-1-phosphate-5-dehydrogenase and an enzyme capable of dephosphorylating a phosphate group in the preparation of dulcitol, preferably in catalyzing the production of dulcitol from T6P; Preferably, the enzyme capable of dephosphorylating a phosphorylation group is selected from dulcitol-6-phosphatase.

8. Dulcitol prepared by the method according to any one of claims 1 to 7.

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