Method for synthesizing L-arabinose, D-xylose or xylitol through enzyme catalysis

Abstract

The invention discloses a method for synthesizing L-arabinose or D-xylose or xylitol through enzyme catalysis, and belongs to the technical field of biological engineering. The invention provides a method for synthesizing L-arabinose through enzyme catalysis, the method takes starch as a raw material, the starch is converted into the L-arabinose through a multi-enzyme cascade reaction, and a reaction system comprises alpha-1, 3, 4-trimethyl-1, 3-pentanediol, alpha-glucosidase, alpha-glucosidase and alpha-glucosidase. The enzyme is prepared from the following raw materials: 1, 4 glucan phosphorylase, UTP-glucose 1-phosphate uridinetransferase, UDP-glucose 6-dehydrogenase, UDP-glucuronic acid decarboxylase, UDP-xylose 4-epimerase, UTP-L-arabinose 1-phosphate uridinetransferase and L-arabinose 1-phosphate phosphatase. The invention further provides a method for converting starch into D-xylose or xylitol through the multi-enzyme cascade reaction, and the method is short in production time, low in cost and capable of being used for large-scale industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a method for synthesizing L-arabinose, or D-xylose, or xylitol by enzyme catalysis. Background Art

[0002] L-arabinose is a naturally occurring five-carbon aldose with a variety of unique physiological functions. L-arabinose has a pure sweetness, about 60% of sucrose, and a glycemic index of 0. It can inhibit the absorption of sucrose by the human body, and has physiological functions such as preventing hypertension and diabetes, regulating sugar and lipid metabolism, reducing fat production, and promoting the proliferation of probiotics. It has now been widely used in food, medicine, meal replacement and other fields.

[0003] At present, the main methods for producing L-arabinose include hydrolysis, enzymatic hydrolysis, and microbial fermentation. The hydrolysis method mainly uses corn cobs, corn husks, sugarcane bagasse, and beet pulp as raw materials, and uses strong acid or strong alkali solutions to degrade some natural polysaccharides in plant tissue cells into a monosaccharide mixture (containing arabinose), which is separated and purified to obtain high-purity L-arabinose; while the enzymatic hydrolysis method mainly uses enzyme preparations to hydrolyze cellulose and hemicellulose in plants to obtain a monosaccharide mixture, and then obtains L-arabinose after filtration, decolorization, ion exchange, evaporation concentration, crystallization and other operations. The common problem faced by the above two processes is that the types of monosaccharides in the hydrolyzate are complex, which increases the difficulty of separation technology and limits large-scale industrial production.

[0004] In response to the problems of hydrolysis and enzymatic hydrolysis, researchers chose to combine traditional hydrolysis with microbial fermentation to prepare high-purity L-arabinose: first, enzymatic hydrolysis or acid hydrolysis was used to obtain a monosaccharide mixture, and microbial fermentation was used to consume non-target monosaccharides in the system, retaining L-arabinose and xylose, etc. This process reduces the difficulty of purification and can obtain a variety of economically valuable sugars, but this process still has certain limitations, such as: the starting raw material composition is complex, the raw material processing is too rough and there is no standardization, which seriously limits production efficiency.

[0005] The document (DOI: 10.1016 / j.biortech.2024.131502) discloses a method for converting D-xylose into L-arabinose by a multi-enzyme cascade reaction, involving enzymes including arabinose isomerase (AraA), ribulose kinase (AraB), sugar phosphatase (AraL), D-xylose 4-epimerase (DX4E) and D-xylose isomerase (XylA). However, the conversion rate of synthesizing L-arabinose by this method is low, and only 34g / L of L-arabinose is produced with 100g / L of D-xylose within 45 hours. Summary of the invention

[0006] In view of the problems of the prior art, the object of the present invention is to provide a method for synthesizing L-arabinose, or D-xylose, or xylitol by enzyme catalysis.

[0007] The present invention provides a method for synthesizing L-arabinose by enzyme catalysis, wherein the method comprises a substrate, a reaction enzyme, an enzyme activator, a cofactor and inorganic phosphorus, and L-arabinose is obtained through a multi-enzyme cascade reaction;

[0008] The substrate is starch or its derivatives;

[0009] The cofactors include nucleotides and oxidized coenzymes;

[0010] The reaction enzymes include α-1,4-glucan phosphorylase, UTP-glucose 1-phosphate uridyl transferase, UDP-glucose 6-dehydrogenase, UDP-glucuronic acid decarboxylase, UDP-xylose 4-epimerase, UTP-L-arabinose 1-phosphate uridyl transferase, L-arabinose 1-phosphate phosphatase and NADH oxidase.

[0011] Further, the α-1,4 glucan phosphorylase is selected from any one or a combination of two or more of the following: derived from Escherichia coli (strain K12), Uniprot number P00490, derived from Thermococcus litoralis, Uniprot number Q9YGA7, derived from Thermococcus kodakarensis, Uniprot number Q5JH18;

[0012] The UTP-glucose 1-phosphate uridyl transferase is selected from any one or a combination of two or more of the following: derived from Sulfurisphaera tokodaii, Uniprot number Q975F9, derived from Thermococcus chitonophagus, Uniprot number A0A170SE13, derived from Hordeum vulgare, Uniprot number Q43772;

[0013] The UDP-glucose 6-dehydrogenase is selected from any one or a combination of two or more of the following: derived from Pyrobaculum islandicum, Uniprot number A1RUM9, derived from Escherichia coli (strain K12), Uniprot number P76373;

[0014] The UDP-glucuronic acid decarboxylase is selected from any one or a combination of two or more of the following: derived from Homosapiens, Uniprot number Q8NBZ7, derived from Thermomicrobium roseum, Uniprot number A0A7C1K4Y9;

[0015] The UDP-D-xylose 4-epimerase is selected from any one or a combination of two or more of the following, or the UDP-D-xylose 4-epimerase is selected from a nucleotide sequence having a sequence identity of 50%-100% with the following enzymes: derived from Arabidopsis thaliana, Uniprot number Q9SA77, derived from Thermodesulfitimonas autotrophica, Uniprot number A0A3N5B0B9, derived from Prunus persica, Uniprot number M4QEJ8;

[0016] The UTP-L-arabinose 1-phosphate uridyl transferase is selected from any one or a combination of two or more of the following, or the UTP-L-arabinose 1-phosphate uridyl transferase is selected from a nucleotide sequence having a sequence identity of 50%-100% with the following enzymes: derived from Arabidopsis thaliana, Uniprot number Q9C5I1, derived from Dioscorea alata, GenBank number KAH7674669.1;

[0017] The L-arabinose 1-phosphate phosphatase is selected from any one or a combination of two or more of the following, or the L-arabinose 1-phosphate phosphatase is selected from a nucleotide sequence having a sequence identity of 30%-100% with the following enzymes: derived from Clostridium acetobutylicum, Uniprot number Q97KR2, derived from Salmonella typhi, Uniprot number Q8Z854, derived from Neisseria meningitidis, Uniprot number Q9JYB3, derived from Escherichia coli (strain K12), Uniprot number P75792, derived from Methanosarcina mazei, Uniprot number Q8PWF4, derived from Acidothermus cellulolyticus, Uniprot number A0LWR5, derived from Bacillus cereus, cereus, Uniprot number Q819K1, from Salmonella paratyphi, Uniprot number C0Q032, from Bacteroides thetaiotaomicron, Uniprot number Q8A759, from Thermoanaerobaculiabacterium, GenBank number: HSG40782.1;

[0018] The NADH oxidase is derived from Anaerocolumna aminovalerica, and the Uniprot number is Q2WFW5;

[0019] Preferably, the α-1,4 glucan phosphorylase is derived from Escherichia coli (strain K12), and its Uniprot number is P00490;

[0020] The UTP-glucose 1-phosphate uridyl transferase is derived from barley Hordeum vulgare, and the Uniprot number is Q43772;

[0021] The UDP-glucose 6-dehydrogenase is derived from Escherichia coli (strain K12), and the Uniprot number is P76373;

[0022] The UDP-glucuronic acid decarboxylase is derived from Thermomicrobium roseum, and its Uniprot number is A0A7C1K4Y9;

[0023] The UDP-xylose 4-epimerase is derived from Arabidopsis thaliana, and the Uniprot number is Q9SA77;

[0024] The UTP-L-arabinose 1-phosphate uridyl transferase is derived from Arabidopsis thaliana, and the Uniprot number is Q9C5I1;

[0025] The L-arabinose 1-phosphate phosphatase is derived from Salmonella typhi (Uniprot number is Q8Z854).

[0026] Furthermore, the preparation of the reaction enzyme comprises the following steps:

[0027] (a) Construction of recombinant plasmid: cloning the target gene into a vector to obtain a recombinant plasmid carrying the target gene;

[0028] (b) constructing an engineered strain: transforming the recombinant plasmid into a host cell to obtain an engineered strain;

[0029] (c) Protease purification: The engineered strain is inoculated into the culture medium, cultured, and the cells are collected by centrifugation, washed, resuspended, broken, and protein purified to obtain the reaction enzyme.

[0030] Further, in step (a), the vector is selected from pET20b vector and pET32a vector;

[0031] In step (b), the host cell is selected from Escherichia coli BL21 (DE3) and Escherichia coli Rosseta-gamiB (DE3);

[0032] In step (c), the culture medium is LB culture medium;

[0033] The culture conditions are: culturing at 37°C, 200-250rpm until the absorbance at 600nm reaches 0.6-1.0, adding an inducer with a final concentration of 0.05-0.15mM, and then culturing at 20-30°C, 200-250rpm for 10-16 hours; preferably: culturing at 37°C, 220rpm until the absorbance at 600nm reaches 0.8, adding an inducer with a final concentration of 0.1mM, and then culturing at 25°C, 220rpm for 14 hours;

[0034] The centrifugal temperature is 2-6°C, preferably 4°C; the centrifugal speed is 6000-10000 rpm, preferably 8000 rpm;

[0035] The washing and resuspension conditions are as follows: washing and resuspension of cells with a buffer solution, wherein the concentration of the buffer solution is 30 to 70 mM, preferably 50 mM; the pH of the buffer solution is 7.0 to 9.0, preferably pH 8.0;

[0036] The crushing equipment is selected from a high-pressure homogenizer or an ultrasonic crusher;

[0037] The protein purification is selected from a thermal precipitation method or a Ni-NTA purification method;

[0038] Furthermore, in step (c), the inducing agent is isopropyl-β-D-thiogalactoside (IPTG).

[0039] Furthermore, the starch or its derivative is starch or its derivative pretreated with starch isomerase;

[0040] The enzyme activator is a compound containing magnesium ions, preferably magnesium chloride;

[0041] The inorganic phosphorus comes from a phosphorus-containing buffer, preferably a phosphate buffer, a sodium hydrogen phosphate-citrate buffer, or a mixture of the two; the pH of the phosphorus-containing buffer is 5.0-9.0, preferably 7.5;

[0042] The nucleotide is uridine triphosphate;

[0043] The oxidized coenzyme is nicotinamide adenine dinucleotide.

[0044] Furthermore, the starch or its derivative is selected from soluble starch, maltodextrin, and starch dextrin; the starch isomerase is selected from any one or a combination of two or more of the following: derived from Escherichia coli (strain K12), Uniprot numbered P15067, and derived from the acidophilic archaeon Cuniculiplasma divulgatum, Uniprot numbered A0A1N5SFX9; preferably, the starch isomerase is derived from the acidophilic archaeon Cuniculiplasma divulgatum, Uniprot numbered A0A1N5SFX9.

[0045] Furthermore, the ratio of starch or its derivative, nucleotide, oxidized coenzyme, α-1,4-glucan phosphorylase, UTP-glucose 1-phosphate uridyl transferase, UDP-glucose 6-dehydrogenase, UDP-glucuronic acid decarboxylase, UDP-xylose 4-epimerase, UTP-L-arabinose 1-phosphate uridyl transferase, L-arabinose 1-phosphate phosphatase, NADH oxidase, enzyme activator and inorganic phosphorus is 5-100 g / L: 2-8 mM: 0.5-4 mM: 0. 1~50U / mL: 0.1~50U / mL: 0.1~50U / mL: 0.1~50U / mL: 0.1~50U / mL: 0.1~50U / mL: 0.1~50U / mL: 0.1~50U / mL: 5~ 15mM: 40-60mM, preferably 10g / L: 5mM: 2mM: 2U / mL: 1U / mL: 2U / mL: 2U / mL: 3U / mL: 1U / mL: 2U / mL: 2U / mL: 10mM: 50mM.

[0046] Furthermore, the method comprises mixing a substrate, a reaction enzyme, an enzyme activator, a cofactor and inorganic phosphorus and reacting them to obtain L-arabinose;

[0047] Alternatively, the method comprises the following steps:

[0048] (1) Using α-1,4-glucan phosphorylase, the substrate and inorganic phosphorus are converted into glucose 1-phosphate;

[0049] (2) UTP-glucose 1-phosphate uridyl transferase is used to convert glucose 1-phosphate and UTP into UDP-glucose and pyrophosphate;

[0050] (3) UDP-glucose 6-dehydrogenase is used to convert UDP-glucose and NAD + Converted into UDP-glucuronic acid and NADH;

[0051] (4) UDP-glucuronic acid is converted into UDP-D-xylose and CO using UDP-glucuronic acid decarboxylase. 2 ;

[0052] (5) using UDP-D-xylose 4-epimerase to convert UDP-D-xylose into UDP-L-arabinose;

[0053] (6) using UTP-L-arabinose 1-phosphate uridyl transferase to reversibly convert UDP-L-arabinose and pyrophosphate into L-arabinose 1-phosphate and UTP;

[0054] (7) Specific L-arabinose 1-phosphate phosphatase is used to catalyze the removal of the phosphate group of L-arabinose 1-phosphate to generate L-arabinose.

[0055] (8) NADH oxidase is used to convert the generated NADH into NAD + , achieving cofactor circulation in the reaction system.

[0056] Furthermore, the reaction temperature is 25 to 85° C., preferably 25 to 45° C., more preferably 30 to 40° C.; the reaction time is 1 to 48 hours, preferably 2 to 24 hours.

[0057] Furthermore, the catalytic reactions in the reaction are carried out simultaneously, and the reaction time is 2 to 48 hours, preferably 8 to 24 hours;

[0058] Alternatively, each catalytic reaction in the reaction is carried out independently of each other, and the reaction time is 1 to 24 hours, preferably 2 to 12 hours.

[0059] Furthermore, the various reaction enzymes and starch isomerase are catalyzed in the form of crude enzyme solution, pure enzyme, whole cell or immobilized enzyme.

[0060] The present invention also provides a method for synthesizing D-xylose by enzyme catalysis, wherein the method comprises mixing a substrate, a reaction enzyme, an enzyme activator, a cofactor and inorganic phosphorus and reacting the mixture to obtain D-xylose; the reaction enzyme is α-1,4-glucan phosphorylase, UTP-glucose 1-phosphate uridyl transferase, UDP-glucose 6-dehydrogenase, UDP-glucuronic acid decarboxylase, UTP-xylose 1-phosphate uridyl transferase, D-xylose 1-phosphate phosphatase and NADH oxidase;

[0061] Alternatively, the method comprises the following steps:

[0062] (I) preparing UDP-D-xylose according to the above steps (1)-(4);

[0063] (II) using UTP-xylose 1-phosphate uridyltransferase DaUSP to reversibly convert UDP-D-xylose and PPi into D-xylose 1-phosphate and UTP;

[0064] (III) Using the specific D-xylose 1-phosphate phosphatase Xyl1PP to catalyze the removal of the phosphate group of D-xylose 1-phosphate, D-xylose is obtained.

[0065] Furthermore, in step (II), the xylose 1-phosphate uridyltransferase USP is derived from Dioscorea alata, with a GenBank number of KAH7674669.1;

[0066] In step (II), the D-xylose 1-phosphate phosphatase Xyl1PP is derived from Escherichia coli (strain K12), and its Uniprot number is P75792.

[0067] The present invention also provides a method for synthesizing xylitol by enzyme catalysis, wherein the method comprises mixing a substrate, a reaction enzyme, an enzyme activator, a cofactor and inorganic phosphorus and reacting the mixture to obtain xylitol; the reaction enzyme is α-1,4-glucan phosphorylase, UTP-glucose 1-phosphate uridyl transferase, UDP-glucose 6-dehydrogenase, UDP-glucuronic acid decarboxylase, UTP-xylose 1-phosphate uridyl transferase, D-xylose 1-phosphate phosphatase and xylose reductase;

[0068] Alternatively, the method comprises the following steps:

[0069] (i) preparing D-xylose according to the above steps (I) to (III);

[0070] (ii) Xylose reductase XYL1 converts D-xylose and NADH into xylitol and NAD + .

[0071] Furthermore, in step (ii), the xylose reductase XYL1 is derived from Scheffersomyces stipitis, with a Uniprot number of P31867.

[0072] The present invention provides a method for synthesizing L-arabinose by enzyme catalysis, which uses starch as a raw material and converts it into L-arabinose through a multi-enzyme cascade reaction, wherein the reaction system comprises α-1,4 glucan phosphorylase (αGP), UTP-glucose 1-phosphate uridyl transferase (UGP), UDP-glucose 6-dehydrogenase (UGDH), UDP-glucuronic acid decarboxylase (UXS), UDP-xylose 4-epimerase (UXE), UTP-L-arabinose 1-phosphate uridyl transferase (USP), and L-arabinose 1-phosphate phosphatase (Ara1PP). Compared with the literature (DOI: 10.1016 / j.biortech.2024.131502), the yield of the present invention is significantly improved, and 25.08g L-arabinose can be produced with 50g / L soluble starch, and the molar conversion rate reaches 60.2%.

[0073] The present invention also provides a method for converting starch into D-xylose or xylitol through a multi-enzyme cascade reaction. The method provided by the present invention has short production time and low cost and can be used for large-scale industrial production.

[0074] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0075] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 : Production pathway for the synthesis of L-arabinose using soluble starch as raw material.

[0077] Figure 2 : Production pathway for synthesizing D-xylose and xylitol using soluble starch as raw material. DETAILED DESCRIPTION

[0078] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercial products. The experimental methods without specific conditions in the following examples are all based on the conditions recommended by the manufacturer.

[0079] In the specific implementation manner of the present invention, the genes used for recombinant expression of each reaction enzyme are obtained by total gene synthesis, and the genes of each reaction enzyme are numbered as follows (see Table 1):

[0080] Table 1 Number of genes for each enzyme

[0081]

[0082]

[0083] The culture medium used in the specific embodiment of the present invention is LB culture medium, and the formula is as follows: 10 g / L peptone, 5 g / L yeast extract and 10 g / L sodium chloride.

[0084] In a specific embodiment of the present invention, the liquid phase detection conditions are: Agilent 1260 Infinity II high performance liquid chromatography (HPLC), RID detector, using Yuexu Xtimate Suger-H (7.8mm*300mm, 5μm) chromatographic column, column temperature 80°C, mobile phase 5mM H 2 SO 4 Aqueous solution, flow rate 0.6mL / min.

[0085] Example 1. Recombinant expression and purification of each reaction enzyme

[0086] α-1,4 glucan phosphorylase (αGP) is EcαGP from Escherichia coli (strain K12), Uniprot number P00490;

[0087] UTP-glucose 1-phosphate uridyl transferase (UGP) was selected from HvUGP from barley (Hordeum vulgare), Uniprot number Q43772;

[0088] UDP-glucose 6-dehydrogenase (UGDH) was EcUGDH from Escherichia coli (strain K12), Uniprot number P76373;

[0089] UDP-glucuronate decarboxylase (UXS) was Tr2UXS from Thermomicrobium roseum , Uniprot number A0A7C1K4Y9;

[0090] UDP-D-xylose 4-epimerase (UXE) is AtUXE from Arabidopsis thaliana , with Uniprot number Q9SA77;

[0091] UTP-L-arabinose 1-phosphate uridyltransferase (USP) is AtUSP from Arabidopsis thaliana , with Uniprot number Q9C5I1;

[0092] L-arabinose 1-phosphate phosphatase (Ara1PP) is StAra1PP from Salmonella typhi, Uniprot number Q8Z854;

[0093] Xylose 1-phosphate uridyltransferase is DaUSP from Dioscorea alata, with GenBank accession number: KAH7674669.1;

[0094] D-xylose 1-phosphate phosphatase is EcXyl1PP from Escherichia coli (strain K12), Uniprot number P75792;

[0095] The xylose reductase is SsXYL1 from Scheffersomyces stipitis , with the Uniprot number P31867;

[0096] NADH oxidase (NOX) was AaNOX from Anaerocolumna aminovalerica , Uniprot number Q2WFW5;

[0097] Starch isomerase (IA) was CdIA from Cuniculiplasma divulgatum, with the Uniprot number A0A1N5SFX9.

[0098] (1) Construction of recombinant plasmids: The above genes were cloned into the pET20b vector by whole gene synthesis to obtain recombinant plasmids carrying the target genes: pET20b-EcαGP, pET20b-TlαGP, pET20b-TkαGP, pET20b-StUGP, pET20b-TcUGP, pET20b-HvUGP, pET20b-PiUGDH, pET20b-EcUGDH, pET20b-HsUXS, pET20b-Tr2UXS, pET20b-AtUXE, pET20b-TaUXE, pET20b-PpUXE, pET20b- AtUSP, pET20b-DaUSP, pET20b-CaAra1PP, pET20b-StAra1PP, pET20b-NmAra1PP, pET20b-EcAra1PP, pET20b-MmAra1PP, pET20b-AcAra1PP, pET20b-BcAra1PP, pET20b-SpAra1PP, pET20b-BtAra1PP, pET20b-TbAra1PP, pET20b-AaNOX, pET20b-EcIA, pET20b-CdIA, pET20b-SsXYL1.

[0099] (2) Construction of engineered strains: The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) to obtain the corresponding engineered strain, and the engineered strain was inoculated into LB medium and cultured at 37°C and 220 rpm until the absorbance at 600 nm reached 0.8, then 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added and cultured at 25°C and 220 rpm for 14 h.

[0100] (3) Protease purification: Collect cells in a centrifuge at 8000 rpm at 4°C, wash cells with 50 mM Tris-HCl (pH 8.0) buffer containing 50 mM NaCl, resuspend cells with 50 mM Tris-HCl (pH 8.0) buffer containing 300 mM NaCl, and disrupt cells with an ultrasonic disruptor. Use Ni-NTA column for protein purification to obtain the required α-1,4-glucan phosphorylase, UTP-glucose 1-phosphate uridyl transferase, UDP-glucose 6-dehydrogenase, UDP-glucuronic acid decarboxylase, UDP-xylose 4-epimerase, UTP-L-arabinose 1-phosphate uridyl transferase, L-arabinose 1-phosphate phosphatase, UTP-D-xylose 1-phosphate uridyl transferase, D-xylose 1-phosphate phosphatase, xylose reductase, NADH oxidase, and starch isomerase.

[0101] (4) Target protease verification: Use polyacrylamide gel electrophoresis (SDS-PAGE) to verify the protein, and use the Bradford protein detection kit to determine the protein concentration.

[0102] Example 2: Catalytic synthesis of L-arabinose using soluble starch as raw material

[0103] 1. After starch isomerase treatment, soluble starch containing only α-1,4 glycosidic bonds is obtained

[0104] CdIA was mixed with starch to obtain a reaction system with a volume of 100 mL consisting of 400 g / L starch, 1 g / L CdIA, 1 mM magnesium chloride, and 50 mM acetate buffer (pH 5.5), and treated at 60° C. for 4 hours.

[0105] 2. Catalytic synthesis of L-arabinose ( Figure 1 )

[0106] The specific operations are as follows:

[0107] The enzymes (αGP, UGP, UGDH, UXS, UXE, USP, Ara1PP, NOX) in Example 1 were mixed with soluble starch to obtain a reaction system with a volume of 1 mL consisting of the following components: 10 g / L (55.5 mM) soluble starch, 5 mM uridine triphosphate (UTP), 2 mM nicotinamide adenine dinucleotide (NAD +), 2U / mL αGP, 1U / mL UGP, 2U / mL UGDH, 2U / mL UXS, 3U / mL UXE, 1U / mL USP, 3U / mL Ara1PP, 2U / mL NOX, 10mM magnesium chloride and 50mM phosphate buffer (pH7.5). After incubation at 37°C for 18 hours, HPLC detection showed that the L-arabinose concentration was 6.18 g (41.2 mM) and the molar conversion rate was 74%.

[0108] 3. To increase the product concentration, further, the catalytic synthesis of L-arabinose at a higher substrate concentration is performed as follows:

[0109] The enzymes (αGP, UGP, UGDH, UXS, UXE, USP, Ara1PP, NOX) in Example 1 were mixed with soluble starch to obtain a reaction system with a volume of 1 mL consisting of the following components: 50 g / L (277.5 mM) soluble starch, 25 mM UTP, 10 mM NAD + , 10U / mL αGP, 5U / mL UGP, 10U / mL UGDH, 10U / mL UXS, 15U / mL UXE, 5U / mL USP, 15U / mL Ara1PP, 10U / mL NOX, 10mM magnesium chloride and 50mM phosphate buffer (pH 7.5). After incubation at 37°C for 24 hours, HPLC was used to detect that the L-arabinose concentration was 25.08g (167mM) and the molar conversion rate was 60.2%.

[0110] Example 3: Catalytic synthesis of D-xylose using soluble starch as raw material

[0111] 1. Soluble starch is obtained after starch isomerase treatment

[0112] The specific steps are as described in Example 2.

[0113] 2. Catalytic synthesis of D-xylose using soluble starch as raw material ( Figure 2 )

[0114] The specific operations are as follows:

[0115] The enzymes (αGP, UGP, UGDH, UXS, DaUSP, Xyl1PP, NOX) in Example 1 were mixed with soluble starch to obtain a reaction system with a volume of 1 mL consisting of the following components: 10 g / L (55.5 mM) soluble starch, 5 mM uridine triphosphate (UTP), 2 mM NAD +, 2U / mLαGP, 1U / mL UGP, 2U / mL UGDH, 1U / mL UXS, 1U / mL DaUSP, 3U / mLXyl1PP, 2U / mL NOX, 10mM magnesium chloride and 50mM phosphate buffer (pH 7.5). After incubation at 37°C for 18 hours, HPLC detection showed that the D-xylose concentration was 6.58g (43.8mM) and the molar conversion rate was 79%.

[0116] Example 4: Enzymatic synthesis of xylitol using soluble starch as raw material

[0117] The enzymes (αGP, UGP, UGDH, UXS, DaUSP, Xyl1PP, XYL1, NOX) in Example 1 were mixed with soluble starch to obtain a reaction system with a volume of 1 mL consisting of the following components ( Figure 2 ): 10g / L (55.5mM) soluble starch, 5mM uridine triphosphate (UTP), 2mM NAD + , 2U / mLαGP, 1U / mL UGP, 2U / mL UGDH, 1U / mL UXS, 1U / mL DaUSP, 3U / mL Xyl1PP, 2U / mL XYL1, 10mM magnesium chloride and 50mM phosphate buffer (pH7.5), incubated at 37°C, and after 18 hours of reaction, HPLC was used to detect that the xylitol concentration was 6.41g (42.7mM) and the molar conversion rate was 77%.

[0118] The control enzyme is prepared as follows.

[0119] Control Example 1: Expression and purification of related enzymes

[0120] α-1,4 glucan phosphorylase, TlαGP from Thermococcus litoralis (Uniprot number Q9YGA7), TkαGP from Thermococcus kodakarensis (Uniprot number Q5JH18);

[0121] UTP-glucose 1-phosphate uridyl transferase, StUGP is derived from Sulfuris phaeratokodaii (Uniprot number Q975F9), TcUGP is derived from Thermococcus chitonophagus (Uniprot number A0A170SE13);

[0122] UDP-glucose 6-dehydrogenase, PiUGDH from Pyrobaculum islandicum (Uniprot number A1RUM9);

[0123] UDP-glucuronate decarboxylase, HsUXS from Homo sapiens (Uniprot number: Q8NBZ7);

[0124] UDP-D-xylose 4-epimerase, TaUXE is derived from Thermodesulfitimonas autotrophica (Uniprot number A0A3N5B0B9), and PpUXE is derived from Prunus persica (Uniprot number M4QEJ8);

[0125] UTP-L-arabinose 1-phosphate uridyltransferase, DaUSP is from Dioscorea alata (GenBank accession number: KAH7674669.1);

[0126] L-arabinose 1-phosphate phosphatase, CaAra1PP from Clostridium acetobutylicum (Uniprot number Q97KR2), NmAra1PP from Neisseria meningitidis (Uniprot number Q9JYB3), EcAra1PP from Escherichia coli (strain K12) (Uniprot number P75792), MmAra1PP from Methanosarcina mazei (Uniprot number Q8PWF4), AcAra1PP from Acidothermus cellulolyticus (Uniprot number A0LWR5), BcAra1PP from Bacillus cereus (Uniprot number A0LWR5), and L-arabinose 1-phosphate phosphatase, CaAra1PP from Clostridium acetobutylicum (Uniprot number Q97KR2), NmAra1PP from Neisseria meningitidis (Uniprot number Q9JYB3), EcAra1PP from Escherichia coli (strain K12) (Uniprot number P75792), MmAra1PP from Methanosarcina mazei (Uniprot number Q8PWF4), cereus (Uniprot number: Q819K1), SpAra1PP is derived from Salmonella paratyphi (Uniprot number: C0Q032), BtAra1PP is derived from Bacteroides thetaiotaomicron (Uniprot number: Q8A759), and TbAra1PP is derived from Thermoanaerobaculia bacterium (GenBank number: HSG40782.1);

[0127] Starch isomerase, EcIA, is derived from Escherichia coli (strain K12), Uniprot number P15067.

[0128] Referring to the method of Example 1, the above enzyme was subjected to the construction of recombinant plasmid, construction of engineering strain, protease purification and target protease verification.

[0129] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0130] Experimental Example 1: Screening of phosphatases

[0131] 1. Experimental methods

[0132] The dephosphorylation activities of phosphatases from different sources on two phosphate compounds, glucose 1 phosphate (G1P) and L-arabinose 1 phosphate (Ara1P), were tested in 100 mM Tris-HCl (pH 7.5) containing 5 mM magnesium chloride at 37°C.

[0133] 2. Experimental results

[0134] The dephosphorylation activity of CaAra1PP from Clostridium acetobutylicum on L-arabinose 1-phosphate was 3.2 U / mg, the dephosphorylation activity of StAra1PP from Salmonella typhi on L-arabinose 1-phosphate was 3.6 U / mg, the dephosphorylation activity of NmAra1PP from Neisseria meningitidis on L-arabinose 1-phosphate was 4.4 U / mg, the dephosphorylation activity of EcAra1PP from Escherichia coli (strain K12) on L-arabinose 1-phosphate was 2.8 U / mg, the dephosphorylation activity of MmAra1PP from Methanosarcina mazei on L-arabinose 1-phosphate was 3.1 U / mg, and the dephosphorylation activity of NmAra1PP from Acidothermus cellulolyticus had a dephosphorylation activity of 2.3 U / mg for L-arabinose 1-phosphate, 2 U / mg for BcAra1PP from Bacillus cereus, 0.5 U / mg for SpAra1PP from Salmonella paratyphi, 1.6 U / mg for BtAra1PP from Bacteroides thetaiotaomicron, and 0.86 U / mg for TbAra1PP from Thermoanaerobaculia bacterium.

[0135] Table 2 Dephosphorylation activity of different phosphatases on two substrates

[0136]

[0137] The above results showed that CaAra1PP, NmAra1PP, EcAra1PP, AcAra1PP and BcAra1PP had dephosphorylation activity towards glucose 1 phosphate and L-arabinose 1 phosphate, and StAra1PP, MmAra1PP, SpAra1PP, BtAra1PP and TbAra1PP had specific dephosphorylation activity towards L-arabinose 1 phosphate, among which StAra1PP had the highest dephosphorylation activity towards L-arabinose 1 phosphate.

[0138] Experimental Example 2: Determination of enzyme activity in the pathway of L-arabinose synthesis catalyzed by different enzymes

[0139] 1. Experimental methods

[0140] (1) Detection of α-1,4-glucan phosphorylase activity

[0141] The different α-1,4-glucan phosphorylases in Example 1 and Control Example 1 were mixed to obtain a reaction system with a volume of 200uL consisting of the following components: 5g / L soluble starch, 0.01g / L α-1,4-glucan phosphorylase, 5mM magnesium chloride, 100mM Tris-HCl (pH 7.5), and reacted at 37°C for 10 minutes. Then, 0.5U / mL of phosphoglucomutase (PGM), 0.5U / mL of glucose 6-phosphate dehydrogenase (G6PDH), 5mM NAD + , 5 mM magnesium chloride, and then react for 2 hours, and then measure the absorbance at 340 nm to calculate the concentration of NADH to detect the amount of G1P generated.

[0142] (2) Detection of UTP-glucose 1-phosphate uridyl transferase activity

[0143] The different UTP-glucose 1-phosphate uridyl transferases in Example 1 and Control Example 1 were mixed to obtain a reaction system with a volume of 200 uL consisting of the following components: 5 mM G1P, 0.01 g / L UTP-glucose 1-phosphate uridyl transferase, 5 mM UTP, 5 mM magnesium chloride, and 100 mM Tris-HCl (pH 7.5), and reacted at 37°C for 10 minutes. Then, 0.5 U / mL UGDH, 5 mM NAD + , 5 mM magnesium chloride, and then react for 2 hours, measure the absorbance at 340 nm, calculate the concentration of NADH, and use this to detect the amount of UDPG generated.

[0144] (3) Detection of UDP-glucose 6-dehydrogenase activity

[0145] The different UDP-glucose 6-dehydrogenases in Example 1 and Control Example 1 were mixed to obtain a reaction system with a volume of 200 uL consisting of the following components: 5 mM UDPG, 0.1 g / L UDP-glucose 6-dehydrogenase, 5 mM NAD + , 5 mM magnesium chloride, 100 mM Tris-HCl (pH 7.5), react at 37°C for 10 minutes, and measure the absorbance at 340 nm in an ELISA reader to measure the NADH concentration.

[0146] (4) Detection of UDP-glucuronic acid decarboxylase activity

[0147] The different UDP-glucuronic acid decarboxylases in Example 1 and Reference Example 1 were mixed to obtain a reaction system with a volume of 200 uL consisting of the following components: 5 mM UDP-glucuronic acid, 0.01 g / L UDP-glucuronic acid decarboxylase, 5 mM magnesium chloride, 100 mM Tris-HCl (pH 7.5), and reacted at 37° C. for 10 minutes. The concentration of UDP-D-xylose was detected by HPLC.

[0148] (5) Detection of UDP-xylose 4-epimerase activity

[0149] The different UDP-xylose 4-epimerases in Example 1 and Control Example 1 were mixed to obtain a reaction system with a volume of 200 uL consisting of the following components: 5 mM UDP-xylose, 0.1 g / L UDP-D-xylose 4-epimerase, 5 mM magnesium chloride, 100 mM Tris-HCl (pH 7.5), and reacted at 37° C. for 10 minutes. The concentration of UDP-L-arabinose was detected by HPLC.

[0150] (6) Detection of UTP-L-arabinose 1-phosphate uridyltransferase activity

[0151] The different UTP-L-arabinose 1-phosphate uridyltransferases in Example 1 and Control Example 1 were mixed to obtain a reaction system with a volume of 200 uL consisting of the following components: 5 mM L-arabinose 1-phosphate, 0.02 g / L UTP-L-arabinose 1-phosphate uridyltransferase, 5 mM UTP, 5 mM magnesium chloride, 100 mM Tris-HCl (pH 7.5), and reacted at 37° C. for 10 minutes. The concentration of UDP-L-arabinose was detected by HPLC.

[0152] (7) Detection of NADH oxidase activity

[0153] The NADH oxidase of Example 1 was mixed to obtain a reaction system with a volume of 200 uL consisting of the following components: 0.25 mM NADH, 0.005 g / L NADH oxidase, 5 mM magnesium chloride, and 100 mM Tris-HCl (pH 7.5). The mixture was reacted at 37° C. for 10 minutes, and the consumption of NADH was detected.

[0154] 2. Experimental results

[0155] (1) It was measured experimentally that the specific enzyme activity of EcαGP derived from Escherichia coli (strain K12) at 37°C was 5.4 U / mg, the specific enzyme activity of TlαGP derived from Thermococcus litoralis at 37°C was 1.3 U / mg, and the specific enzyme activity of TkαGP derived from Thermococcus kodakarensis at 37°C was 2.2 U / mg.

[0156] (2) It was measured experimentally that the specific enzyme activity of StUGP derived from Sulfuris phaeratokodaii at 37°C was 0.3 U / mg, the specific enzyme activity of TcUGP derived from Thermococcus chitonophagus at 37°C was 0.48 U / mg, and the specific enzyme activity of HvUGP derived from Hordeum vulgare at 37°C was 8.85 U / mg.

[0157] (3) It was measured experimentally that the specific enzyme activity of PiUGDH derived from Pyrobaculumislandicum at 37°C was 0.03 U / mg, and the specific enzyme activity of EcUGDH derived from Escherichia coli (strain K12) at 37°C was 0.5 U / mg.

[0158] (4) Experimental results show that the specific enzyme activity of HsUXS from Homo sapiens at 37°C is 0.37 U / mg, and the specific enzyme activity of Tr2UXS from Thermomicrobium roseum at 37°C is 1.2 U / mg.

[0159] (5) Experimental results showed that the specific enzyme activity of AtUXE from Arabidopsis thaliana at 37°C was 9.7 U / mg, the specific enzyme activity of TaUXE from Thermodesulfitimonas autotrophica at 37°C was 3 U / mg, and the specific enzyme activity of UXE from Prunus persica at 37°C was 6.5 U / mg.

[0160] (6) Experimental results showed that the specific enzyme activity of AtUSP from Arabidopsis thaliana at 37°C was 37.6 U / mg, and that of DaUSP from Dioscorea alata at 37°C was 25.5 U / mg.

[0161] (7) Experimental results show that the specific enzyme activity of AaNOX from Anaerocolumna aminovalerica at 37°C is 122 U / mg.

[0162] The above results show that among α-1,4-glucan phosphorylases, EcαGP from Escherichia coli (strain K12) has the best catalytic efficiency; among UTP-glucose 1-phosphate uridyltransferases, HvUGP from barley (Hordeum vulgare) has the best catalytic efficiency; among UDP-glucose 6-dehydrogenases, EcUGDH from Escherichia coli (strain K12) has the best catalytic efficiency; among UDP-glucuronic acid decarboxylases, Tr2UXS from Thermomicrobium roseum has the best catalytic efficiency; among UDP-xylose 4-epimerases, AtUXE from Arabidopsis thaliana has the best catalytic efficiency; and among UTP-L-arabinose 1-phosphate uridyltransferases, AtUSP from Arabidopsis thaliana has the best catalytic efficiency.

[0163] Combining Experimental Examples 1 and 2, it can be seen that EcαGP, HvUGP, EcUGDH, Tr2UXS, AtUXE, AtUSP, StAra1PP and AaNOX are the most efficient in catalyzing the synthesis of L-arabinose.

[0164] In summary, the present invention discloses a method for enzymatically synthesizing L-arabinose, or D-xylose, or xylitol. The present invention uses starch as a raw material and converts it into L-arabinose by a multi-enzyme cascade reaction, and the reaction system comprises α-1,4 glucan phosphorylase (αGP), UTP-glucose 1-phosphate uridyl transferase (UGP), UDP-glucose 6-dehydrogenase (UGDH), UDP-glucuronic acid decarboxylase (UXS), UDP-xylose 4-epimerase (UXE), UTP-L-arabinose 1-phosphate uridyl transferase (USP), L-arabinose 1-phosphate phosphatase (Ara1PP). The present invention also provides a method for converting starch into D-xylose or xylitol by a multi-enzyme cascade reaction, and the method provided by the present invention has short production time and low cost, and can be used for large-scale industrial production.

Claims

1. A method for enzymatically synthesizing L-arabinose, characterized in that: The method comprises the following steps: a substrate, a reaction enzyme, an enzyme activator, a cofactor and inorganic phosphorus are used to obtain L-arabinose through a multi-enzyme cascade reaction; The substrate is starch or its derivatives; The cofactors include nucleotides and oxidized coenzymes; The reaction enzymes include α-1,4-glucan phosphorylase, UTP-glucose 1-phosphate uridyl transferase, UDP-glucose 6-dehydrogenase, UDP-glucuronic acid decarboxylase, UDP-xylose 4-epimerase, UTP-L-arabinose 1-phosphate uridyl transferase, L-arabinose 1-phosphate phosphatase and NADH oxidase.

2. The method according to claim 1, characterized in that The α-1,4 glucan phosphorylase is selected from any one or a combination of two or more of the following: derived from Escherichia coli (strain K12), Uniprot number P00490, derived from Thermococcus litoralis, Uniprot number Q9YGA7, derived from Thermococcus kodakarensis, Uniprot number Q5JH18; The UTP-glucose 1-phosphate uridyl transferase is selected from any one or a combination of two or more of the following: derived from Sulfurisphaera tokodaii, Uniprot number Q975F9, derived from Thermococcus chitonophagus, Uniprot number A0A170SE13, derived from Hordeum vulgare, Uniprot number Q43772; The UDP-glucose 6-dehydrogenase is selected from any one or a combination of two or more of the following: derived from Pyrobaculum islandicum, Uniprot number A1RUM9, derived from Escherichia coli (strain K12), Uniprot number P76373; The UDP-glucuronic acid decarboxylase is selected from any one or a combination of two or more of the following: derived from Homosapiens, Uniprot number Q8NBZ7, derived from Thermomicrobium roseum, Uniprot number A0A7C1K4Y9; The UDP-D-xylose 4-epimerase is selected from any one or a combination of two or more of the following, or the UDP-D-xylose 4-epimerase is selected from a nucleotide sequence having a sequence identity of 50%-100% with the following enzymes: derived from Arabidopsis thaliana, Uniprot number Q9SA77, derived from Thermodesulfitimonas autotrophica, Uniprot number A0A3N5B0B9, derived from Prunus persica, Uniprot number M4QEJ8, The UTP-L-arabinose 1-phosphate uridyl transferase is selected from any one or a combination of two or more of the following, or the UTP-L-arabinose 1-phosphate uridyl transferase is selected from a nucleotide sequence having a sequence identity of 50%-100% with the following enzymes: derived from Arabidopsis thaliana, Uniprot number Q9C5I1, derived from Dioscorea alata, GenBank number KAH7674669.1; The L-arabinose 1-phosphate phosphatase is selected from any one or a combination of two or more of the following, or the L-arabinose 1-phosphate phosphatase is selected from a nucleotide sequence having a sequence identity of 30%-100% with the following enzymes: derived from Clostridium acetobutylicum, Uniprot number Q97KR2, derived from Salmonella typhi, Uniprot number Q8Z854, derived from Neisseria meningitidis, Uniprot number Q9JYB3, derived from Escherichia coli (strain K12), Uniprot number P75792, derived from Methanosarcina mazei, Uniprot number Q8PWF4, derived from Acidothermus cellulolyticus, Uniprot number A0LWR5, derived from Bacillus cereus, cereus, Uniprot number Q819K1, from Salmonella paratyphi, Uniprot number C0Q032, from Bacteroides thetaiotaomicron, Uniprot number Q8A759, from Thermoanaerobaculiabacterium, GenBank number: HSG40782.1; The NADH oxidase is derived from Anaerocolumna aminovalerica, and the Uniprot number is Q2WFW5; Preferably, the α-1,4 glucan phosphorylase is derived from Escherichia coli (strain K12), and its Uniprot number is P00490; The UTP-glucose 1-phosphate uridyl transferase is derived from barley Hordeum vulgare, and the Uniprot number is Q43772; The UDP-glucose 6-dehydrogenase is derived from Escherichia coli (strain K12), and the Uniprot number is P76373; The UDP-glucuronic acid decarboxylase is derived from Thermomicrobium roseum, and its Uniprot number is A0A7C1K4Y9; The UDP-xylose 4-epimerase is derived from Arabidopsis thaliana, and the Uniprot number is Q9SA77; The UTP-L-arabinose 1-phosphate uridyl transferase is derived from Arabidopsis thaliana, and the Uniprot number is Q9C5I1; The L-arabinose 1-phosphate phosphatase is derived from Salmonella typhi, and its Uniprot number is Q8Z854.

3. The method according to claim 1, characterized in that The starch or its derivative is starch or its derivative pretreated by starch isomerase; The enzyme activator is a compound containing magnesium ions, preferably magnesium chloride; The inorganic phosphorus comes from a phosphorus-containing buffer, preferably a phosphate buffer, a sodium hydrogen phosphate-citrate buffer, or a mixture of the two; the pH of the phosphorus-containing buffer is 5.0-9.0, preferably 7.5; The nucleotide is uridine triphosphate; The oxidized coenzyme is nicotinamide adenine dinucleotide.

4. The method according to claim 3, characterized in that The starch or its derivative is selected from soluble starch, maltodextrin, and starch dextrin; The starch isomerase is selected from any one or a combination of two or more of the following: derived from Escherichia coli (strain K12), Uniprot numbered P15067, derived from acidophilic archaeon Cuniculiplasma divulgatum, Uniprot numbered A0A1N5SFX9; preferably, the starch isomerase is derived from acidophilic archaeon Cuniculiplasma divulgatum, Uniprot numbered A0A1N5SFX9.

5. The method according to claim 1, characterized in that The ratio of starch or its derivative, nucleotide, oxidized coenzyme, α-1,4-glucan phosphorylase, UTP-glucose 1-phosphate uridyl transferase, UDP-glucose 6-dehydrogenase, UDP-glucuronic acid decarboxylase, UDP-xylose 4-epimerase, UTP-L-arabinose 1-phosphate uridyl transferase, L-arabinose 1-phosphate phosphatase, NADH oxidase, enzyme activator and inorganic phosphorus is 5-100 g / L: 2-8 mM: 0.5-4 mM: 0.1- 50U / mL: 0.1~50U / mL: 0.1~50U / mL: 0.1~50U / mL: 0.1~50U / mL: 0.1~50U / mL: 0.1~50U / mL: 0.1~50U / mL: 5~1 5mM: 40-60mM, preferably 10g / L: 5mM: 2mM: 2U / mL: 1U / mL: 2U / mL: 2U / mL: 3U / mL: 1U / mL: 2U / mL: 2U / mL: 10mM: 50mM.

6. The method according to claim 1, characterized in that The method comprises mixing a substrate, a reaction enzyme, an enzyme activator, a cofactor and inorganic phosphorus and reacting the mixture to obtain L-arabinose; Alternatively, the method comprises the following steps: (1) Using α-1,4-glucan phosphorylase, the substrate and inorganic phosphorus are converted into glucose 1-phosphate; (2) UTP-glucose 1-phosphate uridyl transferase is used to convert glucose 1-phosphate and UTP into UDP-glucose and pyrophosphate; (3) UDP-glucose 6-dehydrogenase is used to convert UDP-glucose and NAD + Converted into UDP-glucuronic acid and NADH; (4) using UDP-glucuronic acid decarboxylase to convert UDP-glucuronic acid into UDP-D-xylose and CO2; (5) using UDP-D-xylose 4-epimerase to convert UDP-D-xylose into UDP-L-arabinose; (6) using UTP-L-arabinose 1-phosphate uridyl transferase to reversibly convert UDP-L-arabinose and pyrophosphate into L-arabinose 1-phosphate and UTP; (7) Specific L-arabinose 1-phosphate phosphatase is used to catalyze the removal of the phosphate group of L-arabinose 1-phosphate to generate L-arabinose. (8) NADH oxidase is used to convert the generated NADH into NAD + , achieving cofactor circulation in the reaction system.

7. The method according to claim 6, characterized in that The reaction temperature is 25 to 85° C., preferably 25 to 45° C., more preferably 30 to 40° C.; the reaction time is 1 to 48 hours, preferably 2 to 24 hours.

8. The method according to claim 7, characterized in that In the reaction, each catalytic reaction is carried out simultaneously, and the reaction time is 2 to 48 hours, preferably 8 to 24 hours; Alternatively, each catalytic reaction in the reaction is carried out independently of each other, and the reaction time is 1 to 24 hours, preferably 2 to 12 hours.

9. A method for synthesizing D-xylose by enzyme catalysis, characterized in that: The method comprises the following steps: mixing a substrate, a reaction enzyme, an enzyme activator, a cofactor and inorganic phosphorus and reacting the mixture to obtain D-xylose; the reaction enzymes are α-1,4-glucan phosphorylase, UTP-glucose 1-phosphate uridyl transferase, UDP-glucose 6-dehydrogenase, UDP-glucuronic acid decarboxylase, UTP-xylose 1-phosphate uridyl transferase, D-xylose 1-phosphate phosphatase and NADH oxidase; Alternatively, the method comprises the following steps: (I) preparing UDP-D-xylose according to steps (1) to (4) of claim 7; (II) using UTP-xylose 1-phosphate uridyltransferase USP to reversibly convert UDP-D-xylose and PPi into D-xylose 1-phosphate and UTP; (III) Using the specific D-xylose 1-phosphate phosphatase Xyl1PP to catalyze the removal of the phosphate group of D-xylose 1-phosphate, D-xylose is obtained.

10. A method for synthesizing xylitol by enzyme catalysis, characterized in that: The method comprises the following steps: mixing a substrate, a reaction enzyme, an enzyme activator, a cofactor and inorganic phosphorus and reacting the mixture to obtain xylitol; the reaction enzymes are α-1,4-glucan phosphorylase, UTP-glucose 1-phosphate uridyl transferase, UDP-glucose 6-dehydrogenase, UDP-glucuronic acid decarboxylase, UTP-xylose 1-phosphate uridyl transferase, D-xylose 1-phosphate phosphatase and xylose reductase; Alternatively, the method comprises the following steps: (i) preparing D-xylose according to steps (I) to (III) of claim 9; (ii) Xylose reductase XYL1 converts D-xylose and NADH into xylitol and NAD + .

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