Uridine diphosphate-glucose dehydrogenase mutant and application thereof in synthesis of uridine diphosphate-glucuronic acid

Abstract

The invention relates to a uridine diphosphate-glucose dehydrogenase mutant and an application of the uridine diphosphate-glucose dehydrogenase mutant in synthesis of uridine diphosphate-glucuronic acid. The amino acid sequence of the uridine diphosphate-glucose dehydrogenase mutant D7P166M / I169A is as shown in SEQ ID NO. 2, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO. 1. The invention also provides an application of the mutant in preparation of uridine diphosphate-glucuronic acid, and a method for preparing uridine diphosphate-glucuronic acid by using the mutant. Experimental determination shows that the melting temperature of the mutant is increased by 30 DEG C compared with that of a wild type, the activity is improved by 20%, and a powerful tool is provided for enzymatic synthesis of uridine diphosphate-glucuronic acid compounds. Compared with a conventional synthesis pathway, the method for synthesizing uridine diphosphate-glucuronic acid through immobilized enzyme flow provided by the invention can be used for stably and efficiently synthesizing for a long time, and is beneficial to reducing the cost and realizing industrial synthesis.

Description

Technical Field

[0001] The present invention relates to a uridine diphosphate-glucose dehydrogenase mutant and its application in the synthesis of uridine diphosphate-glucuronic acid, belonging to the technical fields of bioengineering and biosynthesis. Background Art

[0002] Uridine diphosphate-glucuronic acid (UDP-GlcA) is a product formed by the condensation of the hemiacetal hydroxyl group at the 1-position of glucuronic acid and the phosphate group at the end of uridine diphosphate, after removing a molecule of water. It is a high-energy activated form of glucuronic acid and an important metabolic intermediate with various physiological functions. First, as a glycosyl donor, it participates in the glucuronidation reaction in vivo. The glucuronic acid group in UDP-GlcA is transferred to the substrate to form a glucuronic acid derivative, thereby enhancing the water solubility of the substrate and promoting its excretion. In addition, UDP-GlcA is also involved in the synthesis process of glycosaminoglycans. As a glycosyl donor, UDP-GlcA is linked in the polysaccharide chain through a glycosidic bond. Especially in the de novo synthesis of heparin, UDP-GlcA and UDP-GlcNAc jointly participate in the extension of the polysaccharide chain. Therefore, the research and synthesis of UDP-GlcA are of great significance.

[0003] Currently, there are mainly two methods for synthesizing UDP-GlcA: chemical synthesis and biological synthesis. Among them, biological synthesis is further divided into microbial synthesis and enzymatic synthesis. Chemical synthesis often requires the use of organic reagents such as acyl chlorides and pyridines. However, nucleotide sugars have low solubility in organic solvents, often have low reaction rates, poor selectivity, and difficult subsequent purification. These factors make it difficult to synthesize nucleotide sugars by chemical methods and are not conducive to subsequent research and synthesis. Although microbial synthesis solves the problems of large pollution and low yield in chemical synthesis to a certain extent, it involves the editing and regulation of engineering cell genes, and the fermentation culture conditions are relatively harsh, and the purification of the product solution is complex. It is still difficult to reduce the cost. Enzymatic synthesis uses enzymatic reactions, which are faster, more selective, and usually have mild reaction conditions and are environmentally friendly compared to other reactions. However, the free enzymes used in enzymatic synthesis are more susceptible to the environment and are easily inactivated or even lose activity under non-optimal conditions. At the same time, the enzymes cannot be recovered and have poor reusability, making the use cost of free enzymes relatively high, which limits their application in industrial production.

[0004] The enzyme immobilization technology usually refers to immobilizing the enzyme on an insoluble carrier to convert it into a heterogeneous catalyst. While improving the enzyme strength, it also facilitates the recovery in various bioreactors in a flowing form. The recycling of enzymes saves the production cost of enzymes, especially for expensive enzymes, and eliminates the cumbersome preparation and separation processes in the downstream process, greatly expanding the application of enzymes in the industrial field. Constructing an immobilized synthesis system is of great significance for realizing the low-cost and high-efficiency synthesis of products in industry. Summary of the Invention

[0005] Aiming at the problems existing in the current methods for synthesizing uridine diphosphate-glucuronic acid, the present invention provides a mutant of uridine diphosphate-glucose dehydrogenase and its application in the synthesis of uridine diphosphate-glucuronic acid. First, the present invention uses computer-aided technology and activity and stability determination to obtain a mutant of uridine diphosphate-glucose dehydrogenase D7 P166M / I169A with high activity and high stability. Then, using a synthesis route with sucrose, uridine triphosphate (UTP), phosphate, and nicotinamide adenine dinucleotide (NAD+) as starting materials, through three-step reactions, uridine diphosphate-glucuronic acid is finally obtained. And using the technical method of immobilized enzyme, the catalytic enzyme is immobilized and then the flow synthesis technology is used to continuously and efficiently synthesize uridine diphosphate-glucuronic acid.

[0006] The technical solution of the present invention is as follows:

[0007] In the first aspect of the present invention, a mutant of uridine diphosphate-glucose dehydrogenase D7 P166M / I169A is provided, whose amino acid sequence is as shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is as shown in SEQ ID NO.1;

[0008] The mutant of uridine diphosphate-glucose dehydrogenase D7 P166M / I169A has 67 amino acid mutations in the wild-type uridine diphosphate-glucose dehydrogenase TuaD; the specific mutations are: A53E, D221E, E394D, H176E, I283L, L61K, N69T, P337R, P355E, P72A, Q187A, S329D, S358R, T92D, V135I, V240M, V396D, Y82F, A191I, A359K, C30V, D238K, Q63G, S314A, V295F, K252H, M89P, N325D, Q132K, S133E, T163F, A5C, E291R, E363D, K195I, S234H, S275K, T174D, T289V, V28I, A103V, D54E, H190N, Q341K, V390I, V44I, M305L, A77N, E57K, K91P, N197D, R311K, S39K, T378A, T400K, I71L, D299R, D62A, E356N, G446A, K101E, K235D, S144P, S16T, S35E, T303K, V308I; the GeneBank accession number of the wild-type uridine diphosphate-glucose dehydrogenase TuaD is BSU35580.

[0009] Compared with the wild-type uridine diphosphate-glucose dehydrogenase TuaD, the uridine diphosphate-glucose dehydrogenase mutant D7 P166M / I169A has the following mutations: alanine at position 53 is mutated to glutamate, aspartic acid at position 221 is mutated to glutamate, glutamic acid at position 394 is mutated to aspartic acid, histidine at position 176 is mutated to glutamate, isoleucine at position 283 is mutated to leucine, leucine at position 61 is mutated to lysine, asparagine at position 69 is mutated to threonine, proline at position 337 is mutated to arginine, proline at position 355 is mutated to glutamate, proline at position 72 is mutated to alanine, glutamine at position 187 is mutated to alanine, serine at position 329 is mutated to aspartic acid, serine at position 358 is mutated to arginine, threonine at position 92 is mutated to aspartic acid, valine at position 135 is mutated to isoleucine, valine at position 240 is mutated to methionine, valine at position 396 is mutated to aspartic acid, tyrosine at position 82 is mutated to phenylalanine, alanine at position 191 is mutated to isoleucine, alanine at position 359 is mutated to lysine, cysteine at position 30 is mutated to valine, aspartic acid at position 238 is mutated to lysine, glutamine at position 63 is mutated to glycine, serine at position 314 is mutated to alanine, valine at position 295 is mutated to phenylalanine, lysine at position 252 is mutated to histidine, methionine at position 89 is mutated to proline, asparagine at position 325 is mutated to aspartic acid, glutamine at position 132 is mutated to lysine, serine at position 133 is mutated to glutamate, threonine at position 163 is mutated to phenylalanine, alanine at position 5 is mutated to cysteine, glutamic acid at position 291 is mutated to arginine, glutamic acid at position 363 is mutated to aspartic acid, lysine at position 195 is mutated to isoleucine, serine at position 234 is mutated to histidine, serine at position 275 is mutated to lysine, threonine at position 174 is mutated to aspartic acid, threonine at position 289 is mutated to valine, valine at position 28 is mutated to isoleucine, alanine at position 103 is mutated to valine, aspartic acid at position 54 is mutated to glutamate, histidine at position 190 is mutated to asparagine, glutamine at position 341 is mutated to lysine, valine at position 390 is mutated to isoleucine, valine at position 44 is mutated to isoleucine, methionine at position 305 is mutated to leucine, alanine at position 77 is mutated to asparagine, glutamic acid at position 57 is mutated to lysine, lysine at position 91 is mutated to proline, asparagine at position 197 is mutated to aspartic acid, arginine at position 311 is mutated to lysine, serine at position 39 is mutated to lysine, threonine at position 387 is mutated to alanine, threonine at position 400 is mutated to lysine, isoleucine at position 71 is mutated to leucine, aspartic acid at position 299 is mutated to arginine, aspartic acid at position 62 is mutated to alanineThe glutamic acid at the 356th position is mutated to asparagine, the glycine at the 446th position is mutated to alanine, the lysine at the 101st position is mutated to glutamic acid, the lysine at the 235th position is mutated to aspartic acid, the serine at the 144th position is mutated to proline, the serine at the 16th position is mutated to threonine, the serine at the 35th position is mutated to glutamic acid, the glutamic acid at the 303rd position is mutated to lysine, and the valine at the 308th position is mutated to isoleucine.

[0010] In a second aspect of the present invention, there is provided an expression cassette or recombinant vector containing the encoding gene of the uridine diphosphate-glucose dehydrogenase mutant D7 P166M / I169A. There are no particular limitations on the starting vector for the recombinant vector, and any vector known in the art can be used as long as it can replicate in the host. For example, the vector includes but is not limited to plasmids and phages. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases integrate into the genome itself.

[0011] Preferably according to the present invention, the recombinant vector or expression cassette is obtained by ligating the encoding gene of the uridine diphosphate-glucose dehydrogenase mutant D7P166M / I169A with an expression vector, and the expression vector is a plasmid.

[0012] More preferably, the plasmid is pET-28a(+).

[0013] In a third aspect of the present invention, there is provided a recombinant host cell containing the encoding gene of the uridine diphosphate-glucose dehydrogenase mutant D7 P166M / I169A. Wherein the "host cell" has the meaning commonly understood in the art, and it is a host cell capable of introducing the encoding gene of the mutant of the present invention, and is called a recombinant host cell after introduction. The strain of the present invention can be a prokaryotic cell or a eukaryotic cell, preferably a prokaryotic cell, and more preferably Escherichia coli BL21(DE3).

[0014] In a fourth aspect of the present invention, there is provided the use of the above-mentioned uridine diphosphate-glucose dehydrogenase mutant D7 P166M / I169A in the preparation of uridine diphosphate-glucuronic acid.

[0015] In a fifth aspect of the present invention, there is provided a method for immobilized enzyme preparation flow synthesis of uridine diphosphate-glucuronic acid using the above-mentioned uridine diphosphate-glucose dehydrogenase mutant D7P166M / I169A, including the following steps:

[0016] Using sucrose, UTP, NAD +Using [substrate] as the substrate, phosphate buffer and inorganic ions were added to the substrate, and then an immobilized enzyme preparation of sucrose phosphatase LmSPase, pyrophosphorylase BlUSP, pyrophosphatase PmPpA, uridine diphosphate-glucose dehydrogenase mutant D7P166M / I169A, and NADH oxidase TkNOX was used as a catalyst to obtain the target product UDP-GlcA through continuous flow synthesis.

[0017] Preferably according to the present invention, the final concentration of sucrose is 12 mM.

[0018] Preferably according to the present invention, the final concentration of UTP is 10 mM.

[0019] Preferably according to the present invention, the NAD + has a final concentration of 10 mM.

[0020] Preferably according to the present invention, the final concentration of the phosphate buffer is 20 mM.

[0021] Preferably according to the present invention, the inorganic ion is magnesium ion.

[0022] More preferably, the magnesium ion is generated by hydrolysis of magnesium chloride, and the final concentration of the magnesium ion is 10 mM.

[0023] Preferably according to the present invention, the reaction temperature of the biosynthesis is 37 °C, and the reaction residence time using a flow synthesis device is 4 to 6 h.

[0024] Preferably according to the present invention, the immobilized enzyme preparation of sucrose phosphatase LmSPase and pyrophosphorylase BlUSP is prepared as follows: The free sucrose phosphatase LmSPase and pyrophosphorylase BlUSP are diluted with a buffer and then mixed with an immobilized carrier respectively, and immobilized at 4 °C and 70-85 rpm for 16 h, and then left to stand for 8-10 h. After washing, the immobilized enzyme preparation of sucrose phosphatase LmSPase and pyrophosphorylase BlUSP is obtained.

[0025] More preferably, the GenBank accession number of sucrose phosphatase LmSPase is D90314; the GenBank accession number of pyrophosphorylase BlUSP is EEI80102.

[0026] More preferably, the immobilized carrier is resin LXTE-706; the loading amount of sucrose phosphatase LmSPase is 25 mg / g, and the loading amount of pyrophosphorylase BlUSP is 20 mg / g, unit: protein mg / immobilized carrier g; the buffer is PB buffer (pH = 7, concentration 0.02 M).

[0027] Preferably according to the present invention, the immobilized enzyme preparation of pyrophosphatase PmPpA is prepared by the following method:

[0028] The free pyrophosphatase PmPpA is diluted with a buffer solution and then mixed with an immobilized carrier activated with glutaraldehyde, and immobilized at 4°C and 70-85 rpm for 16 h, and then left standing for 8-10 h. After washing, the immobilized enzyme preparation of pyrophosphatase PmPpA is obtained.

[0029] More preferably, the GenBank accession number of the pyrophosphatase PmPpA is AAK03275.1.

[0030] More preferably, the immobilized carrier activated with glutaraldehyde is: the resin LXTE-706 is mixed with a 2% glutaraldehyde solution at a mass-to-volume ratio of 1:10, and shaken and activated at 20-25°C for 60 min.

[0031] More preferably, the immobilized carrier is the resin LXTE-706; the loading amount of the pyrophosphatase PmPpA is 10 mg / g, unit: protein mg / immobilized carrier g; the buffer solution is PB buffer (pH = 7, concentration 0.02 M).

[0032] Preferably according to the present invention, the immobilized enzyme preparation of the uridine diphosphate-glucose dehydrogenase mutant D7 P166M / I169A is prepared by the following method:

[0033] The free uridine diphosphate-glucose dehydrogenase mutant D7 P166M / I169A is diluted with a buffer solution and then mixed with an immobilized carrier, and immobilized at 4°C and 70-85 rpm for 16 h. After standing for 8-10 h, a 1 M glycine solution with pH = 7.0 is added at a volume ratio of 1:10, and incubated at 4°C and 70 rpm for 12 h. After washing, the immobilized enzyme preparation of the uridine diphosphate-glucose dehydrogenase mutant D7 P166M / I169A is obtained.

[0034] More preferably, the immobilized carrier is the resin LXTE-706; the loading amount of the uridine diphosphate-glucose dehydrogenase mutant D7 P166M / I169A is 20 mg / g, unit: protein mg / immobilized carrier g; the buffer solution is PB buffer (pH = 7, concentration 0.02 M, containing 30% glycerol).

[0035] Preferably according to the present invention, the preparation method of the immobilized enzyme preparation of NADH oxidase TkNOX is as follows:

[0036] The free NADH oxidase TkNOX was diluted and mixed with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and an immobilization carrier, and immobilized at 4°C under the conditions of 70-85 rpm for 16 h, and then left standing for 8-10 h. After washing, an immobilized enzyme preparation of NADH oxidase TkNOX was obtained.

[0037] Further preferably, the GenBank accession number of the NADH oxidase TkNOX is BAD84493.1.

[0038] Further preferably, the immobilization carrier is resin LXTE-706; the loading amount of the NADH oxidase TkNOX is 10 mg / g, unit: mg of protein / g of immobilization carrier; the buffer solution is PB buffer solution (pH = 7, concentration 0.02 M).

[0039] Preferably according to the present invention, the optimal ratio of the enzyme amounts of the sucrose phosphatase LmSPase, pyrophosphorylase BlUSP, pyrophosphatase PmPpA, uridine diphosphate-glucose dehydrogenase mutant D7 P166M / I169A and NADH oxidase TkNOX immobilized enzyme is 3:21:1:30:10.

[0040] According to the present invention, the above-mentioned uridine diphosphate-glucose dehydrogenase mutant D7 P166M / I169A can be used to continuously synthesize uridine diphosphate-glucuronic acid by multi-enzyme immobilization. The specific method is as follows:

[0041] An assembled flow synthesis device is included, which includes a substrate container, a constant flow pump, a thermostatic jacket chromatography column and a product container connected in sequence through pipelines. The immobilized enzyme preparations of sucrose phosphatase LmSPase, pyrophosphorylase BlUSP, pyrophosphatase PmPpA, uridine diphosphate-glucose dehydrogenase mutant D7 P166M / I169A and NADH oxidase TkNOX are loaded in the thermostatic jacket chromatography column, and then the reaction substrate is pumped into the chromatography column through the constant flow pump for biosynthesis. After synthesis, the product uridine diphosphate-glucuronic acid is collected in the container.

[0042] The technical features of the present invention:

[0043] In the present invention, protein engineering molecular modification was carried out on uridine diphosphate-glucose dehydrogenase TuaD, and the advantageous mutant D7 P166M / I169A was obtained, which can be used as a potential immobilized tool enzyme. Sucrose phosphorylase (LmSPase) was used to catalyze the phosphorolysis of sucrose to generate glucose-1-phosphate (Glucose-1-phosphate, Glc-1-P), and then uridine diphosphate sugar pyrophosphorylase (BlUSP) was used to mediate the condensation of Glc-1-P and UTP to generate UDP-Glc, and coupling with pyrophosphatase (PmPpA) achieved in-situ hydrolysis of pyrophosphate by-products to regenerate phosphate. UDP-Glc was catalyzed by the uridine diphosphate-glucose dehydrogenase mutant D7P166M / I169A to generate the target product UDP-GlcA, and NADH oxidase (TkNOX) was used to eliminate the inhibitory effect of NADH coenzyme. All the catalytic enzymes required for the whole system were immobilized using immobilized enzyme technology, and UDP-GlcA was continuously and efficiently synthesized using a flow synthesis device.

[0044] Advantages:

[0045] 1. The present invention provides a uridine diphosphate-glucose dehydrogenase mutant D7P166M / I169A with high activity and high stability, which has 67 amino acid site mutations compared with the wild-type uridine diphosphate-glucose dehydrogenase TuaD. Through experimental determination, the melting temperature of this mutant is increased by 30 °C compared with the wild-type, and the activity is increased by 20%, providing a powerful tool for the enzymatic synthesis of uridine diphosphate-glucuronic acid compounds.

[0046] 2. The present invention provides a method for the biosynthesis of uridine diphosphate-glucuronic acid, which uses sucrose, UTP and NAD + as raw materials, and then undergoes three-step catalysis with sucrose phosphorylase LmSPase, pyrophosphorylase BlUSP, pyrophosphatase PmPpA, uridine diphosphate-glucose dehydrogenase TuaD and NADH oxidase TkNOX to achieve the synthesis of uridine diphosphate-glucuronic acid. Compared with the conventional synthesis pathways (sucrose synthase pathway, glucuronic acid pathway), the reaction efficiency is greatly improved.

[0047] 3. All the catalytic enzymes in the method for the biosynthesis of uridine diphosphate-glucuronic acid of the present invention participate in the reaction in the form of immobilized enzyme preparations, which improves the stability of the enzymes, and the immobilized enzyme preparations can be reused multiple times, further reducing the cost and enabling stable and long-term high-efficiency synthesis. And using flow synthesis technology, the synthesis process is convenient to regulate, the conditions are mild, and the process is environmentally friendly, which is suitable for popularization and application in industrial production and is conducive to reducing costs and realizing industrialized synthesis. Description of the Drawings

[0048] Figure 1SDS-Page result graphs of enzymes LmSPase, BlUSP, PmPpA, TuaD, and TkNOX.

[0049] Figure 2 Results of the determination of the enzyme activities and T m values of uridine diphosphate-glucose dehydrogenase TuaD and its mutants D1-D7;

[0050] In the figure, the horizontal axis represents the mutant names, and the vertical axis represents the enzyme activities and T m values.

[0051] Figure 3 Results of the determination of the enzyme activity of the D1 single mutant (TuaD-D1);

[0052] In the figure, the horizontal axis represents the mutant names, and the vertical axis represents the enzyme activities

[0053] Figure 4 Results of the determination of the enzyme activity of the D2 mutant (TuaD-D2);

[0054] In the figure, the horizontal axis represents the mutant names, and the vertical axis represents the enzyme activities.

[0055] Figure 5 Results of the determination of the enzyme activity of the uridine diphosphate-glucose dehydrogenase mutant D7 P166M / I169A;

[0056] In the figure, the horizontal axis represents the enzyme types, and the vertical axis represents the enzyme activities.

[0057] Figure 6 Results of the determination of the enzyme activities of different carrier-immobilized enzyme preparations;

[0058] In the figure, the horizontal axis represents the names of different immobilization carriers, and the vertical axis represents the loaded enzyme activities.

[0059] Figure 7 Results of the loading amount of the immobilized enzyme preparation of the uridine diphosphate-glucose dehydrogenase mutant D7 P166M / I169A;

[0060] In the figure, the horizontal axis represents the enzyme addition amount, and the vertical axis represents the loaded enzyme activities and the enzyme activity recovery rates.

[0061] Figure 8 Results of the loading amount of the immobilized enzyme preparation of NADH oxidase TkNOX;

[0062] In the figure, the horizontal axis represents the enzyme addition amount, and the vertical axis represents the loaded enzyme activities and the enzyme activity recovery rates.

[0063] Figure 9 Comparison of the number of reaction cycles of the immobilized enzyme preparations of wild-type uridine diphosphate-glucose dehydrogenase and the uridine diphosphate-glucose dehydrogenase mutant D7P166M / I169A

[0064] In the figure, the abscissa is the number of reaction cycles and the ordinate is the relative conversion rate.

[0065] Figure 10 is the number of reaction cycles of the immobilized enzyme of LmSPase and the immobilized enzyme of BlUSP;

[0066] In the figure, the left figure is LmSPase and the right figure is BlUSP.

[0067] Figure 11 is the comparison result of the enzyme activity loaded by the immobilized enzyme preparation of wild-type uridine diphosphate-glucose dehydrogenase and uridine diphosphate-glucose dehydrogenase mutant D7P166M / I169A;

[0068] In the figure, the abscissa is the name of different immobilization carriers and the ordinate is the enzyme activity loaded.

[0069] Figure 12 is the flow chart for synthesizing uridine diphosphate-glucuronic acid using the uridine diphosphate-glucuronosyltransferase mutant D3 / 144T / 146L.

[0070] Figure 13 is the optimization of the dosage ratio of the immobilized enzymes of LmSPase, BlUSP and PmPpA;

[0071] In the figure, the left figure is the optimization of the ratio (A) of LmSPase and BlUSP, and the right figure is the optimization of the ratio (α) of (A) and PmPpA. The abscissa is the different enzyme dosage ratios and the ordinate is the space-time yield;

[0072] Figure 14 is the optimization of the dosage ratio of the immobilized enzyme in the flow synthesis UDP-GlcA system;

[0073] In the figure, the left figure is the optimization of the ratio (β) of TuaD and TkNOX, and the right figure is the optimization of the ratio of (α) and (β). The abscissa is the different enzyme dosage ratios and the ordinate is the space-time yield;

[0074] Figure 15 is the graph showing the change of the product concentration of UDP-GlcA with time in the flow synthesis;

[0075] In the figure, the abscissa is the reaction time and the ordinate is the product concentration;

[0076] Figure 16 is the HPLC result graph of UDP-GlcA;

[0077] Figure 17 is the mass spectrometry result graph of UDP-GlcA. Specific implementation manners

[0078] The present invention will be described below through specific implementation examples. Unless otherwise specified, the technical means used in the present invention are all methods well-known to those skilled in the art. The following examples are intended to further illustrate the content of the present invention rather than limit the protection scope of the present invention.

[0079] In the examples, the resins LXTE-706 and LXTE-800 were purchased from Xi'an BlueSail New Materials Co., Ltd.

[0080] All the substrate standard reagents used were purchased from Jinan Zhongchu Hengtong Biotechnology Co., Ltd. The required plasmids were commissioned to be synthesized by GenScript Biotech Corporation. The strains and plasmid vectors used are shown in Table 1.

[0081] Table 1. Strains and plasmid vectors

[0082]

[0083] After diluting the reaction solution after the reaction to 0.4 - 5 mM, 100 μL of the volume was taken, and then 200 μL of 3,5-dinitrosalicylic acid reaction solution was added, and color development was carried out in a boiling water bath for 5 min. After the sample was cooled to room temperature, 200 μL was taken into a 96-well microplate, and the absorbance of the sample was read at a wavelength of 540 nm.

[0084] The HPLC method adopted in the present invention is as follows:

[0085] A Shimadzu LC-20A high-performance liquid chromatograph made in Japan was used, the ultraviolet detector was SPD-20A, the chromatographic column was Dikma Polyamino HILIC (5 μm 250×4.6 mm), the mobile phase was 20% acetonitrile aqueous solution (phase A) and an aqueous solution of 0.2 mol / L Na2H2PO4, 20% acetonitrile, and 0.1% triethylamine (phase D). Before use, the mobile phase was filtered through a 0.22 μm filter membrane and degassed by ultrasonic treatment. The flow rate during liquid phase detection was 1 mL / min, the detection wavelength of UDP-Sugar was 254 nm, and the liquid phase analysis program is shown in Table 2.

[0086] Table 2. HPLC analysis program for UDP-Sugar

[0087]

[0088] In the present invention, the sucrose phosphatase (LmSPase) is derived from the strain Leuconostoc mesenteroides with the GenBank accession number D90314; the pyrophosphorylase (BlUSP) is derived from the strain Bifidobacterium longum ATCC55813 with the GenBank accession number EEI80102.1; the pyrophosphatase (PmPpA) is derived from the strain Pasteurella multocida with the GenBank accession number AAK03275.1; the UDP-Glc dehydrogenase (TuaD) is derived from the strain Bacillus subtilis with the GenBank accession number BSU35580; the NADH oxidase (TkNOX) is derived from the strain Thermococcus kodakarensis KOD1 with the GenBank accession number BAD84493.1.

[0089] Example 1: Calculation of thermostable and activity-enhanced TuaD mutants by the PROSS algorithm

[0090] The amino acid sequence of wild-type uridine diphosphate-glucose dehydrogenase (TuaD) (GeneBank accession number BSU35580) was input into the Swiss-Model website for homologous structure prediction under default conditions. The predicted TuaD amino acid sequence and protein crystal structure were input into the PROSS online tool (https: / / pross.weizmann.ac.il) to calculate 7 TuaD mutants with enhanced stability and activity. The mutants were named D1 - D7 from the lowest to the highest mutation rate, and their mutation sites are shown in Table 3.

[0091] Table 3. Mutation sites of TuaD mutants D1 - D7

[0092]

[0093]

[0094] Example 2: Preparation, activity and stability determination of TuaD mutants

[0095] 1. The expression and purification methods of the recombinant proteins of the enzymes LmSPase, BlUSP, PmPpA, TuaD and TkNOX are as follows:

[0096] After the amino acid sequence of the recombinant protein was optimized for E. coli codons, the nucleotide sequence was synthetically generated by Nanjing Genscript Biotech Corporation and cloned into the corresponding vector. Then, it was chemically transformed into E. coli BL21(DE3) competent cells. Single colonies were picked and inoculated into 20 mL of LB liquid medium containing 50 μg / mL of the corresponding resistant antibiotic, and activated at 37°C and 225 rpm for 16 h. The activated bacterial solution was inoculated and expanded to 1 L of LB liquid medium containing 50 μg / mL of the corresponding resistant antibiotic at an inoculation amount of 2% (v / v), and shaken at 37°C and 225 rpm for 2.5 h until OD 600 reached 0.6 - 0.8. IPTG with a final concentration of 0.2 mM was added, and induction was carried out at 20°C and 225 rpm for 20 h to obtain the bacterial solution.

[0097] The obtained bacterial solution was centrifuged at 4°C and 8000 rpm for 10 min to collect the bacterial cells. Each liter of bacterial cells was resuspended in 30 mL of loading buffer containing 20 mM imidazole. The resuspended bacterial cells were disrupted in an ice-water bath. The disruption program was 15 s of working, 45 s of intermittent, an amplitude of 60%, a power of 1500 kJ, and the effective disruption time was calculated as 2.5 min per liter of bacteria. The disrupted bacterial cells were centrifuged at 4°C and 12000 rpm for 25 min, and the supernatant was filtered through a 0.45 μm filter membrane. Nickel ion affinity chromatography column was used for protein purification. After loading, the impurities were washed with the loading buffer containing 20 mM imidazole until no impurity protein flowed out, and then the target protein was eluted with the elution buffer containing 250 mM imidazole. The purified protein solution was ultrafiltered and concentrated to a concentration of more than 10 mg / mL, and the protein concentration was measured using the Bradford Protein Concentration Assay Kit from Beyotime Biotechnology Co., Ltd. 30% glycerol was added to the purified protein solution and mixed well. After aliquoting, it was stored at -80°C in the refrigerator.

[0098] SDS-Page electrophoresis was performed on the obtained enzymes LmSPase, BlUSP, PmPpA, TuaD, and TkNOX, and the results are as Figure 1 shown.

[0099] It can be Figure 1 seen that the enzymes LmSPase, BlUSP, PmPpA, TuaD, and TkNOX were all successfully heterologously expressed.

[0100] Then, according to the same method, mutants D1 - D7 of uridine diphosphate-glucose dehydrogenase (TuaD) were prepared according to the mutation information in Table 1.

[0101] 2. Activity determination of uridine diphosphate-glucose dehydrogenase TuaD and its mutants D1 - D7

[0102] Using UDP-Glc as a substrate, the enzyme activities and T m values of free uridine diphosphate-glucose dehydrogenase TuaD and its mutants D1-D7 were determined. The component contents of the free enzyme and immobilized enzyme reaction systems are shown in Table 4. The enzyme activities and T m values of free uridine diphosphate-glucose dehydrogenase TuaD and its mutants D1-D7 are shown as follows. Figure 2 Shown.

[0103] Table 4. TuaD Activity Assay System

[0104]

[0105] The free enzyme reaction system was preheated in a 37 °C water bath for 5 min after mixing except for the enzyme solution. Then, 190 μL was taken and added to a 96-well plate. After adding 10 μL of the diluted enzyme solution, it was quickly placed in a preheated microplate reader to measure the absorbance change at 340 nm to calculate the enzyme activity. The measurement was carried out for 3 min, once every 10 s, and the point at 1 min was taken for calculation. The immobilized enzyme reaction system was mixed and then reacted in a thermostatic shaking metal bath at 1000 rpm for 20 min. Each experiment was set up with three parallels. The content of the reaction product was analyzed by HPLC.

[0106] It can be seen from Figure 2 that as the mutation rate increases, the activities of TuaD mutants generally show an upward trend. However, among them, mutant D2 lost its activity. Based on this result, the inventors of this application reasonably speculated that among the 8 amino acid residues that differ between D2 and D1 proteins, there may be amino acid sites crucial for activity.

[0107] 2. Stability Determination of Uridine Diphosphate-Glucose Dehydrogenase TuaD and Its Mutants D1-D7

[0108] Differential scanning fluorimetry was used to measure the melting temperature (T m) to characterize its stability. The SYPRO Orange dye was diluted to 2% of the original concentration with protein elution buffer, and the protein sample concentration was diluted to 1 mg / mL. Next, 19 μL of the protein solution was added to the wells to be tested in a white 96-well PCR plate and mixed with 1 μL of the diluted dye. Three parallels were set for each test group. Detection was performed using a real-time fluorescence PCR instrument. The ROX fluorescence channel was set as the detection channel, and the temperature scanning range was set between 25°C and 95°C, with a heating rate of 0.1°C / second. The sample was placed in the instrument to start real-time detection of the fluorescence signal. According to the collected fluorescence intensity data, a fluorescence intensity curve varying with temperature was plotted. By calculating the first derivative of the curve, a first derivative curve was obtained, and the temperature corresponding to the peak was the T m value, and the results were the same as those shown in Figure 2 .

[0109] As can be seen from Figure 2 , the stability of the TuaD mutants D1 - D7 was significantly improved compared to the wild type, and with the increase of the mutation rate, the T m value was higher.

[0110] 3. According to the activity determination results of the TuaD mutants, the mutation sites newly added in the TuaD mutant D2 compared to the TuaD mutant D1 were introduced into the TuaD mutant D1. Eight D1 single mutants (TuaD - D1) were constructed on the basis of the uridine diphosphate - glucose dehydrogenase mutant D1, namely D1 - A191I, D1 - A359K, D1 - C30V, D1 - D238K, D1 - M166P, D1 - Q63G, D1 - S314A, and D1 - V295F. Then, their nucleotide sequences were artificially synthesized and heterologously expressed according to the method described in point 1 of this example, and the enzyme activities of the eight D1 single mutants (TuaD - D1) were measured according to the method described in point 2 of this example. At the same time, the TuaD mutant D1 was used as a control, and the results are as shown in Figure 3 .

[0111] As can be seen from Figure 3 , the key amino acid residue of the TuaD mutant is the 166th position.

[0112] 4. Usually, a single-site mutation will not cause such a significant change in enzyme activity. Therefore, it is further speculated that the loss of enzyme activity of the TuaD mutant D2 is likely due to the synergistic effect of the mutation at the 166th position and an existing mutation site in the TuaD mutant D1, which together lead to the loss of enzyme activity. By analyzing the three-dimensional structure of the mutant, some were found around the 166th position The 169th amino acid within the range is the mutation site in TuaD mutant D1 and it is combined with the 166th amino acid and introduced into TuaD mutant D2. Based on uridine diphosphate-glucose dehydrogenase mutant D2, three D2 mutants (TuaD-D2) were constructed, namely D2 P166M, D2 I169A, and D2 P166M / I169A. Then, after artificially synthesizing their nucleotide sequences according to the method described in point 1 of this example and performing heterologous expression, the enzyme activities of the three D2 mutants (TuaD-D2) were measured according to the method described in point 2 of this example. At the same time, using TuaD mutant D2 as a control, the results are as Figure 4 shown.

[0113] As Figure 4 shown, after the 166th and 169th amino acids were restored, the enzyme activity of TuaD mutant D2 was significantly restored, confirming the synergistic adverse effects of the 166th and 169th amino acids.

[0114] 5. Based on the previous findings, subsequently, the mutation sites at the 166th and 169th positions were restored in TuaD mutant D7 to enhance the enzyme activity of TuaD mutant D7. Based on uridine diphosphate-glucose dehydrogenase mutant D7, uridine diphosphate-glucose dehydrogenase mutant D7 P166M / I169A was constructed. Its amino acid sequence is as shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is as shown in SEQ ID NO.1.

[0115] Then, after artificially synthesizing its nucleotide sequence according to the method described in point 1 of this example and performing heterologous expression, the enzyme activity of uridine diphosphate-glucose dehydrogenase mutant D7 P166M / I169A was measured according to the method described in point 2 of this example. At the same time, using wild-type uridine diphosphate-glucose dehydrogenase as a control, the results are as Figure 5 shown.

[0116] As Figure 5 can be seen, the enzyme activity of the D7 P166M / I169A mutant was increased by approximately 20% compared to wild-type uridine diphosphate-glucose dehydrogenase.

[0117] Example 3: Preparation of immobilized enzyme preparation for synthesizing uridine diphosphate-glucuronic acid

[0118] 1. Preparation of each enzyme's immobilized enzyme preparation

[0119] ①. The preparation method of the immobilized enzyme preparations of sucrose phosphatase LmSPase and pyrophosphorylase BlUSP is as follows:

[0120] The resin carrier was washed repeatedly with buffer three times. After diluting sucrose phosphatase LmSPase and pyrophosphorylase BlUSP with buffer, the diluted sucrose phosphatase LmSPase and pyrophosphorylase BlUSP solutions were respectively mixed with the resin carrier. The ratio of the resin carrier to the enzyme solution was 1:10 (i.e., 1 g of resin was added to 10 mL of enzyme solution) to obtain an immobilization system. The entire immobilization system was added to a conical flask, the protein concentration in the supernatant was measured, and immobilization was carried out at 4 °C and 80 rpm for 16 h, followed by continued static standing for 10 h. After immobilization, the remaining protein content in the supernatant was measured to calculate the loading amount, and it was washed three times with buffer and then filtered by suction to obtain the immobilized enzyme preparations of sucrose phosphatase LmSPase and pyrophosphorylase BlUSP.

[0121] ②. The preparation method of the immobilized enzyme preparation of pyrophosphatase PmPpA is as follows:

[0122] (1) Prepare a 2% glutaraldehyde solution with buffer, mix the 2% glutaraldehyde solution with the resin carrier (the ratio of the carrier to the glutaraldehyde solution is 1:10, i.e., 1 g of resin is added to 10 mL of glutaraldehyde solution), shake and activate at 25 °C for 60 min, wash the carrier three times with buffer to wash off the glutaraldehyde, and filter by suction to remove water to obtain the resin carrier activated by glutaraldehyde.

[0123] (2) The resin carrier activated by glutaraldehyde was washed repeatedly with buffer three times. After diluting pyrophosphatase PmPpA with buffer, the diluted pyrophosphatase PmPpA solution was mixed with the resin carrier activated by glutaraldehyde. The ratio of the resin carrier to the enzyme solution was 1:10 (i.e., 1 g of resin was added to 10 mL of enzyme solution) to obtain an immobilization system. The entire immobilization system was added to a conical flask, the protein concentration in the supernatant was measured, and immobilization was carried out at 4 °C and 80 rpm for 16 h, followed by continued static standing for 10 h. After immobilization, the remaining protein content in the supernatant was measured to calculate the loading amount, and it was washed three times with buffer and then filtered by suction to obtain the immobilized enzyme preparation of pyrophosphatase PmPpA.

[0124] ③. The preparation method of the immobilized enzyme preparation of uridine diphosphate-glucose dehydrogenase mutant D7 P166M / I169A is as follows:

[0125] The resin support was washed repeatedly with buffer three times. After diluting uridine diphosphate-glucose dehydrogenase mutant D7 P166M / I169A with buffer containing 30% glycerol, the diluted uridine diphosphate-glucose dehydrogenase mutant D7 P166M / I169A solution was mixed with the resin support. The ratio of the resin support to the enzyme solution was 1:10 (i.e., 1 g of resin was added to 10 mL of enzyme solution) to obtain an immobilization system. The whole immobilization system was added to a conical flask, the protein concentration in the supernatant was measured, and immobilization was carried out at 4 °C and 80 rpm for 16 h, and then left to stand for another 10 h. After standing, the protein concentration in the supernatant was measured, and then the supernatant protein solution was aspirated. A 1 M glycine solution with pH 7.0 was added in a ratio of 1:10 and incubated at 4 °C and 70 rpm for 12 h. Finally, it was washed three times with buffer, and after suction filtration, an immobilized enzyme preparation of uridine diphosphate-glucose dehydrogenase mutant D7 P166M / I169A was obtained.

[0126] ④. The preparation method of the immobilized enzyme preparation of the NADH oxidase TkNOX is as follows:

[0127] The resin support was washed repeatedly with buffer three times. After diluting NADH oxidase TkNOX with buffer, the diluted NADH oxidase TkNOX solution was mixed with EDC and the resin support. The ratio of the resin support to the enzyme solution was 1:10 (i.e., 1 g of resin was added to 10 mL of enzyme solution) to obtain an immobilization system. The whole immobilization system was added to a conical flask, the protein concentration in the supernatant was measured, and immobilization was carried out at 4 °C and 80 rpm for 16 h, and then left to stand for another 10 h. After immobilization, the remaining protein content in the supernatant was measured to calculate the loading amount, and it was washed three times with buffer, and after suction filtration, an immobilized enzyme preparation of NADH oxidase TkNOX was obtained.

[0128] 2. Screening of immobilized enzyme carriers

[0129] Using resin LXTE-600, LXTE-700S, LXTE-703, LXTE-706, LXTE-800, LXTE-902 as carriers respectively, the immobilized enzyme preparations of sucrose phosphatase LmSPase, pyrophosphorylase BlUSP, pyrophosphatase PmPpA, uridine diphosphate-glucose dehydrogenase mutant D7 P166M / I169A and NADH oxidase TkNOX with different carriers were prepared according to the methods described in the manufacturer's instructions. Among them, since LXTE-706 contains both epoxy groups and amino groups, two methods were used for immobilization of this resin, namely LXTE-706(epoxy) and LXTE-706(-NH2). Then, with the systems in Tables 4, 6, 7, 8, 9, the enzyme activities of the immobilized enzyme preparations were measured to compare and select the best immobilization carrier.

[0130] Among them, the structures of resins LXTE-600, LXTE-700S, LXTE-703, LXTE-706, LXTE-800, and LXTE-902 are shown in Table 5; the method for measuring the enzyme activity of uridine diphosphate-glucose dehydrogenase mutant D7 P166M / I169A is as described in point 2 of Example 1.

[0131] Table 5. Resin Structures and Their Names

[0132]

[0133] Table 6. Activity Assay System for Immobilized LmSPase Enzyme Preparation

[0134]

[0135] After the reaction system is mixed evenly, it is placed in a thermostatic shaking metal bath for reaction at 1000 rpm for 20 min. Three parallels are set for each group of experiments. The content of the reaction product is analyzed by the DNS colorimetric method.

[0136] Table 7. Activity Assay System for Immobilized BlUSP Enzyme Preparation

[0137]

[0138] After the reaction system is mixed evenly, it is placed in a thermostatic shaking metal bath for reaction at 1000 rpm for 20 min. Three parallels are set for each group of experiments. The reaction product is analyzed by HPLC method.

[0139] Table 8. Activity Assay System for Immobilized PmPpA Enzyme

[0140]

[0141] After the reaction system is mixed evenly, it is placed in a thermostatic shaking metal bath for reaction at 1000 rpm for 5 min. Three parallels are set for each group of experiments. After the reaction product is diluted, it is analyzed and quantified using a malachite green phosphate detection kit.

[0142] Table 9. Activity Assay System for Immobilized TkNOX Enzyme

[0143]

[0144] After the reaction system is mixed evenly, it is placed in a thermostatic shaking metal bath for reaction at 1000 rpm at 60 °C for 5 min, and the reaction is quenched with 1% acetic acid. Three parallels and one blank group are set for each group of experiments. In the blank group, the immobilized enzyme is replaced with a blank carrier. The enzyme activity is calculated by measuring the difference in absorbance at 340 nm between the reaction completion and the blank group using an enzyme-labeled instrument.

[0145] The enzyme activity measurement results of the above various immobilized enzyme preparations are asFigure 6 as shown

[0146] It can be seen from Figure 6 that in most cases, the LXTE-902 carrier has the highest enzyme activity loading. However, due to its poor immobilization stability and considering that the reaction needs to be carried out for a long time, the optimal immobilization carriers for sucrose phosphatase LmSPase, pyrophosphorylase BlUSP, and uridine diphosphate-glucose dehydrogenase mutant D7 P166M / I169A are LXTE-706 ultimately; the optimal immobilization carrier for pyrophosphorylase PmPpA is also LXTE-706, but glutaraldehyde is required for assisted immobilization; the optimal immobilization carrier for NADH oxidase is LXTE-800.

[0147] 3. Optimization of the immobilized enzyme loading

[0148] The loading of some enzymes was optimized. The amount of enzyme added was adjusted with reference to the immobilization system, and the enzyme activity loading and enzyme activity recovery rate after immobilization were measured. The results are shown in Tables 10, 11 and Figure 7 、 8 as shown

[0149] Table 10. Optimization results of the immobilized enzyme loading of LmSPase

[0150]

[0151] Table 11. Optimization results of the immobilized enzyme loading of BlUSP

[0152]

[0153] It can be seen from Tables 10 - 11 and Figure 7 - 8 that the optimal loading of the immobilized enzyme of LmSPase is 25 mg / g; the optimal loading of the immobilized enzyme of BlUSP is 20 mg / g; the optimal loading of the immobilized enzyme preparation of uridine diphosphate-glucose dehydrogenase mutant D7 P166M / I169A is 20 mg / g; the optimal loading of the immobilized enzyme of TkNOX is 10 mg / g.

[0154] 4. Determination of the number of recycling times of the immobilized enzyme preparation

[0155] Each immobilized enzyme was used for catalytic reaction according to the reaction system. After one reaction, the immobilized enzyme was washed 2 times with the recovered buffer solution, and the substrate solution was added again for a new round of recycling. Three parallels were set for each group of experiments to determine the recycling stability of the immobilized enzyme preparations of LmSPase, BlUSP, and uridine diphosphate-glucose dehydrogenase mutant D7 P166M / I169A. After measuring the enzyme activity of the free enzyme and the immobilized enzyme, the number of reaction cycles of the immobilized enzymes of WT and D7P166M / I169A was compared. The results are as Figure 9 and Figure 10 shown

[0156] It can be seen from Figure 9 that the immobilized enzyme preparation of uridine diphosphate-glucose dehydrogenase mutant D7 P166M / I169A can stably repeat the reaction multiple times, and the immobilized D7P166M / I169A shows significant stability advantages. After the 4th reaction, the conversion rate is basically stable at about 70%.

[0157] It can be seen from Figure 10 that the immobilized enzyme preparations of LmSPase and BlUSP can stably repeat the reaction multiple times.

[0158] Then, the enzyme loading activities of wild-type uridine diphosphate-glucose dehydrogenase and the immobilized enzyme preparation of uridine diphosphate-glucose dehydrogenase mutant D7P166M / I169A were measured, and the results are as Figure 11 shown.

[0159] It can be seen from Figure 11 that, compared with wild-type uridine diphosphate-glucose dehydrogenase, the comprehensive enzyme loading activity of the immobilized uridine diphosphate-glucose dehydrogenase mutant D7 P166M / I169A increased by about 100%.

[0160] Example 4. Flow synthesis of UDP-GlcA using the immobilized enzyme preparation

[0161] 1. As Figure 12 shown, a method for synthesizing uridine diphosphate-glucuronic acid using uridine diphosphate-glucose dehydrogenase mutant D7 P166M / I169A includes the following steps:

[0162] First, add sucrose, UTP, Mg 2+ and NAD + to the PB buffer, and then add LmSPase, BlUSP, PmPpA, TkNOX and uridine diphosphate-glucose dehydrogenase mutant D7 P166M / I169A to form a reaction system;

[0163] In the reaction system, the concentration of PB buffer (pH = 7) is 40 mM, the concentration of magnesium chloride is 10 mM, the concentration of sucrose is 20 mM, the concentration of UTP is 20 mM, the concentration of NAD + is 20 mM, the concentration of LmSPase is 0.2 mg / mL, the concentration of BlUSP is 0.4 mg / mL, the concentration of PmPpA is 0.1 mg / mL, the concentration of TkNOX is 0.4 mg / mL, and the concentration of uridine diphosphate-glucose dehydrogenase mutant D7P166M / I169A is 0.8 mg / mL;

[0164] Then, the reaction system was catalytically reacted at 37 °C for 6 h to obtain uridine diphosphate-glucuronic acid. After the reaction, the production amount of uridine diphosphate-glucuronic acid was detected by HPLC.

[0165] 2. Optimization of the flow synthesis ratio of immobilized enzyme preparations

[0166] Take each of the prepared immobilized enzyme preparations in Example 3. After mixing the immobilized enzyme preparations in different enzyme amount ratios, they were loaded into a 1 mL small gravity chromatography column. The substrate solution was continuously dropped into the chromatography column, the flow rate of each column was measured, and the product concentration in the effluent was measured after three column volumes had flowed out. First, optimize the ratio of the LmSPase immobilized enzyme preparation and the BlUSP immobilized enzyme preparation, and record the optimal result as (A). Then, load (A) and the PmPpA immobilized enzyme preparation in different ratios to obtain the optimal enzyme ratio, denoted as (α). Then, optimize the enzyme amount ratio of the TuaD mutant D7 P166M / I169A immobilized enzyme preparation and the TkNOX immobilized enzyme, and record the optimal result as (β). Finally, load (β) and (α) in different ratios to obtain the optimal enzyme ratio for the total system. The reaction systems are shown in Tables 12, 13, and 14, and the results are as Figure 13 、 14 shown.

[0167] Table 12. Reaction system for optimizing the flow synthesis enzyme ratio (α)

[0168]

[0169] Table 13. Reaction system for optimizing the flow synthesis enzyme ratio (β)

[0170]

[0171] Table 14. Reaction system for optimizing the UDP-GlcA enzyme ratio in flow synthesis

[0172]

[0173] As Figure 13 、 14 shown, the best reaction effect is achieved when the enzyme amount ratio of the entire UDP-GlcA flow synthesis system is LmSPase:BlUSP:PmPPA:TuaD:TkNOX = 3:21:1:30:10.

[0174] 3. Flow synthesis of UDP-GlcA with immobilized enzyme preparations

[0175] An assembled flow synthesis device includes a substrate container, a constant flow pump, a thermostatic jacket chromatography column, and a product container that are sequentially connected by pipelines; according to the optimized ratio and the loading amount shown in Table 15, the immobilized enzymes are mixed and then mixed with PB buffer solution, and filled into the thermostatic jacket chromatography column to form a fixed bed. Then, under the action of the constant flow pump, the substrate solution in the substrate container continuously flows through the fixed bed. The effluent is collected into the product container at regular intervals, and the product concentration is measured. The reaction system is shown in Table 16. Each time the collected reaction solution is quantitatively analyzed by HPLC for about 20 mL column volume, the flow rate is 4.0 mL / h, and the flow reaction is carried out at 37 °C. The change of the product concentration of the flow-synthesized UDP-GlcA with time is as Figure 15 shown.

[0176] Table 15. Enzyme loading amount for flow synthesis of UDP-GlcA and its shake flask culture volume

[0177]

[0178]

[0179] Table 16. Reaction system for flow synthesis of UDP-GlcA

[0180]

[0181] As Figure 15 shown, the entire flow synthesis system stably operated for about 220 hours, and the concentration of UDP-GlcA in the effluent was stable at about 10 mM. Calculating the entire process at a concentration of 10 mM, about 5.7 g of UDP-GlcA was generated in total, the space-time yield was about 1.3 g / (L·h), and the conversion rate of UTP was close to 100%, showing good operation stability and catalytic efficiency.

[0182] The UDP-GlcA prepared in this example was detected by high performance liquid chromatography (HPLC) and mass spectrometry, and the results are as Figure 16 , 17 shown.

[0183] As Figure 16 and 17 show, the multi-enzyme immobilized continuous flow synthesis method provided by the present invention successfully prepared uridine diphosphate-glucuronic acid (UDP-GlcA).

[0184] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A uridine diphosphate-glucose dehydrogenase mutant D7P166M / I169A, characterized in that, Its amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1; The uridine diphosphate-glucose dehydrogenase mutant D7P166M / I169A has 67 amino acid mutations in the wild-type uridine diphosphate-glucose dehydrogenase TuaD; the specific mutations are: A53E, D221E, E394D, H176E, I283L, L61K, N69T, P337R, P355E, P72A, Q187A, S329D, S358R, T92D, V135I, V240M, V396D, Y82F, A191I, A359K, C30V, D238K, Q63G, S314A, V295F, K252H, M89P, N325D, Q132K, S133E, T163F, A5C, E291R, E363D, K195I, S234H, S275K, T174D, T289V, V28I, A103V, D54E, H190N, Q341K, V390I, V44I, M305L, A77N, E57K, K91P, N197D, R311K, S39K, T378A, T400K, I71L, D299R, D62A, E356N, G446A, K101E, K235D, S144P, S16T, S35E, T303K, V308I; the GeneBank accession number of the wild-type uridine diphosphate-glucose dehydrogenase TuaD is BSU35580.

2. An expression cassette or recombinant vector containing the encoding gene of the uridine diphosphate-glucose dehydrogenase mutant D7P166M / I169A according to claim 1.

3. A recombinant host cell containing the encoding gene of the uridine diphosphate-glucose dehydrogenase mutant D7P166M / I169A according to claim 1.

4. Use of the uridine diphosphate-glucose dehydrogenase mutant D7P166M / I169A according to claim 1 in the preparation of uridine diphosphate-glucuronic acid.

5. A method for the flow synthesis of uridine diphosphate-glucuronic acid using the uridine diphosphate-glucose dehydrogenase mutant D7P166M / I169A immobilized enzyme preparation according to claim 1, characterized in that, Including the following steps: Using sucrose, UTP, NAD + as substrates, adding phosphate buffer and inorganic ions to the substrates, and then using the immobilized enzyme preparations of sucrose phosphatase LmSPase, pyrophosphorylase BlUSP, pyrophosphatase PmPpA, uridine diphosphate-glucose dehydrogenase mutant D7P166M / I169A and NADH oxidase TkNOX as catalysts, the target product UDP-GlcA is obtained by continuous flow synthesis; Among them, the final concentration of the sucrose is 12 mM; the final concentration of the UTP is 10 mM; the final concentration of the NAD + is 10 mM; the final concentration of the phosphate buffer solution is 20 mM; the inorganic ion is a magnesium ion, which is generated by hydrolysis of magnesium chloride, and the final concentration of the magnesium ion is 10 mM; the reaction temperature of the biosynthesis is 37 °C, and the residence time of the reaction using the flow synthesis device is 4 to 6 h; the enzyme amount ratio of the sucrose phosphatase LmSPase, pyrophosphorylase BlUSP, pyrophosphatase PmPpA, uridine diphosphate-glucose dehydrogenase mutant D7P166M / I169A, and NADH oxidase TkNOX immobilized enzymes is 3:21:1:30:

10.

6. The method according to claim 5, wherein The immobilized enzyme preparations of the sucrose phosphatase LmSPase and pyrophosphorylase BlUSP are prepared as follows: The free sucrose phosphatase LmSPase and pyrophosphorylase BlUSP are diluted with a buffer and then mixed with an immobilized carrier respectively, and immobilized at 4°C and 70-85 rpm for 16 h, and then left standing for 8-10 h. After washing, the immobilized enzyme preparations of the sucrose phosphatase LmSPase and pyrophosphorylase BlUSP are obtained; Among them, the GenBank accession number of the sucrose phosphatase LmSPase is D90314; the GenBank accession number of the pyrophosphorylase BlUSP is EEI80102; the immobilized carrier is resin LXTE-706; the loading amount of the sucrose phosphatase LmSPase is 25 mg / g, and the loading amount of the pyrophosphorylase BlUSP is 20 mg / g, unit: protein mg / immobilized carrier g; the buffer solution is PB buffer solution.

7. The method according to claim 5, wherein The immobilized enzyme preparation of the pyrophosphatase PmPpA is prepared according to the following method: Dilute the free pyrophosphatase PmPpA with a buffer solution and mix it with the immobilized carrier activated by glutaraldehyde. Immobilize it at 4°C and 70 - 85 rpm for 16 h, then continue to stand for 8 - 10 h. After washing, the immobilized enzyme preparation of the pyrophosphatase PmPpA is obtained; Among them, the GenBank accession number of the pyrophosphatase PmPpA is AAK03275.1; the immobilized carrier activated by glutaraldehyde is: mix resin LXTE-706 and 2% glutaraldehyde solution according to a mass-volume ratio of 1:10, and shake and activate at 20 - 25°C for 60 min; the immobilized carrier is resin LXTE-706; the loading amount of the pyrophosphatase PmPpA is 10 mg / g, unit: protein mg / immobilized carrier g; the buffer solution is PB buffer solution.

8. The method according to claim 5, characterized in that, The immobilized enzyme preparation of the uridine diphosphate-glucose dehydrogenase mutant D7P166M / I169A is prepared according to the following method: Dilute the free uridine diphosphate-glucose dehydrogenase mutant D7P166M / I169A with a buffer solution and mix it with the immobilized carrier. Immobilize it at 4°C and 70 - 85 rpm for 16 h, then stand for 8 - 10 h. Then add a 1 M glycine solution with pH = 7.0 according to a volume ratio of 1:10, and continue to incubate at 4°C and 70 rpm for 12 h. After washing, the immobilized enzyme preparation of the uridine diphosphate-glucose dehydrogenase mutant D7P166M / I169A is obtained; Among them, the immobilized carrier is resin LXTE-706; the loading amount of the uridine diphosphate-glucose dehydrogenase mutant D7P166M / I169A is 20 mg / g, unit: protein mg / immobilized carrier g; the buffer solution is PB buffer solution.

9. The method according to claim 5, characterized in that, The preparation method of the immobilized enzyme preparation of the NADH oxidase TkNOX is as follows: Dilute the free NADH oxidase TkNOX and mix it with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and the immobilized carrier. Immobilize it at 4°C and 70 - 85 rpm for 16 h, then continue to stand for 8 - 10 h. After washing, the immobilized enzyme preparation of the NADH oxidase TkNOX is obtained; Among them, the GenBank accession number of the NADH oxidase TkNOX is BAD84493.1; the immobilized carrier is resin LXTE-706; the loading amount of the NADH oxidase TkNOX is 10 mg / g, unit: protein mg / immobilized carrier g; the buffer solution is PB buffer solution (pH = 7, concentration is 0.02 M).

10. A method for continuously synthesizing uridine diphosphate-glucuronic acid by multi-enzyme immobilization using the uridine diphosphate-glucose dehydrogenase mutant D7P166M / I169A described in claim 1, characterized in that, The specific method is as follows: Assemble a flow synthesis device, including a substrate container, a constant flow pump, a thermostatic jacket chromatography column and a product container connected in sequence through pipelines. Load the immobilized enzyme preparations of sucrose phosphatase LmSPase, pyrophosphorylase BlUSP, pyrophosphatase PmPpA, uridine diphosphate-glucose dehydrogenase mutant D7P166M / I169A and NADH oxidase TkNOX in the thermostatic jacket chromatography column, and then pump the reaction substrate into the chromatography column through the constant flow pump for biosynthesis. After synthesis, collect the product uridine diphosphate-glucuronic acid in the container.

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