Uridine diphosphate-glucuronyl transferase mutant and application thereof in catalytic synthesis of glucuronide

Abstract

The invention relates to a uridine diphosphate-glucuronyl transferase mutant and an application of the uridine diphosphate-glucuronyl transferase mutant in catalytic synthesis of glucuronide. The amino acid sequence of the uridine diphosphate-glucuronyl transferase mutant 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 provides a uridine diphosphate-glucuronyl transferase mutant with high activity and high stability. Compared with wild uridine diphosphate-glucuronyl transferase, the uridine diphosphate-glucuronyl transferase mutant has mutation of 10 amino acid sites. According to experimental determination, the melting temperature of the mutant is increased by 24 DEG C compared with that of a wild type, the activity is increased to 1.65 times, the expression quantity is increased to 4.2 times, and a powerful tool is provided for enzymatic synthesis of glucuronide compounds.

Description

TECHNICAL FIELD

[0001] The present application relates to a uridine diphosphate-glucuronosyl transferase mutant and its application in catalyzing synthesis of glucuronide, and belongs to the technical field of bioengineering and biosynthesis. BACKGROUND

[0002] Glycosides are widely present in organisms and are important components of organisms. Glucuronic acid is a component unit of active substances such as chondroitin sulfate and heparin in organisms, and glucuronidation is a main way and path of hormone and drug metabolism. Studies have shown that changes in β-glucuronidase activity are related to many diseases, so β-glucuronidase activity has become a detection index for many diseases. Therefore, glucuronide compounds such as p-nitrophenyl-β-D-glucuronide (pNP-GlcA) and 4-methylumbelliferyl-β-D-glucuronide (MUGA) are widely used as substrates for glycosidase and starting substrates for enzymatic synthesis of glycosaminoglycan compounds in enzyme activity determination, disease diagnosis and sugar drug synthesis.

[0003] Currently, glucuronide synthesis mainly has two ways of chemical method and enzymatic method, and commercially available glucuronide compounds such as pNP-GlcA and MUGA are chemically synthesized, which has complex synthesis steps and high cost. Enzymatic synthesis uses uridine 5'-diphosphoglucuronic acid (UDP-GlcA) as a glycosyl donor to catalyze the synthesis of glucuronide under the catalysis of glucuronosyltransferase (UGAT), and the glycosyl donor has high cost, which leads to high cost of enzymatic synthesis of glucuronide. In addition, two previously reported UGATs with wide substrate range have problems of difficulty in obtaining and poor stability, which limits the large-scale and efficient synthesis of glucuronide compounds. SUMMARY

[0004] In view of the problems in the current enzymatic synthesis of glucuronide compounds, the present application provides a uridine diphosphate-glucuronosyl transferase mutant and its application in catalyzing synthesis of glucuronide. The present application uses computer-aided technology and activity and stability determination to obtain a glucuronosyltransferase mutant with high activity and high stability. Then, using cheap glucuronic acid (GlcA) as a substrate, glucuronide compounds are finally obtained through three-step reactions. At the same time, ATP and reaction by-products UDP are used to recycle uridine triphosphate (UTP), improve reaction conversion rate, and reduce synthesis cost. Finally, the system is expanded to realize large-scale and efficient preparation of glucuronide compounds such as pNP-GlcA and MUGA.

[0005] The technical scheme of the present application is as follows:

[0006] In a first aspect of the present application, a uridine diphosphate-glucuronosyl transferase mutant D3 / 144T / 146L is provided, wherein the amino acid sequence is shown as SEQ ID NO. 2, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 1.

[0007] The uridine diphosphate-glucuronosyl transferase mutant D3 / 144T / 146L is a mutant of wild-type uridine diphosphate-glucuronosyl transferase GcaC, wherein 10 amino acids are mutated, and the mutations are S41P, T58A, P89A, A112R, N144T, V158R, S166P, A185P, V203T, and A339H.

[0008] The uridine diphosphate-glucuronosyl transferase mutant D3 / 144T / 146L is a mutant of wild-type uridine diphosphate-glucuronosyl transferase GcaC, wherein 10 amino acids are mutated, and the mutations are S41P, T58A, P89A, A112R, N144T, V158R, S166P, A185P, V203T, and A339H.

[0009] In a second aspect of the present application, an expression cassette or a recombinant vector containing the gene encoding the uridine diphosphate-glucuronosyl transferase mutant D3 / 144T / 146L is provided. The recombinant vector is not particularly limited, and can be any vector known in the art, as long as it can replicate in a host. For example, the vector includes, but is not limited to, a plasmid, a bacteriophage. 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.

[0010] According to the present application, preferably, the recombinant vector or the expression cassette is obtained by linking the gene encoding the uridine diphosphate-glucuronosyl transferase mutant D3 / 144T / 146L to an expression vector, and the expression vector is a plasmid.

[0011] Further preferably, the plasmid is pET-28a(+).

[0012] A third aspect of the invention provides a recombinant host cell containing the encoding gene of the uridine diphosphate-glucuronyl transferase mutant D3 / 144T / 146L. The term "host cell" has the meaning commonly understood in the art; it refers to a host cell capable of receiving the encoding gene of the mutant of the present invention, and is called a recombinant host cell after such reception. The bacterial strain of the present invention can be a prokaryotic or eukaryotic cell, preferably a prokaryotic cell, and more preferably *Escherichia coli* BL21(DE3).

[0013] A fourth aspect of the present invention provides the use of the above-mentioned uridine diphosphate-glucuronyltransferase mutant D3 / 144T / 146L in the preparation of glucuronide.

[0014] According to a preferred embodiment of the present invention, the glucuronide is p-nitrophenyl-β-D-glucuronic acid, 4-methylumbelliferone-β-D-glucuronic acid, p-acetaminophenyl-β-D-glucuronic acid, p-methoxyphenyl-β-D-glucuronic acid, or ferulic acid 4-O-β-D-glucuronic acid.

[0015] A fifth aspect of the present invention provides a method for preparing glucuronide using the above-mentioned uridine diphosphate-glucuronyltransferase mutant D3 / 144T / 146L, comprising the following steps:

[0016] GlcA, ATP, UTP, and phenolic receptor substrate were first added to Tris-HCl buffer, followed by glucuronide kinase (GlcAK), UDP-glucuronide pyrophosphorylase (USP), inorganic pyrophosphorylase (PpA), nucleoside diphosphate kinase (NDK), and uridine diphosphate-glucuronyl transferase mutant D3 / 144T / 146L to form a reaction system. The reaction system was then catalyzed at 35–40 °C for 10–16 h to obtain glucuronide.

[0017] According to a preferred embodiment of the present invention, the molar concentration ratio of GlcA, ATP, UTP, and phenolic receptor substrate in the reaction system is (1-1.5):2.5:0.1:1; and the mass concentration ratio of glucuronide kinase (GlcAK), UDP-glucuronide pyrophosphorylase (USP), inorganic pyrophosphorylase (PpA), nucleoside diphosphate kinase (NDK), and uridine diphosphate-glucuronyl transferase mutant D3 / 144T / 146L in the reaction system is 2:(2-10):2:1:1.

[0018] More preferably, the molar concentration ratio of GlcA, ATP, UTP, and phenolic receptor substrate in the reaction system is 1.25:2.5:0.1:1; and the mass concentration ratio of glucuronide kinase (GlcAK), UDP-glucuronide pyrophosphorylase (USP), inorganic pyrophosphorylase (PpA), nucleoside diphosphate kinase (NDK), and uridine diphosphate-glucuronyl transferase mutant D3 / 144T / 146L in the reaction system is 2:6:2:1:1.

[0019] According to a preferred embodiment of the present invention, the phenolic acceptor substrate is p-nitrophenol (pNP), 4-methylumbelliferone (MU), acetaminophen, p-methoxyphenol, or ferulic acid.

[0020] According to a preferred embodiment of the present invention, the GenBank accession number of the glucuronide kinase (GlcAK) is OAP06484.1;

[0021] The GenBank accession number for the UDP-glucuronide pyrophosphorylase (USP) is EEI80102.1;

[0022] The GenBank accession number for the inorganic pyrophosphatase (PpA) is AAK03275.1;

[0023] The GenBank accession number for the nucleoside diphosphate kinase (NDK) is WP_204218539.1.

[0024] According to a preferred embodiment of the present invention, the reaction system is catalytically reacted at 37°C for 12 hours; the glucuronide is p-nitrophenyl-β-D-glucuronic acid, 4-methylumbelliferone-β-D-glucuronic acid, p-acetaminophenyl-β-D-glucuronic acid, p-methoxyphenyl-β-D-glucuronic acid, or ferulic acid 4-O-β-D-glucuronic acid.

[0025] Beneficial effects

[0026] 1. This invention provides a highly active and stable uridine diphosphate-glucuronyl transferase mutant, D3 / 144T / 146L, which exhibits a mutation at 10 amino acid sites compared to the wild-type uridine diphosphate-glucuronyl transferase. Experimental measurements show that this mutant has a 24°C higher melting temperature, 1.65-fold increased activity, and 4.2-fold increased expression level compared to the wild-type, providing a powerful tool for the enzymatic synthesis of glucuronide compounds.

[0027] 2. This invention provides a method for preparing glucuronides, using GlcA as a substrate and GlcAK, USP, uridine diphosphate-glucuronyl transferase mutant D3 / 144T / 146L, PpA, and NDK as catalytic enzymes. In the preparation process, UTP regeneration is coupled with glucuronide synthesis, using one unit of UTP to catalyze the synthesis of ten units of glycoside compounds, significantly reducing synthesis costs. Ultimately, in a 20 mM reaction system, the yield of p-nitrophenyl-β-D-glucuronide (pNP-GlcA) can reach 5.95 g / L, and the yield of 4-methylumbelliferone-β-D-glucuronide (MUGA) can reach 3.03 g / L, demonstrating significant application potential. Attached Figure Description

[0028] Figure 1 The results show the expression levels of wild-type uridine diphosphate-glucuronyltransferase GcaC and its mutants D1–D8.

[0029] Figure 2 The results show the activity assays of wild-type uridine diphosphate-glucuronyl transferase GcaC and its mutants D1–D8.

[0030] Figure 3 Results of glucuronide transfer activity assay for GcaC mutant D6 and its single and combined mutants.

[0031] Figure 4 The results are from the activity assay of the uridine diphosphate-glucuronyl transferase mutant D3 / 144S / 46M.

[0032] Figure 5 The results show the glucuronidation activity assay of the combined saturated mutants D3 / 144T / 146L of uridine diphosphate-glucuronyl transferase.

[0033] Figure 6 The figures show the activity and stability of uridine diphosphate-glucuronyl transferase GcaC and its mutants; in the figure, the left axis represents the total enzyme activity of the protein expressed per liter of bacterial culture, and the right axis represents the protein melting temperature (T). m ).

[0034] Figure 7 This is a schematic diagram of the enzymatic synthesis pathway of β-glucuronide.

[0035] Figure 8 This shows the changes in each component during the reaction process of the p-nitrophenyl-β-D-glucuronic acid synthesis system.

[0036] Figure 9 These are mass spectrometry results for a series of β-glucuronide compounds. Detailed Implementation

[0037] The technical solution of the present invention will be further described below with reference to the embodiments and accompanying drawings, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the present invention are all methods known to those skilled in the art.

[0038] In this invention, the glucuronide kinase (GlcAK) is derived from the strain Arabidopsis thaliana, with the amino acid sequence GenBank accession number OAP06484.1; the UDP-glucuronide pyrophosphorylase (USP) is derived from the strain Bifidobacterium longum ATCC55813, with the amino acid sequence GenBank accession number EEI80102.1; the inorganic pyrophosphorylase (PpA) is derived from the strain Pasteurella multocida, with the amino acid sequence GenBank accession number AAK03275.1; and the nucleoside diphosphate kinase (NDK) is derived from the strain Desulfurella amilsii, with the amino acid sequence GenBank accession number WP_204218539.1.

[0039] The expression and purification methods of the GlcAK, USP, PpA, and NDK recombinant proteins used in the examples are as follows:

[0040] The amino acid sequences of GlcAK, USP, PpA, and NDK proteins were optimized using E. coli codons and synthesized by Nanjing GenScript Biotech Co., Ltd. The synthesized proteins were then cloned into the pET-21a(+) vector and chemically transformed into E. coli BL21(DE3) competent cells. Single colonies were picked and inoculated into 20 ml of LB broth containing 50 μg / ml carbenicillin at 37°C and 225 rpm for 16 h. The activated bacterial culture was then inoculated at a rate of 1.5% (v / v) into 1 L of LB broth containing 50 μg / ml carbenicillin and cultured at 37°C and 225 rpm with shaking for 2.5 h until OD (Organic Difference) was reached. 600 =0.6~0.8, add IPTG to a final concentration of 0.2mM, and induce for 18h at 22℃ and 225rpm to obtain bacterial culture.

[0041] The obtained bacterial culture was centrifuged at 8000 rpm for 10 min at 4 °C, and the bacterial cells were collected. Each liter of bacterial cells was resuspended in 30 ml of loading buffer containing 20 mM imidazole. The resuspended cells were then lysed in an ice-water bath using a 15 s cycle, 45 s interval, 60% amplitude, 1500 kJ power, with an effective lysis time of 5 min per liter of cells. The lysed cells were centrifuged at 12000 rpm for 20 min at 4 °C, and the supernatant was filtered through a 0.22 μM filter. Protein purification was performed using a nickel affinity chromatography column. After loading, the cells were washed with loading buffer containing 20 mM imidazole, followed by elution with elution buffer containing 250 mM imidazole to obtain the target protein. The purified protein solution was concentrated by ultrafiltration, and the protein concentration was determined using the Bradford Protein Assay Kit from Beyotime Biotechnology Co., Ltd. 10% glycerol was added to the purified protein solution, mixed well, aliquoted, and stored at -80 °C.

[0042] In the following examples, the detection method for β-glucuronide compounds was as follows: After the reaction was completed, the reaction solution was boiled for 5 min, filtered through a 0.22 μm filter membrane, and detected by HPLC. The HPLC detection conditions were as follows: Shim-pack GIS C18 column (4.6 × 250 mm, 5 μm); UV detection wavelengths: pNP-GlcA 310 nm, MUGA 320 nm, p-acetaminophenyl-β-D-glucuronic acid 245 nm, p-methoxyphenyl-β-D-glucuronic acid 285 nm, ferulic acid 4-O-β-D-glucuronic acid 300 nm; mobile phase: phase A was 0.1% formic acid aqueous solution, and phase B was acetonitrile; elution method: gradient elution, phase B concentration 20%–100% for 0–10 min, and phase B concentration 20% for 10.01–15 min; flow rate: 1 ml / min; sample loading volume: 5 μL.

[0043] Example 1: PROSS algorithm for calculating thermally stable GcaC mutants

[0044] The amino acid sequence of wild-type glucuronyl transferase GcaC (GeneBank accession number UKT59746.1) was input into the AlphaFold website, and structure prediction was performed under default conditions. The predicted GcaC amino acid sequence and protein crystal structure were input into the PROSS online tool (https: / / pross.weizmann.ac.il), and eight GcaC mutants that enhance stability were calculated. They were named D1 to D8 from low to high mutation rate, and their mutation sites are shown in Table 1.

[0045] Table 1. Mutation sites of GcaC mutants D1–D8

[0046]

[0047] Continued from Table 1: Mutation sites of GcaC mutants D1–D8

[0048]

[0049] Example 2: Expression and purification of GcaC and mutant recombinant proteins

[0050] 1. The amino acid sequence of the GcaC mutant D1 was optimized by E. coli codons, then artificially synthesized by Nanjing GenScript Biotech Co., Ltd., and cloned into the pET-28a(+) vector. It was then chemically transformed into E. coli BL21(DE3) competent cells and cultured on LB solid medium until single colonies grew.

[0051] Single colonies were picked and placed in 20 ml of LB broth containing 100 μg / ml kanamycin, and activated at 37°C and 225 rpm for 12 h. The activated bacterial culture was then inoculated at a rate of 1.5% (v / v) into 1 L of LB broth containing 100 μg / ml kanamycin, and cultured at 37°C and 225 rpm with shaking for 2.5 h until OD reached. 600 =0.6~0.8, add IPTG to a final concentration of 0.2mM, and induce for 16h at 16℃ and 225rpm to obtain bacterial culture.

[0052] The obtained bacterial culture was centrifuged at 8000 rpm for 10 min at 4 °C, and the bacterial cells were collected. Each liter of bacterial cells was resuspended in 30 ml of loading buffer containing 20 mM imidazole. The resuspended cells were then lysed in an ice-water bath using a 15 s cycle, 45 s interval, 60% amplitude, 1500 kJ power, with an effective lysis time of 5 min per liter of cells. The lysed cells were then centrifuged at 12000 rpm for 20 min at 4 °C, and the supernatant was filtered through a 0.22 μM filter. Protein purification was performed using a nickel affinity chromatography column. After loading, the cells were washed with loading buffer containing 20 mM imidazole, followed by elution with elution buffer containing 250 mM imidazole to obtain the target protein. 10% glycerol was added to the purified protein solution, mixed thoroughly, aliquoted, and stored at -80 °C.

[0053] Wild-type uridine diphosphate-glucuronyltransferase GcaC and GcaC mutants D2–D8 were expressed and purified using this method.

[0054] 2. The purified wild-type uridine diphosphate-glucuronyl transferase GcaC and GcaC mutants D1–D8 were analyzed by SDS-PAGE. Subsequently, protein concentration was determined using the Bradford protein assay kit from Beyotime Biotechnology Co., Ltd., and protein expression levels were calculated. The results are as follows: Figure 1 As shown.

[0055] The solution components used in this process are as follows:

[0056] LB medium: tryptone 10 g / L, sodium chloride 10 g / L, yeast extract 5 g / L;

[0057] Sample loading buffer: Tris 50mM, sodium chloride 300mM, imidazole 20mM, adjusted to pH 7.5 with HCl;

[0058] Elution buffer: Tris 50mM, sodium chloride 300mM, imidazole 250mM, adjusted to pH 7.5 with HCl.

[0059] Depend on Figure 1 It can be seen that the expression levels of multiple mutants calculated by PROSS were significantly higher than those of wild-type GcaC, with the D3 mutant showing the highest expression level at 362.5 mg / L, which is 4 times higher than that of wild-type.

[0060] Example 3: Determination of the transfer activity and stability of GcaC mutants D1–D8

[0061] 1. Assay of glucuronide (UDP-GlcA) transfer activity in GcaC mutants D1–D8

[0062] Using UDP-GlcA as the glycosyl donor and pNP as the glycosyl acceptor, a 200 μl reaction system was prepared according to the concentrations shown in Table 2. The reaction was carried out in a water bath at 37°C for 10 min, and then terminated by boiling for 5 min. The reaction solution was then filtered through a 0.22 μm filter membrane and analyzed by HPLC according to the method described in Table 3. The mobile phase A was 0.1% formic acid aqueous solution, and the mobile phase B was chromatographic acetonitrile. The flow rate was 1 mL / min, and the detection wavelength was 310 nm. The results are as follows: Figure 2 As shown.

[0063] Depend on Figure 2 It is known that the activity of GcaC mutants generally decreases with increasing mutation rate. However, the activity of D7 mutants is different and does not follow this trend. Based on this observation, we can reasonably speculate that among the eight amino acid residues that differ between D6 and D7 proteins, there may be amino acid sites that are crucial to activity.

[0064] Enzyme activity is defined as the activity of transferring 1 μmol of glucuronic acid to the acceptor substrate per minute, which is defined as 1 U, and the unit is μmol / min.

[0065] Table 2. Reaction systems for determining UDP-GlcA transfer activity in GcaC mutants D1–D8

[0066]

[0067] Table 3. HPLC Analysis Procedure

[0068]

[0069] 2. Stability determination of GcaC mutants D1–D8

[0070] The melting temperatures (T0) of wild-type uridine diphosphate-glucuronyl transferase GcaC and GcaC mutants D1–D8 were determined using differential scanning calorimetry. m To characterize its stability, SYPRO Orange dye was used. Protein samples were diluted to 2 mg / ml, and the dye was diluted 50× with Tris-HCl buffer. The protein solution and diluted dye were then mixed at a ratio of 19:1 in a 96-well plate to prepare 20 μL samples. Each detection group was divided into three replicates. The prepared system was placed in a real-time fluorescence PCR instrument, the ROX fluorescence channel was selected, the scanning temperature range was 25℃~95℃, and the heating rate was 1℃ / 10s. Fluorescence intensity versus temperature curves were plotted using data processing software, and the first derivative of the curves was used to calculate the protein Tg. m The values ​​and results are shown in Table 4.

[0071] Table 4. Melting temperatures of GcaC and its mutants (T) m Measurement results

[0072]

[0073] As shown in Table 4, the stability of GcaC mutants D1 to D8 is significantly improved compared to the wild type.

[0074] 3. Based on the glucuronide transfer activity assay results of the GcaC mutant, amino acids different from those in GcaC mutant D7 and GcaC mutant D6 were introduced into GcaC mutant D6 to construct single mutant and combinatorial mutants of D6. After correct sequencing, protein expression, purification, and glucuronide transfer activity assays were performed. The results are as follows: Figure 3 As shown.

[0075] Depend on Figure 3 The results show that the key amino acid residues in the GcaC mutant are the combination of positions 144 and 146.

[0076] 4. Based on the glucuronidation activity assay results of the GcaC mutant, the combined mutation site 144S / 146M, which can increase the D6 activity to the D7 activity level, was introduced into the highly stable and highly expressed D3, constructing the uridine diphosphate-glucuronidation transferase mutant D3 / 144S / 146M. After correct sequencing, protein expression, purification, and glucuronidation activity assays were performed to verify the effect of this combined site on the D3 mutant activity. The results are as follows: Figure 4 As shown.

[0077] Depend on Figure 4 It can be seen that the D3 / 144S / 146M mutant obtained by combining mutations at positions 144 and 146 has significantly improved activity compared to D3, and its activity is comparable to that of the wild type.

[0078] Example 4: Construction and screening of a saturated library of uridine diphosphate-glucuronyltransferase mutants D3 / 144S / 146M

[0079] Using the D3 / 144S / 146M mutant plasmid as a template, a D3 / 144 / 146 dual-site saturated mutant library was constructed using the degenerate codon NNK, which covers 20 amino acids. The DNA library was transferred to Bl21(DE3) competent cells, and single colonies were inoculated into 1 mL of LB broth containing 100 ng / μL kanamycin and activated at 37°C with shaking at 225 rpm for 12 h. The activated bacterial culture was then inoculated into 1 mL of LB broth containing 100 ng / μL kanamycin at a 1.5% (v / v) inoculation rate and cultured at 37°C with shaking at 225 rpm for 2.5 h until OD600 = 0.6–0.8. Protein induction was initiated by adding 0.2 mM IPTG to each well and cultured at 16°C with shaking at 225 rpm for 16 h. After induction, the cells were collected by centrifugation at 4200 rpm for 15 min.

[0080] The bacterial cell pellet was freeze-thawed twice (-80℃ for 15 min, incubated at 37℃ for 30 min), and then resuspended in 200 μL of Lysis Buffer. The suspension was incubated at 37℃ for 1 h to complete cell lysis. After lysis, the 96-well plate was centrifuged at 4200 rpm and 4℃ for 10 min, and the supernatant was collected. The Lysis buffer contained 50 mM Tris-HCl buffer (pH = 7.5), 150 mM NaCl, 100 mg / L lysozyme, and 25 mg / L DNase I.

[0081] Add 50 mM Tris-HCl buffer (pH = 7.5) to the lysed supernatant to dilute it to 1 / 8 of its original concentration. Prepare 50 μL of the reaction mixture into 384-well plates according to the system concentrations shown in Table 5. Determine the activity by detecting the rate of change in absorbance at 405 nm at 37°C using a microplate reader kinetic method. Subsequently, the mutants with enhanced activity were sequenced and purified, and their activity was detected according to the activity detection method described in Example 3. The results are as follows: Figure 5 As shown.

[0082] Depend on Figure 5 It can be seen that, through saturated combination mutation, a total of 6 mutants with enhanced activity were obtained, among which D3 / 144T / 146L and D3 / 144L / 146T had an activity that was 1.65 times that of D3.

[0083] Table 5. Assay system for combined saturated mutant protein activity

[0084]

[0085]

[0086] Following the method described in Example 3, the expression levels and melting temperatures of D3 / 144T / 146L and D3 / 144L / 146T were determined. The expression levels were 375 mg / L and 334 mg / L, respectively, and the melting temperatures were 74.74℃ and 71.88℃, respectively. The results are as follows: Figure 6 As shown.

[0087] Depend on Figure 6 It can be seen that, through multiple rounds of mutation, the present invention constructs a uridine diphosphate-glucuronyl transferase mutant D3 / 144T / 146L with high activity, high stability and high expression level.

[0088] The uridine diphosphate-glucuronyl transferase mutant D3 / 144T / 146L constructed using this invention can efficiently synthesize β-glucuronide. The synthetic route is as follows: Figure 7 As shown.

[0089] Example 5: One-pot synthesis of p-nitrophenyl-β-D-glucuronic acid

[0090] A one-pot method for synthesizing p-nitrophenyl-β-D-glucuronic acid using the uridine diphosphate-glucuronyltransferase mutant D3 / 144T / 146L, comprising the following steps:

[0091] GlcA, ATP, UTP, and pNP were first added to Tris-HCl buffer, followed by GlcAK, USP, PpA, NDK, and the uridine diphosphate-glucuronidase mutant D3 / 144T / 146L to form the reaction system. In the reaction system, the concentrations of Tris-HCl buffer (pH=7.5) were 50 mM, magnesium chloride 10 mM, GlcA 25 mM, ATP 50 mM, UTP 2 mM, pNP 20 mM, GlcAK 0.2 mg / ml, USP 0.6 mg / ml, PpA 0.2 mg / ml, NDK 0.1 mg / ml, and the uridine diphosphate-glucuronidase mutant D3 / 144T / 146L 0.1 mg / ml.

[0092] The reaction system was then catalytically reacted at 37 °C for 12 h to obtain p-nitrophenyl-β-D-glucuronic acid (pNP-GlcA). The amount of pNP-GlcA generated was determined by HPLC after the reaction was complete.

[0093] The pNP-GlcA yield was 94.45%, with a production volume of 5.95 g / L. The changes in the components of the reaction system during the reaction process are as follows: Figure 8 As shown.

[0094] Example 6: One-pot synthesis of 4-methylumbelliferone-β-D-glucuronic acid

[0095] A one-pot method for synthesizing 4-methylumbelliferone-β-D-glucuronic acid using the uridine diphosphate-glucuronyltransferase mutant D3 / 144T / 146L comprises the following steps:

[0096] GlcA, ATP, UTP, and MU were first added to Tris-HCl buffer, followed by GlcAK, USP, PpA, NDK, and the uridine diphosphate-glucuronidase mutant D3 / 144T / 146L to form the reaction system. In the reaction system, the concentrations of Tris-HCl buffer (pH=7.5) were 50 mM, magnesium chloride 10 mM, GlcA 25 mM, ATP 50 mM, UTP 2 mM, MU 20 mM, GlcAK 0.2 mg / ml, USP 0.6 mg / ml, PpA 0.2 mg / ml, NDK 0.1 mg / ml, and the uridine diphosphate-glucuronidase mutant D3 / 144T / 146L 0.1 mg / ml.

[0097] The reaction system was then catalytically reacted at 37 °C for 12 h to obtain 4-methylumbelliferone-β-D-glucuronic acid (MUGA). The amount of MUGA generated was detected by HPLC after the reaction was completed.

[0098] The MUGA yield was 43.83%, and the output was 3.03 g / L.

[0099] Example 7: One-pot synthesis of p-acetaminophenyl-β-D-glucuronic acid

[0100] A one-pot method for synthesizing p-acetaminophenyl-β-D-glucuronic acid using the uridine diphosphate-glucuronyltransferase mutant D3 / 144T / 146L, comprising the following steps:

[0101] GlcA, ATP, UTP, and acetaminophen were first added to Tris-HCl buffer, followed by GlcAK, USP, PpA, NDK, and the uridine diphosphate-glucuronidase mutant D3 / 144T / 146L to form the reaction system. In the reaction system, the concentrations of Tris-HCl buffer (pH=7.5) were 50 mM, magnesium chloride 10 mM, GlcA 25 mM, ATP 50 mM, UTP 2 mM, acetaminophen 20 mM, GlcAK 0.2 mg / ml, USP 0.6 mg / ml, PpA 0.2 mg / ml, NDK 0.1 mg / ml, and the uridine diphosphate-glucuronidase mutant D3 / 144T / 146L 0.1 mg / ml.

[0102] The reaction system was then catalytically reacted at 37 °C for 12 h to obtain p-acetaminophenyl-β-D-glucuronic acid. The amount of p-acetaminophenyl-β-D-glucuronic acid produced was determined by HPLC after the reaction was complete.

[0103] The yield of acetaminophenyl-β-D-glucuronic acid was 36.12%, with a production rate of 2.36 g / L.

[0104] Example 8: One-pot synthesis of p-methoxyphenyl-β-D-glucuronic acid

[0105] A one-pot method for synthesizing p-methoxyphenyl-β-D-glucuronic acid using the uridine diphosphate-glucuronyltransferase mutant D3 / 144T / 146L comprises the following steps:

[0106] GlcA, ATP, UTP, and p-methoxyphenol were first added to Tris-HCl buffer, followed by GlcAK, USP, PpA, NDK, and the uridine diphosphate-glucuronidase mutant D3 / 144T / 146L to form the reaction system. In the reaction system, the concentrations of Tris-HCl buffer (pH=7.5) were 50 mM, magnesium chloride 10 mM, GlcA 25 mM, ATP 50 mM, UTP 2 mM, p-methoxyphenol 20 mM, GlcAK 0.2 mg / ml, USP 0.6 mg / ml, PpA 0.2 mg / ml, NDK 0.1 mg / ml, and the uridine diphosphate-glucuronidase mutant D3 / 144T / 146L 0.1 mg / ml.

[0107] The reaction system was then catalytically reacted at 37 °C for 12 h to obtain p-methoxyphenyl-β-D-glucuronic acid. The amount of p-methoxyphenyl-β-D-glucuronic acid produced was determined by HPLC after the reaction was complete.

[0108] The yield of p-methoxyphenyl-β-D-glucuronic acid was 47.46%, with a production rate of 2.85 g / L.

[0109] Example 9: One-pot synthesis of ferulic acid 4-O-β-D-glucuronic acid

[0110] A one-pot method for synthesizing p-ferulate 4-O-β-D-glucuronic acid using the uridine diphosphate-glucuronyltransferase mutant D3 / 144T / 146L comprises the following steps:

[0111] GlcA, ATP, UTP, and ferulic acid were first added to Tris-HCl buffer, followed by GlcAK, USP, PpA, NDK, and the uridine diphosphate-glucuronidase mutant D3 / 144T / 146L to form the reaction system. In the reaction system, the concentrations of Tris-HCl buffer (pH=7.5) were 50 mM, magnesium chloride 10 mM, GlcA 25 mM, ATP 50 mM, UTP 2 mM, ferulic acid 20 mM, GlcAK 0.2 mg / ml, USP 0.6 mg / ml, PpA 0.2 mg / ml, NDK 0.1 mg / ml, and the uridine diphosphate-glucuronidase mutant D3 / 144T / 146L 0.1 mg / ml.

[0112] The reaction system was then catalytically reacted at 37 °C for 12 h to obtain p-ferulic acid 4-O-β-D-glucuronic acid. The amount of p-ferulic acid 4-O-β-D-glucuronic acid produced was determined by HPLC after the reaction was complete.

[0113] The yield of ferulic acid 4-O-β-D glucuronic acid was 23.45%, with a production rate of 1.74 g / L.

[0114] The mass spectra of p-nitrophenyl-β-D-glucuronic acid, 4-methylumbelliferone-β-D-glucuronic acid, p-acetaminophenyl-β-D-glucuronic acid, p-methoxyphenyl-β-D-glucuronic acid, or ferulic acid 4-O-β-D-glucuronic acid prepared in Examples 5-9 of this invention are as follows: Figure 9 As shown.

[0115] Depend on Figure 9 As can be seen, the structures of all compounds are correct. This invention successfully synthesized glucuronides such as p-nitrophenyl-β-D-glucuronic acid, 4-methylumbelliferone-β-D-glucuronic acid, p-acetaminophenyl-β-D-glucuronic acid, p-methoxyphenyl-β-D-glucuronic acid, or ferulic acid 4-O-β-D-glucuronic acid using the uridine diphosphate-glucuronyltransferase mutant D3 / 144T / 146L.

[0116] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A uridine diphosphate-glucuronyl transferase mutant, characterized in that, The amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence encoding the gene is shown in SEQ ID NO.

1.

2. An expression cassette or recombinant vector containing the gene encoding the uridine diphosphate-glucuronyl transferase mutant of claim 1.

3. A recombinant host cell containing the gene encoding the uridine diphosphate-glucuronyl transferase mutant.

4. The use of the uridine diphosphate-glucuronyl transferase mutant according to claim 1 in the preparation of glucuronide.

5. The application as described in claim 4, characterized in that, The glucuronide is p-nitrophenyl-β-D-glucuronic acid, 4-methylumbelliferone-β-D-glucuronic acid, p-acetaminophenyl-β-D-glucuronic acid, p-methoxyphenyl-β-D-glucuronic acid, or ferulic acid 4-O-β-D-glucuronic acid.

6. A method for preparing glucuronide using the uridine diphosphate-glucuronyltransferase mutant of claim 1, characterized in that, The steps are as follows: GlcA, ATP, UTP and phenolic receptor substrate were first added to Tris-HCl buffer, followed by glucuronide kinase, UDP-glucuronide pyrophosphorylase, inorganic pyrophosphorylase, nucleoside diphosphate kinase and the uridine diphosphate-glucuronyl transferase mutant described in claim 1 to form a reaction system; then the reaction system was catalyzed at 35-40°C for 10-16 h to obtain glucuronide.

7. The preparation method according to claim 6, characterized in that, The molar concentration ratio of GlcA, ATP, UTP, and phenolic receptor substrate in the reaction system is (1-1.5):2.5:0.1:1; the mass concentration ratio of glucuronide kinase UDP-glucuronide pyrophosphorylase, inorganic pyrophosphorylase, nucleoside diphosphate kinase, and uridine diphosphate-glucuronyl transferase mutant in the reaction system is 2:(2-10):2:1:

1.

8. The preparation method according to claim 7, characterized in that, The molar concentration ratio of GlcA, ATP, UTP, and phenolic receptor substrate in the reaction system is 1.25:2.5:0.1:1; the mass concentration ratio of glucuronide kinase, UDP-glucuronide pyrophosphorylase, inorganic pyrophosphorylase, nucleoside diphosphate kinase, and uridine diphosphate-glucuronyl transferase mutant in the reaction system is 2:6:2:1:

1.

9. The preparation method according to claim 6, characterized in that, The phenolic acceptor substrate is p-nitrophenol, 4-methylumbelliferone, p-acetaminophen, p-methoxyphenol, or ferulic acid; The GenBank accession number for the glucuronide kinase is OAP06484.1; The GenBank accession number for the UDP-glucuronide pyrophosphorylase is EEI80102.1; The GenBank accession number for the inorganic pyrophosphatase is AAK03275.1; The GenBank accession number for the nucleoside diphosphate kinase is WP_204218539.

1.

10. The preparation method according to claim 6, characterized in that, The reaction system was catalytically reacted at 37°C for 12 hours; the glucuronic acid glycoside was p-nitrophenyl-β-D-glucuronic acid, 4-methylumbelliferone-β-D-glucuronic acid, p-acetaminophenyl-β-D-glucuronic acid, p-methoxyphenyl-β-D-glucuronic acid, or ferulic acid 4-O-β-D-glucuronic acid.

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