Process for the preparation of fucose and intermediates thereof and use thereof

Abstract

A method for preparing fucose and intermediates thereof and applications thereof. In the method for synthesizing GDP-fucose, GDP-fucose is generated by catalytic reaction of sucrose synthase, CDP-threose 2-epimerase, GDP-mannose dehydratase and isomerase with guanosine diphosphate and sucrose as substrates. In the method for synthesizing fucosyllactose, fucosyllactose is obtained by catalytic action of Hp13FucT2 enzyme with GDP-fucose and pNP-lactose as initial substrates. In the method for preparing L-fucose by enzyme, L-fucose is generated by catalytic reaction of fucosidase with fucosyllactose as substrate. The E. coli expression system has the characteristics of high expression efficiency, large expression amount, low cost and easy operation, and the expressed fucosidase has the advantages of high purity and high yield.

Description

Technical Field

[0001] This application relates to methods for preparing fucose and its intermediates, and their applications. Background Technology

[0002] L-fucose, also known as 6-deoxy-L-galactose, has the molecular formula C6H2O. 12 O5, with a molecular weight of 164.16, is a hexose. In nature, fucose is mainly L-fucose. L-fucose is widely found in polysaccharides in humans, animals, plants, and microorganisms. For example, it exists in human breast milk as a sulfated oligosaccharide, in the eggs of sea urchins and frogs, as well as in plant polysaccharides such as tragacanth gum, potatoes, kiwifruit, soybeans, winged beans, and canola, and in the extracellular polysaccharides of various seaweeds.

[0003] L-fucose possesses various biological activities. For example, it has anti-inflammatory effects; adding appropriate amounts of L-fucose to medications can effectively reduce inflammation. Because L-fucose plays an important targeting role in cell adhesion, it can bind to drugs, guiding them to the site of action. L-fucose also regulates blood lipids and cholesterol. Adding it to lactic acid drinks, cakes, and cheeses instead of sucrose preserves the original flavor while meeting nutritional and health needs. While L-fucose cannot be absorbed by intestinal wall cells, it can be utilized by beneficial intestinal bacteria, increasing their number and maintaining intestinal balance. It can be added as a prebiotic to lactic acid drinks. Adding appropriate amounts of L-fucose to soy products also helps regulate blood lipids and cholesterol and maintain intestinal bacterial balance. Furthermore, L-fucose is widely used in chewing gum, chocolate, various candies, bread, dried fruit, biscuits, jams, and eight-treasure porridge.

[0004] L-fucose can promote fibroblast growth, thus preventing collagen loss and skin sagging caused by radiation and ultraviolet radiation. L-fucose can also reduce the activity of proteolytic enzymes in the skin, thereby reducing skin damage. Therefore, applying L-fucose to cosmetics can moisturize the skin, protect it, promote cell proliferation, increase skin elasticity, and slow down skin aging.

[0005] Fucosyl lactose (FL), the most abundant functional oligosaccharide in breast milk, possesses important physiological functions such as anti-infection, immune regulation, and promotion of infant brain development. FL has significant applications in the food industry. As early as 2015, fucoidan was approved by the U.S. Food and Drug Administration and the European Union for use as a nutritional supplement in infant formula, milk powder, and beverages. To date, FL has been obtained primarily through several methods: natural product extraction, chemical synthesis, enzymatic synthesis, and whole-cell synthesis. Among these, enzymatic synthesis offers significant advantages in terms of the spatial and stereochemical specificity of the catalytic reaction. It is simple to operate, environmentally friendly, produces fewer byproducts, yields a single, easily purified product, and allows for the development of new substrates, thus reducing costs. This has made it a synthetic method that has attracted considerable attention in recent years.

[0006] Currently, the main method for producing FL is based on whole-cell synthesis using engineered strains such as *E. coli* and *Saccharomyces cerevisiae*. However, whole-cell synthesis is complex and highly susceptible to the influence of the bacterial cells. Similarly, FL can be isolated from breast milk by chromatographic extraction, but this method yields fucoidosyllactose, which is both expensive and time-consuming. FL can also be synthesized chemically, but the side chains of FL require protection and deprotection during synthesis, leading to low efficiency.

[0007] The synthesis of FL requires GDP-fucose as a precursor, and the precursor of GDP-fucose is L-fucose. L-fucose can be synthesized chemically, such as from D-galactose, L-arabinose, D-mannose, or D-galacturonic acid. Comparatively, D-galactose is the most suitable raw material for L-fucose production. However, chemical synthesis often requires numerous protection / deprotection steps using selective techniques, making these methods lengthy and cumbersome. Furthermore, chemical synthesis requires laborious chromatographic separation to separate intermediates from byproducts, resulting in low yields and high costs, limiting its application in L-fucose preparation. Currently, fucose is mainly obtained through natural extraction. Oligosaccharides containing fucose are isolated from biomass such as algae through extraction. These oligosaccharides are then hydrolyzed to produce a complex mixture containing fucose and related sugars and / or their derivatives. Fucose is then purified and recovered from this mixture using methods such as ion exchange chromatography, dialysis, and stepwise crystallization. However, fucose obtained in this way is expensive and not easily industrialized.

[0008] Invention disclosure

[0009] This application provides a method for preparing fucose and its intermediates, and their applications.

[0010] This application discloses a method for the multi-stage, multi-enzyme cascade catalytic synthesis of GDP-fucose, comprising the following steps:

[0011] 1) Using guanosine diphosphate and sucrose as substrates, GDP-glucose is generated through a sucrose synthase-catalyzed reaction;

[0012] 2) GDP-mannose is generated from GDP-glucose as a substrate via a reaction catalyzed by CDP-Tives 2-epimerase;

[0013] 3) GDP-mannose is generated by the reaction of DP-mannose dehydrating enzyme and isomer reductase using GDP-mannose as a substrate.

[0014] The sucrose synthase, CDP-Tives 2-epimerase, GDP-mannose dehydratase, and isomer reductase all catalyze the reaction in the form of crude enzyme solution, crude enzyme powder, pure enzyme, or whole cells.

[0015] The sucrose synthase is derived from tomato (Solanum locopersicum), with the amino acid sequence number NP_001234655; the CDP-tivesose 2-epimerase is derived from Salmonella enterica, with the amino acid sequence number DW4091412.1; the GDP-mannose dehydratase is derived from Salmonella enterica, with the amino acid sequence number ACN45760.1; and the isomer reductase is derived from Salmonella enterica, with the amino acid sequence number WP_001041701.1.

[0016] This application also provides a method for synthesizing fucoidan using a single-pot multi-enzyme method, characterized by comprising the step of using GDP-fucose and pNP-lactose prepared according to claim 1 as initial substrates to obtain fucoidan under the catalysis of Hp13FucT2 enzyme.

[0017] Hp13FucT2 is a fucosyltransferase derived from Helicobacter pylori, and its amino acid sequence has the GenBank sequence number WP_000487428.1.

[0018] The enzymatic synthesis method of fucoidyl lactose involves using sucrose, GDP and pNP-lactose as initial substrates, and obtaining fucoidyl lactose under the catalysis of five enzymes: sucrose synthase, CDP-tives 2-epimerase, GDP-mannose dehydratase, isomer reductase and fucoidyltransferase.

[0019] Further steps include the following:

[0020] 1) Take Tris-HCl, GDP solution, sucrose solution, MgCl2 solution and SlSUS enzyme solution, mix them and react;

[0021] 2) Add SeC2E enzyme solution and NAD to the reaction solution in step 1). + Solution, mixture, reaction;

[0022] 3) Add pNP-lactose solution, NADPH solution, SeGMD enzyme solution, SeGER enzyme solution and Hp13FucT2 enzyme solution to the reaction solution in step 2), mix, react, and generate pNP-fucosyllactose.

[0023] 4) The product from step 3) is purified and separated to obtain pNP-fucosylated lactose.

[0024] Furthermore, in step 1), the molar ratio of GDP, sucrose, MgCl2 and Tris-HCl is 5:50:1:25; the amount of SlSUS enzyme added is 20 μL of SlSUS enzyme (2.0 mg / mL) per 1 mole of sucrose.

[0025] Furthermore, the reaction conditions in step 1) are 37°C for 12 hours.

[0026] Furthermore, in step 2), the amount of SeC2E enzyme solution added is 18 μL of SeC2E enzyme (100 mg / mL) per 1 mole of sucrose; NAD + The molar ratio of the solution added to GDP is 80:500.

[0027] Furthermore, the reaction conditions in step 2) are 45°C for 12 hours.

[0028] Furthermore, in step 3), the molar ratio of pNP-lactose:NADPH:GDP is 40:80:500;

[0029] Furthermore, in step 3), the amount of SeGMD enzyme solution added is 6 μl of SeGMD enzyme (25 mg / mL) per 1 mole of sucrose; the amount of SeGER enzyme solution added is 4 μl of SeGER enzyme (38 mg / mL) per 1 mole of sucrose; and the amount of Hp13FucT2 enzyme solution added is 15 μl of Hp13FucT2 enzyme (20 mg / mL) per 1 mole of sucrose.

[0030] Furthermore, the reaction conditions in step 3) are 12 hours at 25°C.

[0031] This invention provides an enzymatic method for preparing L-fucose, using fucosyl lactose as a substrate and generating L-fucose through a fucosidase-catalyzed reaction.

[0032] The fucosidase Eo3066 is derived from the aquatic bacterium Emticicia oligotrophica, with GenBank annotation number UniProt ID I2ERT6.

[0033] This application also provides a bioenzyme composition for preparing GDP-fucose, the bioenzyme composition comprising sucrose synthase, CDP-tives 2-epimerase, GDP-mannose dehydrating enzyme, and isomerase; the sucrose synthase is derived from tomato (Solanum locopersicum), with the amino acid sequence serial number NP_001234655; the CDP-tives 2-epimerase is derived from Salmonella enterica, with the amino acid sequence serial number DW4091412.1; the GDP-mannose dehydrating enzyme is derived from Salmonella enterica, with the amino acid sequence serial number ACN45760.1; and the isomerase is derived from Salmonella enterica, with the amino acid sequence serial number WP_001041701.1.

[0034] This application also provides a bioenzyme composition for preparing fucoidan lactose, comprising a bioenzyme composition for preparing GDP-fucose and a fucosyltransferase Hp13FucT2, wherein the fucosyltransferase Hp13FucT2 is derived from Helicobacter pylori and its amino acid sequence has the GenBank sequence number WP_000487428.1.

[0035] This application also provides a bioenzyme composition for preparing L-fucose, comprising a bioenzyme composition for preparing fucosyl lactose and fucosidase Eo3066, wherein the fucosidase Eo3066 is derived from the aquatic bacterium Emticiciaoligotrophica, GenBank annotation number UniProt ID I2ERT6. Attached Figure Description

[0036] Figure 1 This is the SDS-PAGE analysis result of the enzymes involved in the single-pot multi-enzyme method.

[0037] Figure 2 TLC detection of SlSUS enzymatic reaction products.

[0038] Figure 3 UPLC detection of SeC2E enzymatic reaction products.

[0039] Figure 4 UPLC detection of the products of the enzymatic reaction of SeGMD and SeGER.

[0040] Figure 5 Analysis of HpFucT enzymatic reaction products based on UPLC.

[0041] Figure 6 The results of HpFucT enzyme coupling analysis for GDP-fucose products.

[0042] Figure 7 This is the SDS-PAGE analysis result of the enzymes involved in the single-pot multi-enzyme method.

[0043] Figure 8 TLC analysis of the SlSUS enzymatic reaction products.

[0044] Figure 9 UPLC detection of SeC2E enzymatic reaction products.

[0045] Figure 10 UPLC results for the enzymatic reaction products of SeGMD, SeGER, and Hp13FucT2.

[0046] Figure 11 The results are based on UPLC analysis of the enzymatic reaction products of Hp13FucT2.

[0047] Figure 12 Figure showing the liquid phase separation results after the elution process to improve the results.

[0048] Figure 13 The image shows the LC-MS detection results of the reaction system after separation by a preparative column.

[0049] Figure 14 SDS-PAGE results for SeC2E S122A and SeC2E Y165F.

[0050] Figure 15 The figure shows the effect of site-directed mutagenesis of SeC2E on the transformation rate.

[0051] Figure 16 For the assay of fucosidase activity, 15 mL of reaction solution contained 8.5 μL of pH 7.4 PBS buffer, 1.5 μL of pNP-α-L-fucose (10 mM stock solution), and 5 μL of purified recombinant fucosidase. A reaction system without recombinant enzyme served as a negative control. The negative control (-) reaction system contained no enzyme.

[0052] Figure 17To detect the products of the fucosidase enzymatic reaction, 5 μL of pH 7.4 PBS buffer, 30 μL of recombinant fucosidase, and 5 μL (~10 μM) of purified pNP-fucosyllactose were added to a 50 μL reaction system, and the reaction was carried out at 37°C for 16 hours. The reaction products were detected by HPLC.

[0053] Figure 18 To concentrate fucosidase for enzymatic release of fucose, purified fucosidase was concentrated 5-fold and reacted in a 50 μL reaction system at 37°C for 16 hours. Eo3066 fucosidase completely hydrolyzes pNP-fucosyllactose, releasing fucose.

[0054] The best way to implement an invention

[0055] The following embodiments are provided to facilitate a better understanding of this application, but do not limit the scope of this application. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were all purchased from conventional biochemical reagent stores. All quantitative experiments in the following embodiments were performed in triplicate, and the results were averaged.

[0056] Example 1: Gene cloning, heterologous expression, purification, and SDS-PAGE identification of four enzymes

[0057] 1. Recombinant plasmid transformed into E. coli BL21

[0058] This application identifies genes for enzymes (SUS, C2E, GMD, GER) related to GDP-fucose synthesis from bacteria of different origins; sucrose synthase (SlSUS), which breaks down sucrose, is derived from Solanum lycopersicum; CDP-tivesose 2-epimerase (SeC2E), which can epimerize glucose activated by nucleotides into mannose, is derived from Salmonella enterica; GDP-mannose dehydratase (SeGMD) and isomer reductase (SeGER), which can convert GDP-mannose into GDP-fucose, are also derived from Salmonella enterica. The gene sequences of the sucrose synthase SlSUS (NP_001234655.2), CDP-tivesose 2-epimerase SeC2E (DW4091412.1), GDP-mannose dehydratase SeGMD (ACN45760.1), and isomerase SeGER (WP_001041701.1) were obtained from the database and synthesized. They were then cloned into the pET30a plasmid. Specifically, the coding sequence of SlSUS was ligated at a blunt end to the multiple cloning site of pET30a to obtain pET30a-SlSUS; the coding sequence of SeC2E was ligated between the Nde I / Xho I sites of pET30a (replacing the original sequence between the sites) to obtain pET30a-SeC2E; and the coding sequence of SeGMD was ligated between the Kpn I / Xho I sites of pET30a. pET30a-SeGMD was obtained by ligating the coding sequence of SeGER between the Kpn I / Xho I sites of pET30a, resulting in pET30a-SeGER. The four recombinant plasmids (pET30a-SlSUS, pET30a-SeC2E, pET30a-SeGMD, and pET30a-SeGER) were then transformed into *E. coli* BL21, as detailed below:

[0059] (1) The recombinant plasmid powder returned from gene synthesis was centrifuged and diluted with water to 1 ng / μL;

[0060] (2) Take 1 μL of the diluted recombinant plasmid and transfer it into 100 μL of competent cells;

[0061] (3) Adjust the water bath temperature during the 25-minute interval, quickly place it in a 42°C water bath for 1 minute of heat shock, take it out and quickly put it back into the ice, and let it stand for 2 minutes to reduce the damage to E. coli.

[0062] (4) Take 200 μL of sterilized LB liquid culture medium and add it to each tube of competent cells after heat shock. Incubate at 37°C and 800 rpm for 50 min to allow them to recover.

[0063] (5) Add the shaken bacterial culture evenly to LB solid medium containing 0.1 mg / mL kanamycin. Invert the plate and incubate it in a 37°C incubator for 12-16 h until the plaques grow into single colonies suitable for picking.

[0064] 2. Heterologous expression of recombinant plasmids in Escherichia coli

[0065] (1) Pick a single colony transformed into E. coli BL21 and inoculate it into 5 mL LB liquid medium, add 5 μL of 50 mg / mL kanamycin, and incubate overnight at 37°C and 250 rpm in a constant temperature shaker.

[0066] (2) Transfer 5 mL of overnight culture to 400 mL of fresh, heat-sterilized LB broth for expansion culture. When the OD600 of the fermentation broth reaches between 0.5 and 0.8, add IPTG solution to make the final IPTG concentration 1 mmol / L. Induce expression overnight at 18°C ​​and 250 rpm.

[0067] (3) Take 1 mL of the fermentation broth before and after IPTG induction, centrifuge at 12000 rpm for 5 min, discard the supernatant, and store the cell precipitate at -20℃ as a sample for SDS-PAGE analysis.

[0068] 3. Affinity chromatography purification of recombinant nickel carrier

[0069] (1) Transfer the IPTG-induced bacterial culture to a centrifuge bottle, centrifuge at 4000 rpm for 15 min at 4℃, and discard the supernatant;

[0070] (2) Add 10 mL of Lysis Buffer (cell lysis buffer) to the induced bacterial cells and aspirate thoroughly to dissolve them without any precipitate particles. Transfer the mixture to a 50 mL centrifuge tube and add 100 μL of 100 mM PMSF (phenylmethylsulfonyl fluoride). Sonicate for 20 min using an ultrasonic disruptor to induce cell lysis and release the target protein. Ultrasonic disruption conditions: 1.5 s disruption / 2.5 s interval, 15 min, power 200 W;

[0071] (3) The cell suspension after disruption was centrifuged again at 12,000 rpm for 15 min at 4°C, and the supernatant was collected and placed on ice for later use.

[0072] Cell lysis buffer preparation: Weigh 6.06g of Tris and 5.84g of sodium chloride and dissolve them in 800mL of ultrapure water. Add 10mL of Triton X-100, stir well, adjust the pH to 8.0 with hydrochloric acid, and bring the volume to 1L. Store at 4℃. Due to the short half-life, add the buffers for various nickel affinity chromatography steps just before lysis. The reagents are prepared as follows: ① Washing buffer: Weigh 6.06g of Tris and 2.92g of sodium chloride and dissolve them in 800mL of ultrapure water. Adjust the pH to 8.0 with dilute HCl, and finally bring the volume to 1L with ultrapure water. Store at 4℃. ② Protein elution buffer preparation: Weigh 3.03g of Tris, 1.46g of sodium chloride, and 17.02g of imidazole and dissolve them in 400mL of ultrapure water. Adjust the pH to 8.0 with hydrochloric acid, and bring the volume to 500mL. Store at room temperature.

[0073] 4. Nickel column purification

[0074] The recombinant protein containing 6×His-tag was purified using Ni2+ affinity chromatography. The purification steps are as follows:

[0075] (1) Fix the purification column on the iron stand and rinse the protein chromatography column with about 5-10 column volumes (about 5 mL per column volume) of washing buffer.

[0076] (2) Load the supernatant obtained by centrifugation after cell disruption in the pretreatment process into a pre-equilibrated chromatography column. Control the loading speed and prolong the time the enzyme flows through the chromatography column. After loading, wash the chromatography column with about 10-20 column volumes of washing buffer to remove non-specific binding of impurity proteins and other substances until the protein concentration A280 in the eluent of the chromatography column no longer decreases (below 0.1).

[0077] (3) Elute the recombinant protein with protein elution buffer, and collect about 15 tubes of column elution buffer at the same time, 1 mL per tube. Measure the A280 value of each tube of elution buffer collected. Take the elution buffer with the relatively high A280 value and add 20%-25% volume of glycerol. Aliquot the elution buffer into 100 μL tubes, freeze them in liquid nitrogen and place them in an ultra-low temperature freezer at -80℃ for later use.

[0078] (4) Once the eluted solution A280 no longer decreases, re-equilibrate the chromatography column with protein binding buffer, and then store the chromatography column in 20% ethanol.

[0079] During the purification process, appropriate amounts of the supernatant and precipitate after crushing and centrifugation should be placed in a -20℃ refrigerator as samples for SDS-PAGE analysis.

[0080] 5. SDS-PAGE analysis of recombinant plasmid expression products

[0081] Prepare the necessary buffer, staining solution, and destaining solution for SDS-PAGE. The solution formulations are as follows:

[0082] SDS-PAGE electrophoresis buffer: Weigh 3.05g Tris and 31.3g glycine, dissolve in 800mL ultrapure water, add 50mL of 10% SDS solution, and add ultrapure water to bring the volume to 1L; 5× loading buffer: Weigh 0.39g DTT, 0.5g SDS, and 0.025g bromophenol blue, dissolve in 2.5mL glycerol and 2.5mL 0.25M Tris / HCl (pH 6.8), aliquot into 1.5mL EP tubes, and store at 4℃; Coomassie Brilliant Blue R-250 staining solution (1L): 2g Coomassie Brilliant Blue R-250, 400mL methanol, 100mL glacial acetic acid, and 500mL water; Destaining solution (1L): Mix 100mL glacial acetic acid, 450mL ethanol, and 450mL water thoroughly; Clean the glass plates and SDS-PAGE gel preparation apparatus, and perform assembly and leak testing. Prepare separating gels with different ratios based on protein mass. Different molecular weight proteins require different protein gel ratios. A 10% separating gel is suitable for detecting proteins with a molecular weight of 30-90 kDa, while a 12% separating gel is suitable for detecting proteins with a molecular weight of 20-80 kDa. Pour the SDS-PAGE gel into the gel preparation apparatus. A water seal can be used on the separating gel to prevent unevenness. After it solidifies, pour in the stacking gel. After standing for 30-60 minutes, when a visible boundary line appears between the separating gel and the water layer, pour off the water on top of the separating gel. Prepare the separating gel and place it in the clamp. Quickly insert a comb of appropriate size. Let the entire SDS-PAGE gel stand for about 30 minutes, then immerse it in the electrophoresis tank for a few minutes and slowly remove the comb.

[0083] Add 100 μL and 400 μL of water to the samples before and after induction, respectively, and mix well. Take 20 μL of each sample. Take 2 μL of the lysis supernatant and dilute it with 18 μL of water. Dilute the precipitate with water to a final volume of 20 μL. Dilute the protein from *Cymbidium goeringii* to a final volume of 20 μL based on its OD value (OD × volume = 20). Add 5 μL of 5× loading buffer to all samples and inactivate the protein at 95°C for 10 minutes to ensure complete protein denaturation. Add the protein marker, pre-induction, post-induction, lysis supernatant, and purified enzyme samples sequentially to the wells of the SDS-PAGE gel. Run electrophoresis at 120 V until the blue indicator band just appears on the separating gel. Remove the gel tray and stain the electrophoresis gel with Coomassie Brilliant Blue R-250 staining solution for about 30 minutes. Discard the Coomassie Brilliant Blue and destain with destaining solution, changing the solution every 2 hours until the bands are clearly visible. Scan the gel and observe the results. Figure 1 ; Figure 1The results showed that the molecular masses indicated by these four bands were consistent with the theoretical values ​​(SlSUS, SeC2E, SeGMD and SeGER, with theoretical values ​​of 96.6 kDa, 37.9 kDa, 42.3 kDa and 36.2 kDa, respectively).

[0084] Example 2: Purification and desalting of four recombinases

[0085] The purified enzyme solution contains a large amount of imidazole, so the protease is desalted using a PD-10 desalting column. The detailed steps are as follows:

[0086] (1) Drain the original solution from the disposable desalting column PD-10;

[0087] (2) Prepare a Tris buffer solution with a pH of 8 and a concentration of 10 mM and wash with the buffer solution for 5 column volumes (this step is to prevent protein precipitation when washing with deionized water).

[0088] (3) Take 2.5 ml of the mixture purified by nickel affinity chromatography and add it to the desalting column to allow free elution;

[0089] (4) Rinse the column with 3ml of buffer and collect the target protein according to the OD260 absorbance value.

[0090] (5) The protein concentrations of purified SlSUS, SeC2E, SeGMD and SeGER were 2.0±0.9 mg / mL, 22±1.5 mg / mL, 25±1.2 mg / mL and 38±2.6 mg / mL, respectively.

[0091] Example 3: TLC detection of SlSUS enzymatic reaction products

[0092] Sucrose synthase catalyzes the breakdown of sucrose into glucose and fructose, and then reacts glucose with GDP to form GDP-glucose. The SlSUS theoretical catalytic principle indicates that the optimal nucleotide is uridine diphosphate (UDP), with the weakest affinity for guanosine diphosphate (GDP). The reaction system included: 5 μL 50 mM Tris-Cl (pH 6.0) (Nanjing Shoude Co., Ltd.), 5 μL 50 mM sucrose, 2.5 μL 5 mM GDP (Sigma-Aldrich), 1 μL 2 mM MgCl2, 20 μL SlSUS (2.0 mg / mL), and 6.5 μL ultrapure water. The reaction was carried out at 37 °C. After the reaction, a small amount of the mixture was centrifuged and diluted. One μL of each solution was dropped onto the bottom of the plate, dried, and placed in the chromatography tank. The TLC developing solvent formulation was: n-butanol / ethanol / water = 5 / 3 / 2 (v / v / v). The developing solvent was mixed thoroughly and added to the chromatography tank beforehand to ensure complete saturation. Because GDP itself has conjugated double bonds that are visible under ultraviolet light, such as Figure 2 As shown, the blank control with water replacing SUS only produced GDP, with no additional substances, while reaction system b with added SUS produced new substances. Sucrose and fructose are invisible under UV light and can only be stained with lichenol. After staining the TLC plate with lichenol and drying it, Figure 2 A shows that the reaction system with SlSUS successfully decomposed sucrose into fructose, while the blank control reaction system did not produce fructose. Combined with UV detection (…), Figure 2 B) This shows that SlSUS has the ability to break down sucrose and use GDP to produce GDP-glucose.

[0093] Example 4: UPLC detection of SeC2E enzymatic reaction products

[0094] The C2E catalytic principle indicates that glucose activated by cytidine diphosphate (CDP-Glc) has the highest affinity, while glucose activated by guanosine diphosphate (GDP) has the lowest affinity. The reaction system consisted of: 3 μL of supernatant from the SlSUS reaction mixture or 2 mM GDP-mannose (Sigma), 5 μL of 50 mM Tris-Cl (pH 7.5) (Nanjing Shoude Co., Ltd.), 1 μL of 2 mM MgCl2, and 3 μL of 2 mM NAD+. + (Shanghai Ruji Biotechnology Development Co., Ltd.), 18 μL SeC2E (22 mg / mL). The reaction was carried out overnight at 45°C. After the reaction, a small amount of the mixture was centrifuged and diluted. 50 μL was injected into a sample vial and detected according to the SeC2E enzyme activity assay method described above. Figure 3 The results showed that when GDP-mannose was used as the substrate, the addition of SeC2E to the reaction system significantly decreased the peak of the GDP-mannose (GDP-Man) peak, and a new peak appeared at 3 min. In contrast, the GDP-mannose peak in the blank control remained unchanged, with no new peaks. When the mixture after overnight reaction with SlSUS was analyzed, a strong peak appeared at the same elution position, suggesting that the peak following GDP-mannose was GDP-glucose (GDP-Glc). Using this concentrated mixture as the substrate, SeC2E was added again, and the reaction was carried out overnight under the same optimal conditions. UPLC analysis showed a significant decrease in the GDP-glucose peak, and the appearance of the GDP-mannose peak. This indicates that SeC2E has catalytic activity for both GDP-glucose and GDP-mannose and can couple with SlSUS to produce GDP-mannose.

[0095] Example 5: UPLC detection of SeGMD and SeGER enzymatic reaction products

[0096] GDP-mannose is first catalyzed by SeGMD to generate GDP-4-dehydro-6-deoxy-mannose, which is then catalyzed by SeGER enzyme with the participation of the cofactor NADPH to form GDP-L-fucose (GDP-L-Fuc). To detect the activities of SeGMD and SeGER enzymes, fucosyltransferase HpFucT and pNP-lactose were added to the reaction system to convert the SeGMD and SeGER reaction product GDP-L-Fuc into pNP-fucosyllactose. The formation of pNP-fucosyllactose was then detected by UPLC.

[0097] The fucosyltransferase HpFucT (serial number: WP_000487428.1) gene derived from Helicobacter pylori was synthesized and cloned into the Nde I / Xho I site of pET30a. It was then transformed into Escherichia coli according to the methods in Examples 1 and 2, expressed and purified, and HpFucT was obtained for analysis of the coupled enzyme reaction products.

[0098] The reaction system for SeGMD and SeGER was as follows: 4 μl of 2 mM GDP-mannose (Sigma), 5 μl of 50 mM Tris (pH 7.5) (Nanjing Shoude Co., Ltd.), 4 μl of 2 mM NADPH (Shanghai Ruji Biotechnology Development Co., Ltd.), 2 μl of 2 mM pNP-lactose (Suzhou Cabotsens Biotechnology Co., Ltd.), 6 μl of SeGMD, 4 μl of SeGER, and 15 μl of HpFucT. The reaction was carried out overnight at 37°C. After the reaction was complete, the reaction was terminated by heating at 95°C for 10 min and centrifuged. The supernatant was appropriately diluted, filtered, and 50 μL was injected into a sample vial for analysis. The UPLC results are as follows. Figure 2-6 The results showed that, compared to the blank control without the addition of SeGMD, SeGER, and HpFucT enzymes, only the pNP-lactose substrate peak was detected without any new peak. However, in the reaction system with the addition of SeGMD, SeGER, and HpFucT enzymes, a new peak was generated 0.3 min before the pNP-lactose peak. The samples were detected by ESI electrochemical chromatography (Shimadzu) using MS, with the mode set to positive ion scan mode and the m / z range set between 200 and 1000. Separation results. Figure 4 and Figure 5 The results were consistent with those of UPLC, showing that the substrate peak near 6 min contained the most [M+Na]+ with a mass-to-charge ratio of 486, which corresponds exactly to the mass-to-charge ratio of pNP-lactose plus Na. + The molecular weight is 463+23. The newly appearing peak contains [M+Na] with a mass-to-charge ratio of 632. + Doing more corresponds to the mass-to-charge ratio of pNP-fucosyl lactose plus Na.+ The molecular weight of 609+23 proves that SeGMD and SeGER are active and can successfully convert GDP-mannose into GDP-fucose. Subsequently, GDP-fucose combines with pNP-lactose under the catalysis of HpFucT enzyme to generate pNP-fucosyllactose. Therefore, it is proved that GMD and GER are both active and can catalyze the synthesis of GDP-fucose.

[0099] Example 6: Synthesis of GDP-fucose via multi-enzyme cascade catalysis

[0100] The 85 μL reaction system for GDP-fucose catalysis by a multi-enzyme cascade consisted of: 5 μL 500 mM Tris-HCl (pH 7.5) (Nanjing Shoude Co., Ltd.), 5 μL 100 mM GDP (Sigma-Aldrich), 5 μL 1 M sucrose, 1 μL 100 mM MgCl2 (Sigma-Aldrich), 14 μL SlSUS (2.0 mg / mL), and reacted at 37 °C for 12 hours. Then, 4 μL 20 mM NAD+ was added. + (Shanghai Ruji Biotechnology Development Co., Ltd.) and 20 μL SeC2E (100 mg / mL) were reacted at 45 °C for 12 hours. Finally, 4 μL 20 mM NADPH (Shanghai Ruji Biotechnology Development Co., Ltd.), 6 μL SeGMD (25 mg / mL), and 4 μL SeGER (38 mg / mL) were added, and the reaction was carried out at 25 °C for 12 hours. Figure 6 As shown, the optimal conversion rate of GDP-fucose can reach 20%, as determined by HpFucT enzyme coupling reaction.

[0101] Example 7: Discovery, expression, purification, and activity identification of enzymes related to the preparation of fucoidosyl lactose.

[0102] A sucrose synthase (NP_001234655.2) was discovered in the Solanum lycopersicum (tomato) genome database that can break down sucrose and transfer one molecule of glucose to GDP to generate GDP-glucose; similarly, in Salmonella... The *Enterica* genome database yielded a CDP-tives 2-epimerase (GenBank sequence number DW4091412.1) that epimerizes GDP-glucose into GDP-mannose, and a GDP-mannose dehydratase (GenBank sequence number ACN45760.1) and isomer reductase (GenBank sequence number WP_001041701.1) that convert GDP-mannose into GDP-fucose. Similarly, the *Helicobacter pylori* genome database yielded a fucosyltransferase (GenBank sequence number WP_000487428.1) that transfers fucosyl groups to a lactose donor to form fucosyllactose.

[0103] A series of molecular biology techniques were used to achieve recombinant expression of the genes of all enzymes required for the reactions in the prokaryotic host *E. coli*. The enzymes were purified by nickel chromatography, and the purification results were detected by SDS-PAGE. Furthermore, thin-layer chromatography, ultra-high performance liquid chromatography, and liquid chromatography-mass spectrometry were used to identify the enzyme-catalyzed reaction products. The specific steps are as follows:

[0104] 2. The recombinant plasmid was transformed into E. coli BL21.

[0105] Genes of enzymes related to GDP-fucose synthesis (SUS, C2E, GMD, GER, Hp13FucT2) were discovered from bacteria of different origins; sucrose synthase (SlSUS), which breaks down sucrose, is derived from Solanum lycopersicum; CDP-tivesose 2-epimerase (SeC2E), which can epimerize glucose activated by nucleotides into mannose, is derived from Salmonella enterica; GDP-mannose dehydratase (SeGMD) and isomer reductase (SeGER), which can convert GDP-mannose into GDP-fucose, are also derived from Salmonella enterica. The gene sequences of the sucrose synthase SlSUS (NP_001234655.2), CDP-tivesose 2-epimerase SeC2E (DW4091412.1), GDP-mannose dehydratase SeGMD (ACN45760.1), isomerase SeGER (WP_001041701.1), and fucosyltransferase Hp13FucT2 (WP_000487428.1) were obtained from the database and synthesized. They were then cloned into the pET30a plasmid. Specifically, the coding sequence of SlSUS was ligated at a blunt end to the multiple cloning site of pET30a to obtain pET30a-SlSUS; the coding sequence of SeC2E was ligated between the Nde I / Xho I sites of pET30a (replacing the original sequence between the sites) to obtain pET30a-SeC2E; and the coding sequence of SeGMD was ligated to the Kpn site of pET30a. pET30a-SeGMD was obtained by ligating the I / Xho I site to pET30a, pET30a-SeGER was obtained by ligating the SeGER coding sequence to the Kpn I / Xho I site of pET30a, and pET30a-Hp3 / 4FucT was obtained by ligating the Hp3 / 4FucT coding sequence to the NdeI / XhoI site of pET30a. These four recombinant plasmids (pET30a-SlSUS, pET30a-SeC2E, pET30a-SeGMD, pET30a-SeGER, and pET30a-Hp3 / 4FucT) were then transformed into *E. coli* BL21. The specific procedures are as follows:

[0106] (1) The recombinant plasmid powder returned from gene synthesis was centrifuged and diluted with water to 1 ng / μL;

[0107] (2) Take 1 μL of the diluted recombinant plasmid and transfer it into 100 μL of competent cells;

[0108] (3) Adjust the water bath temperature during the 25-minute interval, quickly place it in a 42°C water bath for 1 minute of heat shock, take it out and quickly put it back into the ice, and let it stand for 2 minutes to reduce the damage to E. coli.

[0109] (4) Take 200 μL of sterilized LB liquid culture medium and add it to each tube of competent cells after heat shock. Incubate at 37°C and 800 rpm for 50 min to allow them to recover.

[0110] (5) Add the shaken bacterial culture evenly to LB solid medium containing 0.1 mg / mL kanamycin. Invert the plate and incubate it in a 37°C incubator for 12-16 h until the plaques grow into single colonies suitable for picking.

[0111] 2. Heterologous expression of recombinant plasmids in Escherichia coli

[0112] (1) Pick a single colony transformed into E. coli BL21 and inoculate it into 5 mL LB liquid medium, add 5 μL of 50 mg / mL kanamycin, and incubate overnight at 37°C and 250 rpm in a constant temperature shaker.

[0113] (2) Transfer 5 mL of overnight culture to 400 mL of fresh, heat-sterilized LB broth for expansion culture. When the OD600 of the fermentation broth reaches between 0.5 and 0.8, add IPTG solution to make the final IPTG concentration 1 mmol / L. Induce expression overnight at 18°C ​​and 250 rpm.

[0114] (3) Take 1 mL of the fermentation broth before and after IPTG induction, centrifuge at 12000 rpm for 5 min, discard the supernatant, and store the cell precipitate at -20℃ as a sample for SDS-PAGE analysis.

[0115] 3. Affinity chromatography purification of recombinant nickel carrier

[0116] (1) Transfer the IPTG-induced bacterial culture to a centrifuge bottle, centrifuge at 4000 rpm for 15 min at 4℃, and discard the supernatant;

[0117] (2) Add 10 mL of Lysis Buffer (cell lysis buffer) to the induced bacterial cells and aspirate thoroughly to dissolve them without any precipitate particles. Transfer the mixture to a 50 mL centrifuge tube and add 100 μL of 100 mM PMSF (phenylmethylsulfonyl fluoride). Sonicate for 20 min using an ultrasonic disruptor to induce cell lysis and release the target protein. Ultrasonic disruption conditions: 1.5 s disruption / 2.5 s interval, 15 min, power 200 W;

[0118] (3) The cell suspension after disruption was centrifuged again at 12,000 rpm for 15 min at 4°C, and the supernatant was collected and placed on ice for later use.

[0119] Cell lysis buffer preparation: Weigh 6.06g of Tris and 5.84g of sodium chloride and dissolve them in 800mL of ultrapure water. Add 10mL of Triton X-100, stir well, adjust the pH to 8.0 with hydrochloric acid, and bring the volume to 1L. Store at 4℃. Due to the short half-life, add the buffers for various nickel affinity chromatography steps just before lysis. The reagents are prepared as follows: ① Washing buffer: Weigh 6.06g of Tris and 2.92g of sodium chloride and dissolve them in 800mL of ultrapure water. Adjust the pH to 8.0 with dilute HCl, and finally bring the volume to 1L with ultrapure water. Store at 4℃. ② Protein elution buffer preparation: Weigh 3.03g of Tris, 1.46g of sodium chloride, and 17.02g of imidazole and dissolve them in 400mL of ultrapure water. Adjust the pH to 8.0 with hydrochloric acid, and bring the volume to 500mL. Store at room temperature.

[0120] 4. Nickel column purification

[0121] Recombinant proteins containing 6×His-tag were produced using Ni 2+ Purification was performed using affinity chromatography, and the purification steps are as follows:

[0122] (1) Fix the purification column on the iron stand and rinse the protein chromatography column with about 5-10 column volumes (about 5 mL per column volume) of washing buffer.

[0123] (2) Load the supernatant obtained by centrifugation after cell disruption in the pretreatment process into a pre-equilibrated chromatography column. Control the loading speed and prolong the time the enzyme flows through the chromatography column. After loading, wash the chromatography column with about 10-20 column volumes of washing buffer to remove non-specific binding of impurity proteins and other substances until the protein concentration A280 in the eluent of the chromatography column no longer decreases (below 0.1).

[0124] (3) Elute the recombinant protein with protein elution buffer, and collect about 15 tubes of column elution buffer at the same time, 1 mL per tube. Measure the A280 value of each tube of elution buffer collected. Take the elution buffer with the relatively high A280 value and add 20%-25% volume of glycerol. Aliquot the elution buffer into 100 μL tubes, freeze them in liquid nitrogen and place them in an ultra-low temperature freezer at -80℃ for later use.

[0125] (4) Once the eluted solution A280 no longer decreases, re-equilibrate the chromatography column with protein binding buffer, and then store the chromatography column in 20% ethanol.

[0126] During the purification process, appropriate amounts of the supernatant and precipitate after crushing and centrifugation should be placed in a -20℃ refrigerator as samples for SDS-PAGE analysis.

[0127] 5. SDS-PAGE analysis of recombinant plasmid expression products

[0128] Prepare the necessary buffer, staining solution, and destaining solution for SDS-PAGE. The solution formulations are as follows:

[0129] SDS-PAGE electrophoresis buffer: Weigh 3.05g Tris and 31.3g glycine, dissolve in 800mL ultrapure water, add 50mL of 10% SDS solution, and add ultrapure water to bring the volume to 1L; 5× loading buffer: Weigh 0.39g DTT, 0.5g SDS, and 0.025g bromophenol blue, dissolve in 2.5mL glycerol and 2.5mL 0.25M Tris / HCl (pH 6.8), aliquot into 1.5mL EP tubes, and store at 4℃; Coomassie Brilliant Blue R-250 staining solution (1L): 2g Coomassie Brilliant Blue R-250, 400mL methanol, 100mL glacial acetic acid, and 500mL water; Destaining solution (1L): Mix 100mL glacial acetic acid, 450mL ethanol, and 450mL water thoroughly; Clean the glass plates and SDS-PAGE gel preparation apparatus, and perform assembly and leak testing. Prepare separating gels with different ratios based on protein mass. Different molecular weight proteins require different protein gel ratios. A 10% separating gel is suitable for detecting proteins with a molecular weight of 30-90 kDa, while a 12% separating gel is suitable for detecting proteins with a molecular weight of 20-80 kDa. Pour the SDS-PAGE gel into the gel preparation apparatus. A water seal can be used on the separating gel to prevent unevenness. After it solidifies, pour in the stacking gel. After standing for 30-60 minutes, when a visible boundary line appears between the separating gel and the water layer, pour off the water on top of the separating gel. Prepare the separating gel and place it in the clamp. Quickly insert a comb of appropriate size. Let the entire SDS-PAGE gel stand for about 30 minutes, then immerse it in the electrophoresis tank for a few minutes and slowly remove the comb.

[0130] Add 100 μL and 400 μL of water to the samples before and after induction, respectively, and mix well. Take 20 μL of each sample. Take 2 μL of the lysis supernatant and dilute it with 18 μL of water. Dilute the precipitate with water to a final volume of 20 μL. Dilute the protein from *Cymbidium goeringii* to a final volume of 20 μL based on its OD value (OD × volume = 20). Add 5 μL of 5× loading buffer to all samples and inactivate the protein at 95°C for 10 minutes to ensure complete protein denaturation. Add the protein marker, pre-induction, post-induction, lysis supernatant, and purified enzyme samples sequentially to the wells of the SDS-PAGE gel. Run electrophoresis at 120 V until the blue indicator band just appears on the separating gel. Remove the gel tray and stain the electrophoresis gel with Coomassie Brilliant Blue R-250 staining solution for about 30 minutes. Discard the Coomassie Brilliant Blue and destain with destaining solution, changing the solution every 2 hours until the bands are clearly visible. Scan the gel and observe the results. Figure 7 ; Figure 7 The results showed that the molecular masses indicated by these four bands were consistent with the theoretical values ​​(SlSUS, SeC2E, SeGMD, SeGER, and Hp13FucT2 were 96.6 kDa, 37.9 kDa, 42.3 kDa, 36.2 kDa, and 42.5 kDa, respectively).

[0131] The purified protein concentrations of SlSUS, SeC2E, SeGMD, SeGER, and Hp13FucT2 were 2.0±0.9 mg / mL, 22±1.5 mg / mL, 25±1.2 mg / mL, 38±2.6 mg / mL, and 20±1.6 mg / mL, respectively, for subsequent use.

[0132] Example 8: One-pot multi-enzyme synthesis of fucoidyl lactose

[0133] 1) Take 5 μL Tris-HCl (500 mM pH = 7.5), 5 μL GDP solution (100 mM), 5 μL sucrose solution (1 M), 1 μL MgCl2 solution (100 mM) and 14 μL SlSUS enzyme solution (2.0 mg / mL), mix them evenly, and react at 37℃ for 12 h;

[0134] 2) Next, add 20 μL of SeC2E enzyme solution (22 mg / mL) and 4 μL of NAD to the above reaction solution. + The solution (20mM) was mixed thoroughly and reacted at 45℃ for 12h.

[0135] 3) Finally, add 2 μL of pNP-lactose solution (20 mM), 4 μL of NADPH solution (20 mM), 6 μL of SeGMD enzyme solution (25 mg / mL), 4 μL of SeGER enzyme solution (38 mg / mL), and 15 μL of Hp13FucT2 enzyme solution (20 mg / mL) to the reaction system, mix well, and react at 25°C for 12 h.

[0136] After the reaction, the formation of pNP-fucosyl lactose was detected using UPLC-MS. 40 μL of the reaction mixture was heated at 95 °C for 10 min to completely denature the protein. The inactivated reaction mixture was then centrifuged at 12000 rpm for 10 min to separate the denatured protein and supernatant. The supernatant was then diluted 5-fold with ultrapure water. The reaction mixture, a blank control without enzymes (without SeGMD, SeGER, and Hp13FucT2 enzymes, all other steps unchanged), and pNP-lactose standards were added sequentially to different sample vials. Each experimental group was set up in triplicate. The results are as follows: Figure 10As shown, compared to the blank control without the addition of SeGMD, SeGER, and Hp13FucT2 enzymes, only the pNP-lactose substrate peak was detected without any new peak. However, in the reaction system with the addition of SeGMD, SeGER, and Hp13FucT2 enzymes, a new peak was generated 0.3 min before the pNP-lactose peak. The samples were detected by ESI electrochemical chromatography using MS, with the mode set to positive ion scan mode and the m / z range set between 200 and 1000. The separation results are as follows. Figure 11 The results are consistent with those of UPLC, showing that the substrate peak near 6 min contains [M+Na] with a mass-to-charge ratio of 486. + At most, it corresponds exactly to the mass-to-charge ratio of pNP-lactose plus Na. + The molecular weight is 463+23. The newly appearing peak contains [M+Na] with a mass-to-charge ratio of 632. + Doing more corresponds to the mass-to-charge ratio of pNP-fucosyl lactose plus Na. + The molecular weight is 609+23, which proves that SeGMD, SeGER and Hp3 / 4FucT enzymes are active and can successfully catalyze the production of pNP-fucosylated lactose from GDP-mannose and pNP-lactose.

[0137] The above UPLC-MS analysis was performed using a Shimadzu Nexera liquid chromatograph and an MS2020 in series. The system included a SIL-30AC autosampler, an LC-30AD pump, an ESI ion source, and an SPD-UV20AD detector. Mobile phase A was 50 mM ammonium formate (pH 4.5), mobile phase B was 100% acetonitrile, the eluent flow rate was 0.8 mL / min, and the UV detection wavelength was 300 nm. Samples were injected... HyperClone™-ODS C18 reversed-phase column (5μm, The column (250×4.6mm) was equilibrated by rapidly increasing the proportion of mobile phase B from 20% to 60% over 0.01–5 minutes, then rapidly increasing it to 90% over the next minute and holding for 2 minutes. The column was then rapidly decreased to 20% over the next minute and held for 6 minutes. The liquid eluent from the liquid chromatograph was immediately introduced into the mass spectrometer. Dry nitrogen flow rate was 10 L / min, nebulized gas flow rate was 1.5 L / min, DL temperature was 250℃, and a positive ion scan mode was used with an m / z range of 200–1000. Simultaneously, single-particle detection (SMP) mode was used to check the consistency of the molecular weights of the substrate and product peaks.

[0138] Example 9: Preparation and purification of pNP-fucosyl lactose

[0139] 1. Preparation of pNP-fucosyl lactose

[0140] The original reaction system was scaled up from 85 μL to 100 mL. 5.88 mL of Tris-HCl (500 mM, pH 7.5), 5.88 mL of GDP solution (100 mM), 5.88 mL of sucrose solution (1 M), 1.18 mL of MgCl2 solution (100 mM), and 16.47 mL of SlSUS enzyme solution were mixed thoroughly and reacted at 37 °C for 12 h. Then, 23.53 mL of SeC2E enzyme solution and 4.7 mL of NAD were added to the above reaction solution. + The solution (20 mM) was mixed thoroughly and reacted at 45°C for 12 h. Finally, 2.35 mL of pNP-lactose solution (20 mM), 4.7 mL of NADPH solution (20 mM), 7.06 mL of SeGMD enzyme solution, 4.7 mL of SeGER enzyme solution, and 17.65 mL of Hp13FucT2 enzyme solution were added to the reaction system, mixed thoroughly, and reacted at 25°C for 12 h. The required mixture was then added to a 250 mL shake flask and incubated on a shaker. The temperature was adjusted according to the order of enzyme addition. To ensure sufficient contact between the protease and substrate, the shaker speed was adjusted to 100 rpm. After the reaction was complete, the reaction mixture was obtained.

[0141] 2. Isolation and purification of pNP-fucosyl lactose

[0142] The reaction mixture was heated in a water bath at 95°C for 10 min to terminate the reaction. After cooling to room temperature, the mixture was centrifuged in 50 ml centrifuge tubes to remove the denatured and inactivated protein precipitate. A small amount of deionized water was added to the protein precipitate, and the mixture was mixed and centrifuged again to collect the supernatant. This process was repeated 2-3 times to reduce product loss. The supernatant obtained in the above step was collected in a lyophilized bottle and lyophilized in a freeze dryer. Separation was performed using a preparative HPLC column. The preparative column separation method was based on a C18 column with specifications of 250 mm * 4.6 mm * 5 μm. The elution program was 75 min. The proportion of mobile phase B was increased from 10% to 15% within 0.01-40 min, then rapidly increased to 90% within the next minute and held for 9 min. The proportion was then rapidly decreased to 10% within 5 min and held for 20 min. Mobile phase A was 50 mM ammonium formate (pH 4.5), and mobile phase B was 100% acetonitrile. The maximum loading volume of the preparative column was 1 ml, which is 18.9 times the volume of the basic C18 column. To maintain the original column pressure conditions, the flow rate was increased from 1 ml / min to 5 ml / min. The UV detection wavelength was 300 nm. The results are as follows: Figure 12 As shown, when the total elution program duration was 75 min, two similar peaks appeared at 36 min and 38.5 min. These two peaks were collected separately in test tubes and detected again by UPLC. The results are shown below. Figure 13As shown, the peaks corresponding to 36 min and 38.5 min are the substrate pNP-lac and the product pNP-FL, respectively. This demonstrates that by using a preparative column and eluting for 75 min, the time difference between the product and substrate peaks can be increased, thus enabling better separation of pNP-lac and pNP-FL.

[0143] Comparative Example 1: Discovering suitable FucT in a single-pot multi-enzyme method

[0144] Using SlSUS, SeC2E, SeGMD, and SeGER as the first four fixed enzymes in the basic reaction system, with the substrate and coenzyme unchanged, the activities of Hp13FucT2 (prepared in Example 7) and Hp13FucT1 (GenBank accessions: O30511.1 and 2NZW_A; expression and purification methods were the same as in Example 7) as the last enzyme in a one-pot multi-enzyme method were measured using UPLC with a UV detector at a wavelength of 300 nm. Figure 11 It is known that both Hp13FucT2 and Hp13FucT1 can transfer fucosylation to a lactose donor using pNP-lactose as a substrate, generating pNP-FL. The conversion rate was calculated by peak values. The reaction system using Hp13FucT2 as the fucosylation enzyme achieved an overall conversion rate of 20%, while the system using Hp13FucT1 as the fucosylation enzyme had a conversion rate of less than 5%. Therefore, Hp13FucT2 was selected as the fucosylation enzyme for this application.

[0145] Comparative Example 2: Effect of site-directed mutagenesis of the C2E catalytic site on the synthesis of FL using a single-pot multi-enzyme method.

[0146] The SeC2E enzyme was mutated using a point mutation kit to obtain SeC2E S122A and SeC2E Y165F, respectively, with amino acid 122 mutated to alanine and amino acid 165 mutated to phenylalanine. The expression and purification results of SeC2E S122A and SeC2E Y165F were analyzed by SDS-PAGE. Figure 14 As shown, the following conclusions can be drawn: Both SeC2E S122A and SeC2E Y165F mutant genes can be successfully induced and purified in the host, and their molecular weight is consistent with that of the wild type (SDS-PAGE analysis of samples showed that all samples were proteins purified by nickel column).

[0147] The original system was reacted by replacing SeC2E with two different mutants, and the results were analyzed by mass spectrometry as follows: Figure 15As shown, the peak of the target product pNP-FL can be clearly observed in the system after the reaction of wild-type SeC2E. Mutation at site 122 does not change the enzyme activity itself, and the product peak still appears at the same time, with a conversion rate almost identical to that of wild-type SeC2E. However, site-directed mutagenesis at site 165 inactivates the enzyme, making it impossible to detect the formation of pNP-FL. Therefore, in the one-pot multi-enzyme method, wild-type SeC2E is still used for epimerization of GDP-glucose.

[0148] Example 10: Gene cloning, heterologous expression, purification, and enzyme activity assay of fucosidase Eo3066

[0149] 1. Recombinant plasmid transformed into E. coli BL21

[0150] This application identifies fucosidase (GenBank annotation number UniProt ID I2ERT6) from the aquatic bacterium *Emticicia oligotrophica*. The gene sequence of fucosidase Eo3066 (as shown in Sequence 1) was synthesized, and the sequence between the Nde I and Xho I sites of the pET30a plasmid was replaced, while other sequences remained unchanged, to obtain the recombinant plasmid pET30a-Eo3066. This recombinant plasmid was then transformed into *E. coli* BL21 to obtain *E. coli* BL21-Eo3066. The specific process is as follows:

[0151] (1) The recombinant plasmid powder returned from gene synthesis was centrifuged and diluted with water to 1 ng / μL;

[0152] (2) Take 1 μL of the diluted recombinant plasmid and transfer it into 100 μL of competent cells;

[0153] (3) Adjust the water bath temperature during the 25-minute interval, quickly place it in a 42°C water bath for 1 minute of heat shock, take it out and quickly put it back into the ice, and let it stand for 2 minutes to reduce the damage to E. coli.

[0154] (4) Take 200 μL of sterilized LB liquid culture medium and add it to each tube of competent cells after heat shock. Incubate at 37°C and 800 rpm for 50 min to allow them to recover.

[0155] (5) Add the shaken bacterial culture evenly to LB solid medium containing 0.1 mg / mL kanamycin. Invert the plate and incubate it in a 37°C incubator for 12-16 h until the plaques grow into single colonies suitable for picking.

[0156] 2. Heterologous expression of recombinant plasmids in Escherichia coli

[0157] (1) Pick a single colony transformed with E. coli BL21 (i.e. E. coli BL21-Eo3066 containing recombinant plasmid) and inoculate it into 3 mL of LB liquid medium. Add 3 μL of 50 mg / mL kanamycin and incubate overnight at 37 °C and 250 rpm in a constant temperature shaker.

[0158] (2) Take 1 mL of bacterial culture and inoculate it into 400 mL of fresh LB medium. Incubate at 37°C and 250 rpm until OD reaches 1000. 600 The concentration was 0.5, and then IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 1 mM. The mixture was then incubated at 20 °C and 250 rpm for 3 hours.

[0159] (3) After the culture is completed, the cells are collected by centrifugation (5000g, 15min, 4℃). The cells are resuspended in 10mL of lysis buffer (100mM NaCl, 50mM Tris, 1% Triton X-100, and 1mM phenyl-methylsulfonyl fluoride, pH 8.0) and sonicated in an ice bath for 20 minutes (40 on / offcycles with 20μm amplitude for 15s). Then, the cells are centrifuged (20000g, 20min, 4℃) to remove cell debris and the cell lysate is collected.

[0160] Preparation of cell lysis buffer: Weigh 6.06 g of Tris and 5.84 g of sodium chloride and dissolve them in 800 mL of ultrapure water. Add 10 mL of Triton X-100, stir well, adjust the pH to 8.0 with hydrochloric acid, and bring the volume to 1 L. Store at 4 °C.

[0161] 3. Nickel affinity chromatography purification of recombinant vector

[0162] Cell lysis buffer loaded onto a nickel column (Ni 2+ -nitrilotriacetate agarose affinity chromatography column (2 mL bed volume) (Qiagen). After loading the sample, wash thoroughly with 50 mL of wash buffer (50 mM Tris-HCl, 50 mM NaCl, pH 8.0) to remove unadsorbed protein. Elute the recombinant protein with elution buffer (300 mM imidazole, 50 mM Tris-HCl, 50 mM NaCl, pH 8.0). The elution buffer containing the target protein should be approximately 4 mL and can be used directly for subsequent enzyme activity analysis.

[0163] The reagents were prepared as follows: ① Washing and elution buffer: Weigh 6.06g of Tris and 2.92g of sodium chloride and dissolve them in 800mL of ultrapure water. Adjust the pH to 8.0 with dilute HCl, and finally bring the volume to 1L with ultrapure water. Store at 4℃. ② Elution buffer preparation: Weigh 3.03g of Tris, 1.46g of sodium chloride, and 17.02g of imidazole and dissolve them in 400mL of ultrapure water. Adjust the pH to 8.0 with hydrochloric acid, bring the volume to 500mL, and store at room temperature.

[0164] 4. Enzyme activity analysis of recombinant plasmid expression products

[0165] The purified recombinant enzyme fucosidase Eo3066 showed detectable fucosidase activity when pNP-α-L-fucose was used as a substrate.

[0166] Fucosidase activity assay: 15 L of reaction solution contained 8.5 μL PBS buffer (pH 7.4), 1.5 μL pNP-α-L-fucose (10 mM stock solution) (Sigma), and 5 μL purified recombinant fucosidase. A reaction system without recombinant enzyme served as a negative control (-). Results are as follows: Figure 16 As shown in the figure. The results indicate that the recombinant enzyme Eo3066 possesses fucosidase activity and can hydrolyze pNP-α-L-fucose to produce fucose.

[0167] The purified enzyme solution obtained in step 3 above contains a large amount of imidazole, so the protease is desalted using a PD-10 desalting column. The detailed steps are as follows:

[0168] (1) Drain the original solution from the disposable desalting column PD-10;

[0169] (2) Prepare a Tris buffer solution with a pH of 8 and a concentration of 10 mM and wash with the buffer solution for 5 column volumes (this step is to prevent protein precipitation when washing with deionized water).

[0170] (3) Take 2.5 ml of the mixture purified by nickel affinity chromatography and add it to the desalting column to allow free elution;

[0171] (4) Rinse the column with 3ml of buffer and collect the target protein according to the OD260 absorbance value.

[0172] (5) The protein concentration of the purified fucosidase Eo3066 was approximately 1.0 mg / mL.

[0173] Example 11: Detection of products catalyzed by fucosidase Eo3066

[0174] The 50 μL reaction system consisted of 5 μL (~10 μM) purified pNP-fucosyllactose, 5 μL pH 7.4 PBS buffer, and 30 μL (1.0 mg / mL) fucosidase Eo3066 obtained in Example 10. The reaction was carried out at 37°C for 16 hours. A reaction system without fucosidase Eo3066 was used as a control. After the reaction, the product was analyzed using a reverse-phase C18 column (Phenomenex Hyperclone 5 μm 250 × 4.6 mm) at a flow rate of 0.8 mL / min and a UV detector wavelength of 300 nm. Elution conditions were a gradient elution of 20-60% (vol) of acetonitrile in formate buffer (50 mM, pH 4.5) for 6 minutes. Figure 17 As shown, fucosidase Eo3066 can hydrolyze pNP-fucosyllactose (pNP-fucosyllactose) to release fucose.

[0175] Example 12: Preparation of L-fucose by fucosidase

[0176] To prepare L-fucose, purified recombinant fucosidase at a concentration of 1.0 mg / mL was concentrated 5 times using centrifugal concentrators (Vivaspin mini, 10 kDa MWCO) to obtain fucosidase at a concentration of 5 mg / mL.

[0177] The 50 μL reaction system included: 5 μL (~10 μM) purified pNP-fucosyllactose (prepared in Example 9), 5 μL pH 7.4 PBS buffer, and 30 μL (5 mg / mL) recombinant fucosidase, reacted at 37°C for 16 hours. The enzymatic reaction was carried out at 37°C for 16 hours. A reaction system without fucosidase Eo3066 was used as a control. Results are as follows... Figure 18 As shown, Eo3066 fucosidase at a concentration of 5 mg / mL can completely hydrolyze pNP-fucosyllactose in the reaction system, releasing L-fucose.

[0178] Cross-reference to related applications

[0179] This application claims priority to Chinese patent applications filed on August 16, 2023 (application number 202311031350.X), Chinese patent applications filed on August 16, 2023 (application number 202311031352.9), and Chinese patent applications filed on August 16, 2023 (application number 202311031348.2), the entire contents of which are incorporated herein by reference.

[0180] Industrial applications

[0181] 1. The *E. coli* expression system used in this application features high expression efficiency, large expression volume, low cost, and ease of operation. The four enzymes expressed and involved in the cascade reaction exhibit high purity and high yield. This application provides a novel method for the in vitro preparation of low-cost GDP-fucose using guanosine diphosphate and sucrose as raw materials and a cascade catalysis of four enzymes. By utilizing the *E. coli* expression system and inexpensive guanosine diphosphate and sucrose as raw materials, efficient expression of various enzymes in the cascade catalytic reaction is achieved, thereby enabling low-cost, large-scale production of GDP-fucose. This application not only provides inexpensive raw materials for the mass production of human milk oligosaccharides but also provides inexpensive intermediates for the production of natural oligosaccharide drugs.

[0182] 2. The one-pot multi-enzyme method for synthesizing fucoidosyl lactose in this application uses sucrose, GDP and pNP-lactose as initial substrates to successfully generate 3'-fucosyl lactose in vitro through a one-pot multi-enzyme method. This method is simpler and faster than whole-cell synthesis, and therefore has broad commercial application value.

[0183] 3. The intestinal expression system used in this application has the advantages of high expression efficiency, large expression volume, low cost, and easy operation, and the expressed fucosidase has high purity and high yield. This application provides a new method for preparing low-cost L-fucose using fucoidanase as a raw material.

Claims

1. A method for synthesizing GDP-fucose via a multi-stage, multi-enzyme cascade catalysis, characterized in that, Includes the following steps: 1) Using guanosine diphosphate and sucrose as substrates, GDP-glucose is generated through a sucrose synthase-catalyzed reaction; 2) GDP-mannose is generated from GDP-glucose as a substrate via a reaction catalyzed by CDP-Tives 2-epimerase; 3) GDP-mannose is generated from GDP-mannose through a reaction catalyzed by GDP-mannose dehydrating enzyme and isomer reductase; The isomeric reductase is derived from intestinal Salmonella ( Salmonella enterica The sequence number of the amino acid sequence is WP_001041701.

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2. The method according to claim 1, characterized in that, The sucrose synthase, CDP-Tives 2-epimerase, GDP-mannose dehydratase, and isomer reductase all catalyze reactions in the form of crude enzyme solution, crude enzyme powder, pure enzyme, or whole cells.

3. The method according to claim 1, characterized in that, The sucrose synthase is derived from tomatoes ( Solanum lycopersicum The amino acid sequence number is NP_001234655; the GDP-mannose dehydrase is derived from intestinal Salmonella (…). Salmonella enterica The amino acid sequence number is ACN45760.

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4. A method for synthesizing fucoidyl lactose using a single-reactor, multi-enzyme process, characterized in that, The enzymatic synthesis method for fucoidyl lactose involves using sucrose, GDP, and pNP-lactose as initial substrates, and obtaining fucoidyl lactose under the catalysis of five enzymes: sucrose synthase, CDP-tivesose 2-epimerase, GDP-mannose dehydratase, isomer reductase, and fucoidosyltransferase. The isomer reductase is derived from intestinal Salmonella (…). Salmonella enterica The sequence number of the amino acid sequence is WP_001041701.

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5. The method according to claim 4, characterized in that, The fucosyltransferase is derived from Helicobacter pylori (Helicobacter pylori) Helicobacter pylori The amino acid sequence is located in GenBank and its sequence number is WP_000487428.

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6. A method for preparing L-fucose enzymatically, characterized in that: Fucosyl lactose is prepared by the method described in claim 4, and L-fucose is generated by fucosyl lactose as a substrate through a fucosidase-catalyzed reaction.

7. The method according to claim 6, characterized in that, The fucosidase is derived from aquatic bacteria. Emticicia oligotrophica The UniProt annotation number is UniProt ID I2ERT6.

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