Preparation method and application of fucose and intermediate thereof

Abstract

The invention discloses a preparation method and application of fucose and an intermediate thereof. The method for synthesizing the GDP-fucose comprises the following steps: taking guanosine diphosphate and cane sugar as substrates, and carrying out catalytic reaction on cane sugar synthase, CDP-tetrose 2-epimerase, GDP-mannose dehydratase and isoreductase to generate the GDP-fucose. The method for synthesizing the fucosyllactose comprises the following steps: taking GDP-fucose and pNP-lactose as initial substrates, and obtaining the fucosyllactose under the catalytic action of Hp13FucT2 enzyme. According to the method for preparing the L-fucose through the enzyme method, fucosyllactose is used as a substrate, and the L-fucose is generated through a fucosidase catalytic reaction. The adopted large intestine expression system has the characteristics of high expression efficiency, large expression quantity, low cost, easiness in operation and the like, and the expressed fucosidase has the advantages of high purity, high yield and the like.

Description

Preparation method and application of fucose and its intermediates Technical Field

[0001] The present application relates to a preparation method and application of fucose and its intermediates. Background Art

[0002] L-fucose, also known as 6-deoxy-L-galactose, has a molecular formula of C6H 12 O5, with a molecular weight of 164.16, is a hexose sugar. The predominant form of fucose in nature is L-fucose. L-fucose is widely present in polysaccharides in humans, animals, plants, and microorganisms. For example, it exists in the form of sulfated oligosaccharides in human breast milk, sea urchin and frog eggs, as well as in plant polysaccharides such as tragacanth gum, potatoes, kiwifruit, soybeans, winged beans, and canola, as well as in the extracellular polysaccharides of various seaweeds.

[0003] L-fucose has multiple biological activities, including anti-inflammatory properties. Adding an appropriate amount of L-fucose to medications can effectively reduce inflammation. Because L-fucose plays an important targeting role in cell adhesion, drugs can be bound to L-fucose, guiding them to the site of action. L-fucose also regulates blood lipids and cholesterol. Adding it to lactic acid beverages, cakes, and cheese, replacing sucrose, enhances the original flavor while meeting people's nutritional and health needs. L-fucose cannot be absorbed by intestinal lining cells, but it can be used by probiotics, thereby increasing their numbers and maintaining a healthy balance. It can be added to lactic acid beverages as a prebiotic. Adding an appropriate amount of L-fucose to soy products can also help regulate blood lipids and cholesterol and maintain a healthy balance of probiotics in the intestine. Furthermore, L-fucose is widely used in foods such as chewing gum, chocolate, various candies, bread, preserved fruit, biscuits, jam, and eight-treasure porridge.

[0004] L-fucose promotes fibroblast growth, thereby preventing collagen deficiency and sagging caused by radiation and UV exposure. It also reduces the activity of proteolytic enzymes in the skin, thereby minimizing skin damage. Therefore, using L-fucose in cosmetics can help moisturize and protect the skin, promote cell proliferation, increase skin elasticity, and slow down skin aging.

[0005] Fucosyllactose (FL), the most abundant functional oligosaccharide in breast milk, possesses important physiological functions such as anti-infection, immunomodulation, and promoting brain development in infants. FL has significant application value in the food industry. As early as 2015, it was approved by the US Food and Drug Administration and the European Union as a nutritional supplement for infant formula, milk powder, and beverages. To date, FL has been primarily obtained through the following methods: natural product extraction, chemical synthesis, enzymatic synthesis, and whole-cell synthesis. Enzymatic synthesis offers significant advantages in terms of spatial and stereochemical specificity of the catalytic reaction. It is also characterized by simplicity, environmental friendliness, low byproduct count, a single, easily purified product, and the potential for developing new substrates to reduce costs. Consequently, it has become a synthetic method that has garnered significant attention in recent years.

[0006] Current methods for producing FL primarily rely on whole-cell synthesis using engineered strains such as Escherichia coli and Saccharomyces cerevisiae. However, this whole-cell synthesis method is complex and significantly affected by bacterial strains. Similarly, FL can be isolated from breast milk through chromatographic extraction, but the fucosyllactose obtained this way is expensive and time-consuming. FL can also be synthesized chemically, but the side chains of FL need to be protected and deprotected during synthesis, resulting in 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, for example, from D-galactose, L-arabinose, D-mannose, or D-galacturonic acid. D-galactose is the most suitable raw material for its 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 separations to separate intermediates from byproducts, resulting in low yields and high costs, limiting its application in the production of L-fucose. Currently, fucose is primarily sourced from natural sources. Oligosaccharides containing fucose are isolated from biomass, such as algae, through extraction. Hydrolysis of the oligosaccharides produces a complex mixture containing fucose, related sugars, and / or their derivatives. Fucose is then purified and recovered from this mixture through methods such as ion exchange chromatography, dialysis, and fractional crystallization. However, the high cost of fucose obtained in this manner makes it difficult to commercialize.

[0008] Invention Disclosure

[0009] The present application provides a preparation method of fucose and its intermediates and applications thereof.

[0010] The present application provides a method for synthesizing GDP-fucose by multi-stage multi-enzyme cascade catalysis, comprising the following steps:

[0011] 1) Using guanosine diphosphate and sucrose as substrates, sucrose synthase catalyzes the reaction to produce GDP-glucose;

[0012] 2) Using GDP-glucose as substrate, GDP-mannose is generated by CDP-teviose 2-epimerase catalysis;

[0013] 3) Using GDP-mannose as a substrate, GDP-fucose is generated through the catalytic reaction of DP-mannose dehydratase and isomeroreductase.

[0014] The sucrose synthase, CDP-tyrosine 2-epimerase, GDP-mannose dehydratase and isomeroreductase all catalyze in the form of crude enzyme solution, crude enzyme powder, pure enzyme or whole cell.

[0015] Among them, the sucrose synthase is derived from tomato (Solanum locopersicum), and the sequence number of the amino acid sequence is NP_001234655; the CDP-tyvetose 2-epimerase is derived from Salmonella enterica, and the sequence number of the amino acid sequence is DW4091412.1; the GDP-mannose dehydratase is derived from Salmonella enterica, and the sequence number of the amino acid sequence is ACN45760.1; the isomeroreductase is derived from Salmonella enterica, and the sequence number of the amino acid sequence is WP_001041701.1.

[0016] The present application also provides a method for synthesizing fucosyllactose by a one-pot multi-enzyme method, characterized in that it includes the step of using GDP-fucose and pNP-lactose prepared according to claim 1 as initial substrates and obtaining fucosyllactose under the catalysis of Hp13FucT2 enzyme.

[0017] The Hp13FucT2 is a fucosyltransferase derived from Helicbacter pylori, and the GenBank sequence number of the amino acid sequence is WP_000487428.1.

[0018] Among them, the enzymatic synthesis method of fucosyllactose is to use sucrose, GDP and pNP-lactose as initial substrates, and obtain fucosyllactose under the catalysis of five enzymes: sucrose synthase, CDP-teviose 2-epimerase, GDP-mannose dehydratase, isomeroreductase and fucosyltransferase.

[0019] Further, the following steps are included:

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

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

[0022] 3) adding pNP-lactose solution, NADPH solution, SeGMD enzyme solution, SeGER enzyme solution, and Hp13FucT2 enzyme solution to the reaction solution in step 2), mixing, and reacting to generate pNP-fucosyllactose;

[0023] 4) Purifying and separating the product of step 3) to obtain pNP-fucosyllactose.

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

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

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

[0027] Furthermore, the reaction condition in step 2) is 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 SeGMD enzyme (25 mg / mL) per 1 mol of sucrose; the amount of SeGER enzyme solution added is 4 μl SeGER enzyme (38 mg / mL) per 1 mol of sucrose; and the amount of Hp13FucT2 enzyme solution added is 15 μl Hp13FucT2 enzyme (20 mg / mL) per 1 mol of sucrose.

[0030] Furthermore, the reaction condition in step 3) is 25° C. for 12 hours.

[0031] The present invention provides a method for preparing L-fucose by an enzymatic method, wherein fucosyllactose is used as a substrate and L-fucose is generated by a fucosidase-catalyzed reaction.

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

[0033] The present application also provides a bio-enzyme composition for preparing GDP-fucose, which includes sucrose synthase, CDP-tyvesu 2-epimerase, GDP-mannose dehydratase and isomeroreductase; the sucrose synthase is derived from tomato (Solanumlocopersicum), and the sequence number of the amino acid sequence is NP_001234655; the CDP-tyvesu 2-epimerase is derived from Salmonella enterica, and the sequence number of the amino acid sequence is DW4091412.1; the GDP-mannose dehydratase is derived from Salmonella enterica, and the sequence number of the amino acid sequence is ACN45760.1; the isomeroreductase is derived from Salmonella enterica, and the sequence number of the amino acid sequence is WP_001041701.1.

[0034] The present application also provides a biological enzyme composition for preparing fucosyllactose, comprising a biological enzyme composition for preparing GDP-fucose and fucosyltransferase Hp13FucT2, wherein the fucosyltransferase Hp13FucT2 is derived from Helicobacter pylori, and the GenBank sequence number of the amino acid sequence is WP_000487428.1.

[0035] The present application also provides a biological enzyme composition for preparing L-fucose, comprising a biological enzyme composition for preparing fucosyllactose and fucosidase Eo3066, wherein the fucosidase Eo3066 is derived from the aquatic bacterium Emticicia oligotrophica, and the GenBank annotation number is UniProt ID I2ERT6. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 shows the SDS-PAGE analysis results of the related enzymes in the one-pot multi-enzyme method.

[0037] FIG2 is a TLC detection of the SlSUS enzymatic reaction product.

[0038] FIG3 shows the UPLC detection of SeC2E enzymatic reaction products.

[0039] FIG4 shows UPLC detection of SeGMD and SeGER enzymatic reaction products.

[0040] FIG5 is an analysis of HpFucT enzymatic reaction products based on UPLC.

[0041] FIG6 shows the results of HpFucT enzyme coupling analysis of GDP-fucose products.

[0042] FIG7 is the SDS-PAGE analysis result of the enzymes involved in the one-pot multi-enzyme method.

[0043] FIG8 is a TLC analysis of the S1SUS enzymatic reaction products.

[0044] FIG9 shows UPLC detection of SeC2E enzymatic reaction products.

[0045] FIG10 shows the UPLC detection results of the enzymatic reaction products of SeGMD, SeGER, and Hp13FucT2.

[0046] FIG11 shows the analysis results of the enzymatic reaction products of Hp13FucT2 based on UPLC.

[0047] FIG12 is a diagram showing the liquid phase separation results after improving the elution procedure.

[0048] FIG13 is a graph showing the LC-MS detection results of the reaction system after separation by a preparative column.

[0049] FIG14 is a graph showing the SDS-PAGE results of SeC2E S122A and SeC2E Y165F.

[0050] FIG15 is a graph showing the effect of site-directed mutation of SeC2E on conversion rate.

[0051] Figure 16 shows a fucosidase activity assay. A 15 mL 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 negative control was performed without recombinant enzyme. The negative control (-) reaction system contained no enzyme.

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

[0053] Figure 18 shows the enzymatic release of fucose by concentrated fucosidase. Purified fucosidase was concentrated fivefold and then reacted in a 50 μL reaction system at 37°C for 16 hours. Eo3066 fucosidase completely hydrolyzes pNP-fucosyllactose, releasing fucose.

[0054] Best Mode for Carrying Out the Invention

[0055] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples 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. Transform the recombinant plasmid into E. coli BL21

[0058] This application discovered genes for enzymes related to the synthesis of GDP-fucose (SUS, C2E, GMD, GER) from bacteria of different sources; sucrose synthase (SlSUS) that decomposes sucrose comes from Solanum lycopersicum; CDP-tyrosine 2-epimerase (SeC2E) that can diisomorphize glucose after nucleotide activation into mannose comes from Salmonella enterica; GDP-mannose dehydratase (SeGMD) and isomeroreductase (SeGER) that can convert GDP-mannose into GDP-fucose also come from Salmonella enterica. The sucrose synthase SlSUS (NP_001234655.2), CDP-tyvetose 2-epimerase SeC2E (DW4091412.1), GDP-mannose dehydratase SeGMD (ACN45760.1) and isomeroreductase SeGER (WP_001041701.1) gene sequences were obtained from the database and synthesized and cloned into the pET30a plasmid, respectively, by blunt-end ligating the coding sequence of SlSUS to the multiple cloning site of pET30a to obtain pET30a-SlSUS, ligating the coding sequence of SeC2E between the Nde I / Xho I sites of pET30a (replacing the sequence between the original sites) to obtain pET30a-SeC2E, and ligating the coding sequence of SeGMD to the Kpn I / Xho I sites of pET30a to obtain pET30a- The SeGER coding sequence was ligated between the Kpn I and Xho I sites of pET30a to obtain pET30a-SeGMD. The SeGER coding sequence was ligated between the Kpn I and Xho I sites of pET30a to obtain pET30a-SeGER. The four recombinant plasmids (pET30a-S1SUS, pET30a-SeC2E, pET30a-SeGMD, and pET30a-SeGER) were transformed into E. coli BL21, respectively, using the following procedure:

[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) After 25 minutes of standing, adjust the water bath temperature and quickly place the dish in a 42°C water bath for 1 minute. Remove the dish and quickly place it back in ice. Let it stand for 2 minutes to reduce the damage of E. coli.

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

[0063] (5) Evenly add the shaken cultured bacterial solution to LB solid medium containing 0.1 mg / mL kanamycin and spread it. Place the plate upside down in a 37°C constant temperature incubator and incubate for 12-16 hours until the plaques grow into single colonies suitable for picking.

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

[0065] (1) Pick a single colony transferred into E. coli BL21 and inoculate it into 5 mL of 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, sterilized LB liquid medium for expansion. When the OD600 of the fermentation broth reaches between 0.5 and 0.8, add IPTG solution to a final IPTG concentration of 1 mmol / L. Induce expression overnight at 18°C ​​and 250 rpm.

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

[0068] 3. Affinity chromatography column purification of recombinant nickel vector

[0069] (1) Transfer the IPTG-induced bacterial suspension to a centrifuge bottle, centrifuge at 4000 rpm for 15 min at 4°C, and discard the supernatant.

[0070] (2) Add 10 mL of Lysis Buffer to the induced cells and pipette thoroughly to dissolve the cells and remove any precipitated particles. Transfer the mixture to a 50 mL centrifuge tube and add 100 μL of 100 mM PMSF (phenylmethylsulfonyl fluoride). Ultrasonicate for 20 min to disrupt the cells and release the target protein. Ultrasonication conditions: 1.5 s for disruption / 2.5 s for rest, 15 min, 200 W power.

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

[0072] Preparation of cell lysis buffer: Weigh 6.06g Tris and 5.84g sodium chloride and dissolve them in 800mL ultrapure water. Add 10mL Triton X-100, stir well, adjust the pH to 8.0 with hydrochloric acid, and dilute to 1L. Store at 4°C. Prepare various nickel affinity chromatography buffers due to their short half-life, adding them just before disruption. Prepare the various reagents as follows: ① Washing buffer: Weigh 6.06g Tris and 2.92g sodium chloride and dissolve them in 800mL ultrapure water. Adjust the pH to 8.0 with dilute HCl, and finally dilute to 1L with ultrapure water. Store in a refrigerator at 4°C. ② Protein elution buffer: Weigh 3.03g Tris, 1.46g sodium chloride, and 17.02g imidazole and dissolve them in 400mL ultrapure water. Adjust the pH to 8.0 with hydrochloric acid, and dilute to 500mL. Store at room temperature.

[0073] 4. Nickel column purification

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

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

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

[0077] (3) Elute the recombinant protein with protein eluent and collect about 15 tubes of chromatographic column eluate, 1 mL per tube. Measure the A280 value of each collected tube of eluate. Add 20%-25% volume of glycerol to the eluate with a relatively high A280 value, and aliquot 100 μL per tube. Freeze in liquid nitrogen and place in a -80°C ultra-low temperature freezer for later use.

[0078] (4) When the A280 of the eluted solution no longer decreases, the column is re-equilibrated with the protein binding solution, and then the column is stored in 20% ethanol.

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

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

[0081] Prepare the buffer, staining solution, and decolorizing solution required for SDS-PAGE. The solution formulas are as follows:

[0082] SDS-PAGE running buffer: Weigh 3.05 g of Tris and 31.3 g of glycine and dissolve them in 800 mL of ultrapure water. Add 50 mL of 10% SDS solution and adjust the volume to 1 L with ultrapure water. 5× loading buffer: Weigh 0.39 g of DTT, 0.5 g of SDS, and 0.025 g of bromophenol blue and dissolve them in 2.5 mL of glycerol and 2.5 mL of 0.25 M Tris / HCl (pH 6.8). Aliquot into 1.5 mL EP tubes and store at 4°C. Coomassie Brilliant Blue R-250 staining solution (1 L): 2 g of Coomassie Brilliant Blue R-250, 400 mL of methanol, 100 mL of glacial acetic acid, and 500 mL of water. Destaining solution (1 L): Mix 100 mL of glacial acetic acid, 450 mL of ethanol, and 450 mL of water. Clean the glass plate and SDS-PAGE gel preparation apparatus and assemble and test for leaks. Prepare different separation gels based on protein quality. Proteins of different molecular weights require different gel ratios. A 10% separation gel is suitable for detecting proteins with a molecular weight of 30-90 kDa, while a 12% separation gel is suitable for detecting proteins with a molecular weight of 20-80 kDa. Pour the SDS-PAGE gel into the gel preparation apparatus. Seal the separation gel with water to prevent uneven distribution. Once it has solidified, pour the stacking gel. After 30-60 minutes, when a visible boundary appears between the separation gel and the aqueous layer, discard the top layer of the separation gel. Prepare the separation gel and add it to the plywood. Quickly insert a comb of an appropriately sized gel. Let the entire SDS-PAGE gel rest for approximately 30 minutes. Place it in the electrophoresis tank and soak for a few minutes before slowly removing the comb.

[0083] Add 100 and 400 μL of water to the pre- and post-induction samples, respectively, and pipette to mix thoroughly. Take 20 μL of each. Take 2 μL of the lysate supernatant and mix with 18 μL of water. Take the pellet and mix it up to 20 μL with water at the appropriate concentration. For the spring flower protein, add 5 μL of 5× loading buffer based on its OD value (OD x volume = 20). Inactivate all samples at 95°C for 10 minutes to completely denature the proteins. Add a protein marker, pre- and post-induction samples, lysate supernatant, and purified enzyme samples to the wells of an SDS-PAGE gel in that order. Run the electrophoresis at 120 volts until the blue indicator band just emerges from the separating gel. Remove the gel and stain the gel with Coomassie Brilliant Blue R-250 for approximately 30 minutes. Discard the Coomassie Brilliant Blue and destain with destaining buffer, changing the buffer every two hours until the bands are clearly visible. The gel was scanned and the results were observed as shown in Figure 1. The results in Figure 1 show that the molecular masses indicated by the four bands are consistent with the theoretical values ​​(the theoretical values ​​of S1SUS, SeC2E, SeGMD and SeGER are 96.6 kDa, 37.9 kDa, 42.3 kDa and 36.2 kDa, respectively).

[0084] Example 2: Desalting after purification of four recombinant enzymes

[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 in the disposable desalting column PD-10;

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

[0088] (3) 2.5 ml of the mixture purified by nickel affinity chromatography was added to a desalting column to allow free elution;

[0089] (4) Rinse the column with 3 ml of buffer and collect the target protein based on 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 S1SUS enzymatic reaction products

[0092] Sucrose synthase catalyzes the breakdown of sucrose into glucose and fructose, and reacts glucose with GDP to form GDP-glucose. The theoretical catalytic mechanism of SlSUS indicates that the optimal nucleotide is uridine diphosphate (UDP), with the weakest affinity for guanosine diphosphate (GDP). The reaction system consists of: 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), 1 μL 2 mM MgCl2, 20 μL SlSUS (2.0 mg / mL), and 6.5 μL ultrapure water. The reaction is carried out at 37°C. After completion, a small amount of the mixture is centrifuged and diluted. A 1 μL droplet is placed under the plate, air-dried, and placed in a chromatography chamber. The TLC developing solvent is formulated as n-butanol / ethanol / water = 5 / 3 / 2 (v / v / v). The developing solvent is mixed and added to the chromatography chamber in advance to fully saturate the column. Because GDP itself has conjugated double bonds visible under UV light, as shown in Figure 2, the blank control, in which SUS was replaced with water, showed only GDP with no additional substances produced. However, reaction system b, in which SlSUS was added, showed the production of new substances. Sucrose and fructose are invisible under UV light and can only be stained with orcinol. After the TLC plate was stained with orcinol and air-dried, Figure 2A shows that the reaction system with SlSUS successfully decomposed sucrose into fructose, while no fructose was produced in the blank control reaction system. Combined with UV detection (Figure 2B), this demonstrates that SlSUS has the ability to decompose sucrose and utilize GDP to produce GDP-glucose.

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

[0094] The C2E theoretical catalytic principle shows that the affinity for cytidine diphosphate glucose (CDP-Glc) is the highest, while the affinity for guanosine diphosphate (GDP) activated glucose is the lowest. The reaction system is: 3 μL of the supernatant of 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, 3 μL of 2 mM NAD +(Shanghai Ruji Biotechnology Development Co., Ltd.), 18 μL SeC2E (22 mg / mL). The reaction was allowed to proceed overnight at 45°C. After completion, a small amount of the mixture was centrifuged and diluted. 50 μL was pipetted into a sample vial and assayed according to the SeC2E enzyme activity assay described above. Figure 3 shows that when GDP-mannose was used as the substrate, the addition of SeC2E to the reaction system significantly decreased the GDP-mannose (GDP-Man) peak, and a new peak appeared at 3 minutes. In the blank control reaction, the GDP-mannose peak remained unchanged, with no new peaks generated. When the mixture was assayed with the SlSUS reaction mixture overnight, a strong peak appeared at the same peak position. It is speculated that the peak following GDP-mannose is GDP-glucose (GDP-Glc). Using this concentrated mixture as the substrate, SeC2E was added again and the reaction was continued overnight under the same optimal reaction conditions. UPLC analysis revealed a significant decrease in the GDP-glucose peak, while the GDP-mannose product peak appeared. This indicates that SeC2E has catalytic activity towards 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 form GDP-4-dehydro-6-deoxy-mannose. SeGER, with the cofactor NADPH, then catalyzes the conversion of GDP-4-dehydro-6-deoxy-mannose to GDP-L-fucose (GDP-L-Fuc). To test the activity of SeGMD and SeGER, fucosyltransferase HpFucT and pNP-lactose were added to the reaction system. The SeGMD and SeGER reaction product, GDP-L-Fuc, was converted to pNP-fucosyllactose, which was then detected by UPLC.

[0097] The fucosyltransferase HpFucT (SEQ ID NO: WP_000487428.1) gene from Helicobacter pylori was synthesized and cloned into the Nde I / Xho I sites of pET30a. Escherichia coli was transformed and expressed and purified according to the methods of Examples 1 and 2 to obtain HpFucT for analysis of coupled enzymatic reaction products.

[0098] The SeGMD and SeGER reaction system consisted of 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 Kabosens Biotechnology Co., Ltd.), 6 μl of SeGMD, 4 μl of SeGER, and 15 μl of HpFucT. The reaction was incubated overnight at 37°F. After completion, the reaction was terminated by heating at 95°C for 10 minutes and centrifuged. The supernatant was diluted, filtered, and 50 μL was pipetted into a sample vial for analysis. The UPLC results are shown in Figures 2-6. Compared to the blank control without SeGMD, SeGER, or HpFucT, only the pNP-lactose substrate peak was detected, with no new peaks generated. However, the reaction system with SeGMD, SeGER, and HpFucT produced a new peak 0.3 minutes before the pNP-lactose peak. The samples were detected by MS using ESI (Shimadzu Corporation) in positive ion scan mode with an m / z range of 200-1000. The separation results, Figures 4 and 5, are consistent with the UPLC results. The substrate peak near 6 min contained the highest mass-to-charge ratio of 486 [M+Na]+, which corresponds exactly to the mass-to-charge ratio of pNP-lactose plus Na+. + The molecular weight of the new peak is 463+23. The new peak contains a mass-to-charge ratio of 632 [M+Na] + Go long, corresponding to the mass-to-charge ratio of pNP-fucosyllactose plus Na + The molecular weight of SeGMD is 609+23, which 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 form pNP-fucosyllactose, thus proving that both GMD and GER are active and can catalyze the synthesis of GDP-fucose.

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

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

[0101] Example 7: Mining, expression, purification and activity identification of enzymes related to fucosyllactose preparation

[0102] A sucrose synthase (NP_001234655.2) that can decompose sucrose and transfer one molecule of glucose to GDP to generate GDP-glucose was found in the Solanum lycopersicum (tomato) genome database. enterica (Salmonella enterica) genome database was mined to identify CDP-tyvetose 2-epimerase (GenBank sequence number DW4091412.1) that can diisomorphize GDP-glucose into GDP-mannose, as well as GDP-mannose dehydratase (GenBank sequence number ACN45760.1) and isomeroreductase (GenBank sequence number WP_001041701.1) that convert GDP-mannose into GDP-fucose; similarly, fucosyltransferase (GenBank sequence number WP_000487428.1) that can transfer fucosyl to lactose donor to form fucosyllactose was mined from the Helicobacter pylori (Helicobacter pylori) genome database.

[0103] A series of molecular biology techniques are used to recombinantly express the genes for all the enzymes required for the reactions in the prokaryotic host, E. coli. The enzymes are purified using nickel chromatography columns and analyzed using SDS-PAGE. Furthermore, thin-layer chromatography, ultra-high performance liquid chromatography, and liquid chromatography-mass spectrometry are used to identify the products of the enzymatic reactions. The specific steps are as follows:

[0104] 2. Transform the recombinant plasmid into E. coli BL21

[0105] Based on the genes of enzymes related to the synthesis of GDP-fucose (SUS, C2E, GMD, GER, Hp13FucT2) from bacteria of different sources, the authors discovered that sucrose synthase (SlSUS) that decomposes sucrose comes from Solanum lycopersicum; CDP-tyrosine 2-epimerase (SeC2E) that can diisomorphize glucose into mannose after activation of nucleotides comes from Salmonella enterica; GDP-mannose dehydratase (SeGM D) and isomeroreductase (SeGER) that can convert GDP-mannose into GDP-fucose also come from Salmonella enterica. The sucrose synthase SlSUS (NP_001234655.2), CDP-tyveline 2-epimerase SeC2E (DW4091412.1), GDP-mannose dehydratase SeGMD (ACN45760.1), isomeroreductase SeGER (WP_001041701.1) and fucosyltransferase Hp13FucT2 (WP_000487428.1) gene sequences were obtained from the database and synthesized and cloned into the pET30a plasmid, respectively, by blunt-end ligating the coding sequence of SlSUS to the multiple cloning site of pET30a to obtain pET30a-SlSUS, ligating the coding sequence of SeC2E between the Nde I / Xho I sites of pET30a (replacing the sequence between the original sites) to obtain pET30a-SeC2E, and ligating the coding sequence of SeGMD to the Kpn I site of pET30a. The coding sequence of SeGER was ligated between the Kpn I and Xho I sites of pET30a to obtain pET30a-SeGMD. The coding sequence of Hp3 / 4FucT was ligated between the Nde I and Xho I sites of pET30a to obtain pET30a-SeGER. The coding sequence of Hp3 / 4FucT was ligated between the Nde I and Xho I sites of pET30a to obtain pET30a-Hp3 / 4FucT. The four recombinant plasmids (pET30a-SlSUS, pET30a-SeC2E, pET30a-SeGMD, pET30a-SeGER, and pET30a-Hp3 / 4FucT) were transformed into E. coli BL21. The specific process is 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) After 25 minutes of standing, adjust the water bath temperature and quickly place the dish in a 42°C water bath for 1 minute. Remove the dish and quickly place it back in ice. Let it stand for 2 minutes to reduce the damage of E. coli.

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

[0110] (5) Evenly add the shaken cultured bacterial solution to LB solid medium containing 0.1 mg / mL kanamycin and spread it. Place the plate upside down in a 37°C constant temperature incubator and incubate for 12-16 hours until the plaques grow into single colonies suitable for picking.

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

[0112] (1) Pick a single colony transferred into E. coli BL21 and inoculate it into 5 mL of 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, sterilized LB liquid medium for expansion. When the OD600 of the fermentation broth reaches between 0.5 and 0.8, add IPTG solution to a final IPTG concentration of 1 mmol / L. Induce expression overnight at 18°C ​​and 250 rpm.

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

[0115] 3. Affinity chromatography column purification of recombinant nickel vector

[0116] (1) Transfer the IPTG-induced bacterial suspension to a centrifuge bottle, centrifuge at 4000 rpm for 15 min at 4°C, and discard the supernatant.

[0117] (2) Add 10 mL of Lysis Buffer to the induced cells and pipette thoroughly to dissolve the cells and remove any precipitated particles. Transfer the mixture to a 50 mL centrifuge tube and add 100 μL of 100 mM PMSF (phenylmethylsulfonyl fluoride). Ultrasonicate for 20 min to disrupt the cells and release the target protein. Ultrasonication conditions: 1.5 s for disruption / 2.5 s for rest, 15 min, 200 W power.

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

[0119] Preparation of cell lysis buffer: Weigh 6.06g Tris and 5.84g sodium chloride and dissolve them in 800mL ultrapure water. Add 10mL Triton X-100, stir well, adjust the pH to 8.0 with hydrochloric acid, and dilute to 1L. Store at 4°C. Prepare various nickel affinity chromatography buffers due to their short half-life, adding them just before disruption. Prepare the various reagents as follows: ① Washing buffer: Weigh 6.06g Tris and 2.92g sodium chloride and dissolve them in 800mL ultrapure water. Adjust the pH to 8.0 with dilute HCl, and finally dilute to 1L with ultrapure water. Store in a refrigerator at 4°C. ② Protein elution buffer: Weigh 3.03g Tris, 1.46g sodium chloride, and 17.02g imidazole and dissolve them in 400mL ultrapure water. Adjust the pH to 8.0 with hydrochloric acid, and dilute to 500mL. Store at room temperature.

[0120] 4. Nickel column purification

[0121] The recombinant protein containing 6×His-tag was purified by Ni 2+ The purification was performed by affinity chromatography, and the purification steps were as follows:

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

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

[0124] (3) Elute the recombinant protein with protein eluent and collect about 15 tubes of chromatographic column eluate, 1 mL per tube. Measure the A280 value of each collected tube of eluate. Add 20%-25% volume of glycerol to the eluate with a relatively high A280 value, and aliquot 100 μL per tube. Freeze in liquid nitrogen and place in a -80°C ultra-low temperature freezer for later use.

[0125] (4) When the A280 of the eluted solution no longer decreases, the column is re-equilibrated with the protein binding solution, and then the column is stored in 20% ethanol.

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

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

[0128] Prepare the buffer, staining solution, and decolorizing solution required for SDS-PAGE. The solution formulas are as follows:

[0129] SDS-PAGE running buffer: Weigh 3.05 g of Tris and 31.3 g of glycine and dissolve them in 800 mL of ultrapure water. Add 50 mL of 10% SDS solution and adjust the volume to 1 L with ultrapure water. 5× loading buffer: Weigh 0.39 g of DTT, 0.5 g of SDS, and 0.025 g of bromophenol blue and dissolve them in 2.5 mL of glycerol and 2.5 mL of 0.25 M Tris / HCl (pH 6.8). Aliquot into 1.5 mL EP tubes and store at 4°C. Coomassie Brilliant Blue R-250 staining solution (1 L): 2 g of Coomassie Brilliant Blue R-250, 400 mL of methanol, 100 mL of glacial acetic acid, and 500 mL of water. Destaining solution (1 L): Mix 100 mL of glacial acetic acid, 450 mL of ethanol, and 450 mL of water. Clean the glass plate and SDS-PAGE gel preparation apparatus and assemble and test for leaks. Prepare different separation gels based on protein quality. Proteins of different molecular weights require different gel ratios. A 10% separation gel is suitable for detecting proteins with a molecular weight of 30-90 kDa, while a 12% separation gel is suitable for detecting proteins with a molecular weight of 20-80 kDa. Pour the SDS-PAGE gel into the gel preparation apparatus. Seal the separation gel with water to prevent uneven distribution. Once it has solidified, pour the stacking gel. After 30-60 minutes, when a visible boundary appears between the separation gel and the aqueous layer, discard the top layer of the separation gel. Prepare the separation gel and add it to the plywood. Quickly insert a comb of an appropriately sized gel. Let the entire SDS-PAGE gel rest for approximately 30 minutes. Place it in the electrophoresis tank and soak for a few minutes before slowly removing the comb.

[0130] Add 100 and 400 μL of water to the pre- and post-induction samples, respectively, and pipette to mix thoroughly. Take 20 μL of each. Take 2 μL of the lysate supernatant and mix with 18 μL of water. Take the pellet and mix it up to 20 μL with water at the appropriate concentration. For the spring flower protein, add 5 μL of 5× loading buffer based on its OD value (OD x volume = 20). Inactivate all samples at 95°C for 10 minutes to completely denature the proteins. Add a protein marker, pre- and post-induction samples, lysate supernatant, and purified enzyme samples to the wells of an SDS-PAGE gel in that order. Run the electrophoresis at 120 volts until the blue indicator band just emerges from the separating gel. Remove the gel and stain the gel with Coomassie Brilliant Blue R-250 for approximately 30 minutes. Discard the Coomassie Brilliant Blue and destain with destaining buffer, changing the buffer every two hours until the bands are clearly visible. The gel was scanned and the results were observed as shown in Figure 7 ; Figure 7 shows that the molecular masses indicated by the four bands are consistent with the theoretical values ​​(SlSUS, SeC2E, SeGMD, SeGER, and Hp13FucT2 are 96.6 kDa, 37.9 kDa, 42.3 kDa, 36.2 kDa, and 42.5 kDa, respectively).

[0131] The protein concentrations of the purified 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: Synthesis of fucosyllactose by a one-pot multi-enzyme method

[0133] 1) Mix 5 μL of Tris-HCl (500 mM pH = 7.5), 5 μL of GDP solution (100 mM), 5 μL of sucrose solution (1 M), 1 μL of MgCl2 solution (100 mM), and 14 μL of SlSUS enzyme solution (2.0 mg / mL), mix well, and react at 37°C for 12 h.

[0134] 2) Then add 20 μL SeC2E enzyme solution (22 mg / mL) and 4 μL NAD + solution (20 mM), mixed well, and reacted at 45 °C for 12 h;

[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-fucosyllactose was detected using a UPLC-MS-based method. A 40 μL reaction mixture was heated at 95°C for 10 minutes to completely denature the protein. The inactivated reaction system was then centrifuged at 12,000 rpm for 10 minutes to separate the denatured protein and supernatant. The supernatant was removed and diluted fivefold with ultrapure water. The reaction system, an enzyme-free blank control (no SeGMD, SeGER, or Hp13FucT2 enzyme added, all other steps remained unchanged), and a pNP-lactose standard as a negative control were sequentially added to different vials. Each experimental group was set up in triplicate. The results are shown in Figure 10. Compared to the blank control without SeGMD, SeGER, or Hp13FucT2 enzyme, only the pNP-lactose substrate peak was detected, with no new peak generated. However, the reaction system with SeGMD, SeGER, and Hp13FucT2 enzyme added generated a new peak 0.3 minutes before the pNP-lactose peak. The sample was detected by ESI ionization in MS mode, with the positive ion scan mode and the m / z range set between 200-1000. The separation results are shown in Figure 11 and are the same as the UPLC results. The substrate peak near 6 min contains a mass-to-charge ratio of 486 [M+Na] + At most, it corresponds to the mass-to-charge ratio of pNP-lactose plus Na + The molecular weight of the new peak is 463+23. The new peak contains a mass-to-charge ratio of 632 [M+Na] + Go long, corresponding to the mass-to-charge ratio of pNP-fucosyllactose plus Na + The molecular weight of the product was 609+23, which proved that SeGMD, SeGER and Hp3 / 4FucT enzymes were active and could successfully catalyze GDP-mannose and pNP-lactose to generate pNP-fucosyllactose.

[0137] The above UPLC-MS analysis was performed in series with a Shimadzu Nexera liquid chromatograph and MS2020. The system includes 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. HyperCloneTM-ODS C18 reverse phase chromatography column (5μm, The column was equilibrated using a 250×4.6 mm column (250×4.6 mm). The proportion of mobile phase B was increased from 20% to 60% over 0.01-5 minutes, then rapidly increased to 90% over the next minute and held for 2 minutes. The mobile phase B ratio was then rapidly decreased to 20% over the next minute and held for 6 minutes. The liquid effluent from the liquid chromatography was immediately fed into the mass spectrometer. The dry nitrogen flow rate was 10 L / min, the nebulizing gas flow rate was 1.5 L / min, and the DL temperature was 250°C. Positive ion scan mode was used over the m / z range of 200-1000. Single particle detection (SIM) scanning mode was used to verify the molecular mass consistency of the substrate and product peaks.

[0138] Example 9. Preparation, separation and purification of pNP-fucosyllactose

[0139] 1. Preparation of pNP-fucosyllactose

[0140] The original reaction system of 85 μL was expanded to 100 mL in equal proportions. 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 evenly and reacted at 37 ° C for 12 h; then 23.53 mL of SeC2E enzyme solution and 4.7 mL of NADPH were added to the above reaction solution. + Solution (20 mM) was added, mixed evenly, 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 evenly, and reacted at 25°C for 12 h. The required mixture of the above reaction system was added to a 250 mL shake flask and incubated on a shaker. The temperature varied with the order of enzyme addition. The shaker speed was adjusted to 100 rpm to ensure sufficient contact between the protease and the substrate. After the reaction was completed, a reaction mixture was obtained.

[0141] 2. Isolation and Purification of pNP-fucosyllactose

[0142] The reaction mixture was heated at 95°C in a water bath for 10 minutes to terminate the reaction. The mixture was cooled to room temperature and centrifuged in a 50ml centrifuge tube 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 by pipetting and centrifuged to obtain the supernatant. This was repeated 2-3 times to reduce product loss. The supernatant obtained in the above step was collected into a lyophilization bottle and lyophilized in a freeze dryer. Separation was performed using an HPLC preparative column. The preparative column separation method was based on a C18 column with a specification of 250mm*4.6mm*5μm. The elution program was 75 minutes. Within 0.01-40 minutes, the proportion of mobile phase B was increased from 10% to 15%, then rapidly increased to 90% within the next minute, maintained for 9 minutes, and then rapidly decreased to 10% within 5 minutes, maintained for 20 minutes. Mobile phase A consisted of 50 mM ammonium formate (pH 4.5) and mobile phase B consisted of 100% acetonitrile. The preparative column could be loaded with up to 1 ml of sample, resulting in an internal volume 18.9 times that of a basic C18 column. To maintain the original column pressure, the flow rate was increased from 1 ml / min to 5 ml / min, and UV detection was performed at a wavelength of 300 nm. The results, shown in Figure 12, show that with a total elution time of 75 minutes, two similar peaks appeared at 36 and 38.5 minutes. These two peaks were collected in separate tubes and analyzed again by UPLC. The results, shown in Figure 13, indicate that the peaks corresponding to 36 and 38.5 minutes are the substrate pNP-lac and the product pNP-FL, respectively. This demonstrates that the use of a preparative column in a 75-minute elution time can extend the time difference between the product and substrate peaks, enabling better separation of pNP-lac and pNP-FL.

[0143] Comparative Example 1: Discovering Suitable FucT in a One-Pot Multi-Enzyme Method

[0144] Using SlSUS, SeC2E, SeGMD, and SeGER as the first four immobilized enzymes in the basic reaction system, with the substrate and coenzyme remaining unchanged, the activity of Hp13FucT2 (prepared in Example 7) and Hp13FucT1 (GenBank sequence numbers: O30511.1 and 2NZW_A; expression and purification methods were the same as in Example 7) as the final enzyme in the one-pot multi-enzyme method was measured using UPLC with a UV detector at a wavelength of 300 nm. Figure 11 shows that both Hp13FucT2 and Hp13FucT1 transfer fucosyl groups to a lactose donor using pNP-lactose as a substrate, generating pNP-FL. The conversion rate was obtained by calculating the peak value. The overall conversion rate of the reaction system using Hp13FucT2 as the fucosyltransferase reached 20%, while the conversion rate of the system using Hp13FucT1 as the fucosyltransferase was less than 5%. Therefore, Hp13FucT2 was selected as the fucosyltransferase of this application.

[0145] Comparative Example 2: Effect of site-directed mutation of the C2E catalytic site on the one-pot multi-enzyme synthesis of FL

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

[0147] The two mutants were substituted for SeC2E in the original reaction system. Mass spectrometry analysis revealed the following results, as shown in Figure 15. Wild-type SeC2E clearly exhibited the peak of the target product, pNP-FL, in the reaction system. Mutating position 122 did not alter the enzyme's activity, resulting in the appearance of the product peak at the same time and a conversion rate nearly similar to that of wild-type SeC2E. However, site-directed mutagenesis at position 165 inactivated the enzyme, making the formation of pNP-FL undetectable. Therefore, wild-type SeC2E was used for the epimerization of GDP-glucose in the single-pot, multi-enzyme method.

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

[0149] 1. Transform the recombinant plasmid into E. coli BL21

[0150] This application discovered a fucosidase (GenBank annotation number: UniProt ID I2ERT6) from the aquatic bacterium Emticicia oligotrophica. The gene sequence of fucosidase Eo3066 (shown in SEQ ID NO: 1) was synthesized and replaced with the sequence between the Nde I / Xho I sites of the pET30a plasmid, leaving other sequences unchanged, to obtain the recombinant plasmid pET30a-Eo3066. The recombinant plasmid was then transformed into Escherichia 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) After 25 minutes of standing, adjust the water bath temperature and quickly place the dish in a 42°C water bath for 1 minute. Remove the dish and quickly place it back in ice. Let it stand for 2 minutes to reduce the damage of E. coli.

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

[0155] (5) Evenly add the shaken cultured bacterial solution to LB solid medium containing 0.1 mg / mL kanamycin and spread it. Place the plate upside down in a 37°C constant temperature incubator and incubate for 12-16 hours until the plaques grow into single colonies suitable for picking.

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

[0157] (1) A single colony transformed with E. coli BL21 (i.e., E. coli BL21-Eo3066 containing the recombinant plasmid) was selected and inoculated into 3 mL of LB liquid medium. 3 μL of 50 mg / mL kanamycin was added and the culture was incubated overnight at 37°C and 250 rpm in a constant temperature shaker.

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

[0159] (3) After the incubation period, the cells were collected by centrifugation (5000 g, 15 min, 4°C) and resuspended in 10 mL of lysis buffer (100 mM NaCl, 50 mM Tris, 1% Triton X-100, and 1 mM phenyl-methylsulfonyl fluoride, pH 8.0). The cells were ultrasonically lysed in an ice bath for 20 min (40 on / off cycles with 20 μm amplitude for 15 s). The cells were then centrifuged (20000 g, 20 min, 4°C) to remove cell debris and collect the cell lysate.

[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 and stir well. Adjust the pH to 8.0 with hydrochloric acid, make up to 1 L, and store at 4°C.

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

[0162] The cell lysate was loaded onto a nickel column (Ni 2+ -nitrilotriacetate agarose affinity chromatography column, column bed volume 2 mL) (Qiagen). After loading, unadsorbed proteins were fully washed with 50 mL of wash buffer (50 mM Tris-HCl, 50 mM NaCl, pH 8.0). The recombinant protein was eluted with elution buffer (300 mM imidazole, 50 mM Tris-HCl, 50 mM NaCl, pH 8.0). The volume of the eluate containing the target protein was approximately 4 mL, which could be directly used for subsequent enzyme activity analysis.

[0163] Various reagents were prepared as follows: ① Washing and desorption buffer: Weigh 6.06 g of Tris and 2.92 g of sodium chloride and dissolve them in 800 mL of ultrapure water, adjust the pH to 8.0 with dilute HCl, and finally dilute to 1 L with ultrapure water and store in a refrigerator at 4°C; ② Elution buffer: Weigh 3.03 g of Tris, 1.46 g of sodium chloride, and 17.02 g of imidazole and dissolve them in 400 mL of ultrapure water, adjust the pH to 8.0 with hydrochloric acid, dilute to 500 mL, and store at room temperature.

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

[0165] The fucosidase activity of the purified recombinant enzyme Eo3066 was detected when pNP-α-L-fucose was used as substrate.

[0166] Fucosidase activity assay: A 15-L reaction solution contained 8.5 μL of PBS buffer (pH 7.4), 1.5 μL of pNP-α-L-fucose (10 mM stock solution) (Sigma), and 5 μL of purified recombinant fucosidase. A negative control (-) was used without recombinant enzyme. The results are shown in Figure 16. The results demonstrate that recombinant enzyme Eo3066 exhibits 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 in the disposable desalting column PD-10;

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

[0170] (3) 2.5 ml of the mixture purified by nickel affinity chromatography was added to a desalting column to allow free elution;

[0171] (4) Rinse the column with 3 ml of buffer and collect the target protein based on 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 Fucosidase Eo3066-catalyzed Reaction Products

[0174] A 50 μL reaction system included 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 incubated at 37°C for 16 hours. A control reaction was performed without fucosidase Eo3066. After completion of the reaction, the reaction product was analyzed on 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 at 300 nm. Elution conditions were a 6-minute gradient of 20-60% (volume) acetonitrile in 50 mM ammonium formate buffer (pH 4.5). As shown in Figure 17, fucosidase Eo3066 hydrolyzed pNP-fucosyllactose (pNP-fucosyllactose) to release fucose.

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

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

[0177] A 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, and was incubated at 37°C for 16 hours. The enzymatic reaction was carried out at 37°C for 16 hours. A control reaction without fucosidase Eo3066 was used. As shown in Figure 18 , 5 mg / mL Eo3066 fucosidase completely hydrolyzed the pNP-fucosyllactose in the reaction system, releasing L-fucose.

[0178] CROSS-REFERENCE TO RELATED APPLICATIONS

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

[0180] Industrial Applications

[0181] 1. The large intestine expression system used in this application has the characteristics of high expression efficiency, large expression amount, low cost, and easy operation. The four enzymes expressed in the cascade reaction have the advantages of high purity and high yield. This application provides a new method for preparing low-cost GDP-fucose in vitro using guanosine diphosphate and sucrose as raw materials and cascade catalysis of four enzymes. By utilizing the E. coli expression system and cheap guanosine diphosphate and sucrose as raw materials, efficient expression of various enzymes in the cascade catalytic reaction is achieved, thereby achieving low-cost mass production of GDP-fucose; this application not only provides cheap raw materials for the mass production of human milk oligosaccharides, but also provides cheap intermediates for the production of natural oligosaccharide drugs.

[0182] 2. The one-pot multi-enzyme method of the present application for synthesizing fucosyllactose successfully generates 3'-fucosyllactose in vitro using sucrose, GDP, and pNP-lactose as initial substrates. This method is simpler and less time-consuming than whole-cell synthesis, and therefore has broad commercial application value.

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

Claims

1. A method for synthesizing GDP-fucose by multi-stage multi-enzyme cascade catalysis, characterized in that: The steps include: 1) Using guanosine diphosphate and sucrose as substrates, sucrose synthase catalyzes the reaction to generate GDP-glucose; 2) Using GDP-glucose as substrate, GDP-mannose is generated by CDP-Tyvesin 2-epimerase catalysis; 3) Using GDP-mannose as substrate, GDP-fucose is generated through the catalytic reaction of DP-mannose dehydratase and isomeroreductase.

2. The method according to claim 1, characterized in that: The sucrose synthase, CDP-tyvetose 2-epimerase, GDP-mannose dehydratase and isomeroreductase all catalyze in the form of crude enzyme liquid, crude enzyme powder, pure enzyme or whole cell.

3. The method according to claim 1, characterized in that The sucrose synthase is derived from tomato (Solanum locopersicum), and the sequence number of the amino acid sequence is NP_001234655; the CDP-tyveline 2-epimerase is derived from Salmonella enterica, and the sequence number of the amino acid sequence is DW4091412.1; the GDP-mannose dehydratase is derived from Salmonella enterica, and the sequence number of the amino acid sequence is ACN45760.1; the isomeroreductase is derived from Salmonella enterica, and the sequence number of the amino acid sequence is WP_001041701.

1.

4. A method for synthesizing fucosyllactose by a one-pot multi-enzyme method, characterized in that: The method comprises the steps of using the GDP-fucose and pNP-lactose prepared in claim 1 as initial substrates to obtain fucosyllactose under the catalysis of Hp13FucT2 enzyme.

5. The method according to claim 4, characterized in that The Hp13FucT2 is a fucosyltransferase derived from Helicbacter pylori, and the GenBank sequence number of the amino acid sequence is WP_000487428.

1.

6. The method according to claim 4, characterized in that The enzymatic synthesis method of fucosyllactose is to use sucrose, GDP and pNP-lactose as initial substrates, and obtain fucosyllactose under the catalytic action of five enzymes: sucrose synthase, CDP-tyvese 2-epimerase, GDP-mannose dehydratase, isomeroreductase and fucosyltransferase.

7. A method for preparing L-fucose by enzymatic method, characterized in that: The fucosyllactose prepared in claim 4 is used as a substrate to generate L-fucose through a fucosidase-catalyzed reaction.

8. The method according to claim 7, characterized in that The fucosidase Eo3066 is derived from the aquatic bacterium Emticicia oligotrophica, and its GenBank annotation number is UniProt ID I2ERT6.

9. A bioenzyme composition for preparing GDP-fucose, characterized in that: The bio-enzyme composition comprises sucrose synthase, CDP-tyveline 2-epimerase, GDP-mannose dehydratase and isomeroreductase; the sucrose synthase is derived from tomato (Solanum locopersicum), and the sequence number of the amino acid sequence is NP_001234655; the CDP-tyveline 2-epimerase is derived from Salmonella enterica, and the sequence number of the amino acid sequence is DW4091412.1; the GDP-mannose dehydratase is derived from Salmonella enterica, and the sequence number of the amino acid sequence is ACN45760.1; the isomeroreductase is derived from Salmonella enterica, and the sequence number of the amino acid sequence is WP_001041701.

1.

10. A bioenzyme composition for preparing fucosyllactose, characterized in that: The invention comprises the bioenzyme composition for preparing GDP-fucose according to claim 9 and fucosyltransferase Hp13FucT2, wherein the fucosyltransferase Hp13FucT2 is derived from Helicobacter pylori, and the GenBank sequence number of the amino acid sequence is WP_000487428.

1.

11. A bioenzyme composition for preparing L-fucose, characterized in that: The invention comprises the biological enzyme composition for preparing fucosyllactose according to claim 10 and fucosidase Eo3066, wherein the fucosidase Eo3066 is derived from the aquatic bacterium Emticicia oligotrophica, and the GenBank annotation number is UniProt ID I2ERT6.

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