A method for detecting fucose content based on fucose dehydrogenase

Abstract

The present invention discloses a method for detecting fucose content based on fucose dehydrogenase. The present invention provides a method for detecting fucose content, comprising the following steps: + Under the action of NAD, it catalyzes the dehydrogenation reaction of free fucose. + The fucose content is detected by ultraviolet absorption of NADH at a wavelength of 340 nm. The present invention realizes accurate, rapid, economical and convenient detection of fucose in samples for the first time through a green and pollution-free method.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for detecting fucose content based on fucose dehydrogenase. 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 fucose existing in nature is mainly L-fucose. L-fucose is widely present in glycoproteins and polysaccharides in humans, animals, plants and microorganisms, such as lactoferrin and breast milk oligosaccharides in human milk, and also in sea urchin and frog eggs, as well as plant polysaccharides in tragacanth gum, potatoes, kiwifruit, soybeans, winged bean varieties, Canadian rapeseed, and extracellular polysaccharides of various seaweeds.

[0003] L-fucose has a variety of biological activities. For example, it has anti-inflammatory effects. Adding an appropriate amount of L-fucose to medicine can play an effective anti-inflammatory role. L-fucose also has the effect of regulating blood lipids and cholesterol. It can replace sucrose and be added to lactic acid drinks, cakes, and cheeses. It can not only keep the original flavor of the drinks, but also meet people's needs for nutrition and health. L-fucose cannot be absorbed by intestinal wall cells, but can be used by intestinal probiotics, thereby increasing the number of probiotics in the intestine and maintaining the balance of intestinal probiotics. It can be added to lactic acid drinks as a prebiotic. Adding an appropriate amount of L-fucose to soy products can also regulate blood lipids and cholesterol in the body and maintain the balance of probiotics in the intestine. In addition, L-fucose is also widely used in foods such as chewing gum, chocolate, various candies, bread, preserved fruits, biscuits, jams, and eight-treasure porridge.

[0004] L-fucose can promote the growth of fibroblasts, thereby avoiding the lack of skin collagen and skin sagging caused by radiation and ultraviolet rays. L-fucose can also reduce the activity of proteolytic enzymes in the skin, thereby reducing damage to the skin. Therefore, the application of L-fucose in cosmetics can play a role in moisturizing the skin, protecting the skin, promoting cell proliferation, increasing skin elasticity, and slowing down skin aging.

[0005] As the most abundant functional oligosaccharide in breast milk, fucosyllactose (FL) has important physiological functions such as prebiotics, anti-infection, immunomodulation, and promotion of infant brain development. It has obvious effects in improving infant immunity, preventing intestinal diseases, and intellectual development. FL has important application value in the food industry. As early as 2015, fucosyllactose was approved by the US Food and Drug Administration and the European Union to be added as a nutritional supplement to infant formula, milk powder and beverages. In addition, in the glycosylation structure of whey protein and lactoferrin, the sugar chain with fucose plays an important role in anti-pathogenic function.

[0006] At present, fucose in glycoproteins and polysaccharides is mainly detected by HPLC and mass spectrometry, which is time-consuming and costly. Developing a rapid, economical and accurate fucose detection method will be beneficial to the rapid detection of fucose in active glycoproteins, glycoprotein drugs and active polysaccharides. Summary of the invention

[0007] The purpose of the present invention is to provide a method for detecting fucose content based on fucose dehydrogenase.

[0008] In a first aspect, the present invention claims a method for detecting fucose content.

[0009] The method for detecting fucose content claimed in the present invention may include the following steps: + Under the action of NAD, it catalyzes the dehydrogenation reaction of free fucose. + Reduced to NADH; the fucose content was detected by ultraviolet absorption of NADH at a wavelength of 340nm.

[0010] Furthermore, the method may include the following steps:

[0011] (A1) Draw a standard curve: Add fucose dehydrogenase, coenzyme NAD into a series of free fucose standard solutions with known concentrations. + and reaction buffer to obtain a series of reaction systems; the series of reaction systems were incubated at 37° C. for 30 min and then OD was detected 340nm value; then according to the concentration of free fucose standard and its corresponding OD 340nm The standard curve is obtained by plotting the concentration of algae standard as the horizontal axis and the OD 340nm Draw a standard curve.

[0012] (A2) After replacing the free fucose standard solution in (A1) with the sample to be tested containing free fucose, the operation is performed according to (A1), and then the OD 340nm The value is substituted into the standard curve obtained in (A1), and the content of free fucose in the sample to be tested is further calculated.

[0013] The sample to be tested containing free fucose may be a sample that originally contains free fucose, or may be obtained by subjecting a fucosylated glycoconjugate sample to acid hydrolysis. In the latter case, the content of fucose in the fucosylated glycoconjugate sample may be detected according to the method.

[0014] Furthermore, the acid hydrolysis may be hydrochloric acid hydrolysis.

[0015] Furthermore, the acid hydrolysis condition may be incubation at 80° C. for 12-16 h (eg, 12 hours).

[0016] In one embodiment of the present invention, acid hydrolysis is performed using 6M hydrochloric acid (incubated at 80° C. for 12-16 hours, such as 12 hours).

[0017] Furthermore, after the acid hydrolysis step, an alkali neutralization step may be further included. In one embodiment of the present invention, the pH value is adjusted to 7-8 using a 6M NaOH solution.

[0018] Furthermore, after the alkali neutralization step, an ultrafiltration centrifugation step (such as 12000 rpm and 4° C. centrifugation) may be included to avoid interference of impurities in the sample on subsequent detection.

[0019] Wherein, the fucosylated glycoconjugate sample can be glycoprotein, oligosaccharide, milk powder or liquid milk. In one embodiment of the present invention, the glycoprotein is horseradish peroxidase (HRP), lactoferrin or amylase.

[0020] Further, in step (A2), the OD of the standard curve obtained in (A1) is substituted 340nm The value is any of the following: (1) When the color of the sample to be tested containing free fucose is the same as that of the free fucose standard solution (both are colorless), substitute the OD 340nm The value is the OD measured after the reaction of the sample containing free fucose 340nm (2) When the color of the sample to be tested containing free fucose is inconsistent with that of the free fucose standard solution (the free fucose standard solution is colorless, and the sample to be tested containing free fucose turns brown due to acid hydrolysis), substitute the OD 340nm The value is the OD measured after the reaction of the sample containing free fucose 340nm The OD values ​​were compared with those of the negative control group. 340nm The negative control group is a control in which the fucose dehydrogenase (not inactivated) is replaced with an equal volume of water or an inactivated fucose dehydrogenase solution (in order to avoid the influence of color background).

[0021] In the method, the fucose dehydrogenase may be any of the following:

[0022] (a1) a protein with an amino acid sequence as shown in SEQ ID No. 1 or positions 1 to 344 of SEQ ID No. 1 or positions 1 to 346 of SEQ ID No. 1;

[0023] (a2) a protein derived from Cyclobacterium marinum having the same function as the amino acid sequence defined in (a1) by substitution and / or deletion and / or addition of one or more amino acid residues;

[0024] (a3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity with the amino acid sequence defined in any one of (a1) to (a2) and having the same function as that of Cyclobacterium marinum;

[0025] (a4) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein defined in any one of (a1) to (a3).

[0026] In the above-mentioned proteins, identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using an identity search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as a matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences, the identity value (%) can be obtained.

[0027] The 80% or more identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The 85% or more identity may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The 90% or more identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The greater than 95% identity may be at least 95%, 96%, 97%, 98% or 99% identity.

[0028] In one embodiment of the present invention, the fucose dehydrogenase is a protein shown in SEQ ID No. 1 obtained by expressing in an E. coli expression system. Specifically, the gene shown in SEQ ID No. 2 is introduced into E. coli via a pET30a plasmid, and then induced to express by IPTG (e.g., using IPTG with a final concentration of 1 M at 18° C., 180 rpm for 18-20 hours), and purified by a nickel column to obtain the protein shown in SEQ ID No. 1.

[0029] In the method, the pH of the reaction buffer may be 8. In one embodiment of the present invention, the reaction buffer is Tris·HCl with a pH of 8.

[0030] Furthermore, in the reaction system, the final concentration of the fucose dehydrogenase may be 0.47-0.72 mg / mL.

[0031] Furthermore, in the reaction system, the coenzyme NAD + The final concentration can be 2 mM.

[0032] Furthermore, in the reaction system, the final concentration of Tris·HCl may be 50 mM.

[0033] In a second aspect, the present invention claims a kit of reagents for detecting fucose.

[0034] The kit of reagents for detecting fucose claimed in the present invention may include: the fucose dehydrogenase and the coenzyme NAD + .

[0035] Furthermore, the reagent set may also include all or part of the following: Tris·HCl and hydrochloric acid, such as 1M Tris·HCl, pH 8 and 6M hydrochloric acid.

[0036] Furthermore, the kit may further include NaOH, such as 6M NaOH solution.

[0037] In a third aspect, the present invention claims the use of the fucose dehydrogenase described in the first aspect above or the kit of reagents described in the second aspect above in any of the following:

[0038] (B1) Detection of fucose content;

[0039] (B2) preparing a product for detecting fucose content;

[0040] (B3) detecting the free fucose content in the sample to be tested;

[0041] (B4) preparing a product for detecting the free fucose content in a sample to be tested;

[0042] (B5) detecting the fucose content in the fucosylated glycoconjugate sample;

[0043] (B6) Preparing a product for detecting the fucose content in a fucosylated glycoconjugate sample.

[0044] Wherein, the fucosylated glycoconjugate sample may be glycoprotein, oligosaccharide, milk powder or liquid milk.

[0045] In one embodiment of the present invention, the glycoprotein is horseradish peroxidase (HRP), lactoferrin or amylase.

[0046] In the above aspects, the fucose is L-fucose.

[0047] Beneficial effects of the present invention: The present invention realizes accurate, rapid, economical and convenient detection of fucose in samples for the first time through a green and pollution-free method. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The figure is the working principle and flow chart of the enzymatic detection of L-fucose of the present invention.

[0049] Figure 2 This is the SDS-PAGE electrophoresis analysis of recombinant CmFucDH. M is the protein molecular weight standard; 1 is the purified protein; 2 is the ultrasonic cell lysate; 3 is the induced bacteria; 4 is the bacteria before induction. The red box is the target protein.

[0050] Figure 3 The results of the activity test of recombinant CmFucDH on L-fucose.

[0051] Figure 4 The results of activity detection of recombinant CmFucDH on different sugars.

[0052] Figure 5 The standard curve for the enzymatic detection of L-fucose is established. A is the real-time reaction curve of the enzyme marker for the determination of different concentrations of fucose at a wavelength of 340 nm; B is the standard curve for the determination of L-fucose concentration in the range of 0-0.1 mM; C is the standard curve for the determination of L-fucose concentration in the range of 0-0.5 mM.

[0053] Figure 6The present invention is an application of the method for detecting free fucose based on fucose dehydrogenase in actual sample detection. A is the real-time reaction curve of enzymatic determination of L-fucose in amylase; B is the real-time reaction curve of enzymatic determination of L-fucose in lactoferrin; C is the real-time reaction curve of enzymatic determination of L-fucose in horseradish peroxidase; D is the real-time reaction curve of enzymatic determination of L-fucose in a commercially available milk powder; E is the real-time reaction curve of enzymatic determination of L-fucose in human milk; F is the real-time reaction curve of enzymatic determination of L-fucose in goat milk; G is the real-time reaction curve of enzymatic determination of L-fucose in bovine milk; H is the bar graph of enzymatic detection of L-fucose content in solid powder samples; I is the bar graph of enzymatic detection of L-fucose content in liquid milk samples. DETAILED DESCRIPTION

[0054] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0055] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0056] The working principle and flow chart of the enzymatic method for detecting L-fucose of the present invention are as follows: Figure 1 shown.

[0057] Example 1. Expression, purification and enzyme activity determination of fucose dehydrogenase

[0058] 1. Transform the recombinant plasmid into E.coli BL21

[0059] The invention mines fucose dehydrogenase from Cyclobacterium marinum (amino acid sequence is shown in the 1st to 344th positions of SEQ ID No.1); the coding gene sequence of the fucose dehydrogenase (named as CmFucDH) (shown in the 1st to 1032nd positions of SEQ ID No.2) is synthesized to replace the sequence between the Nde I / Xho I sites of the pET30a plasmid, and other sequences remain unchanged to obtain the recombinant plasmid pET30a-CmFucDH. The recombinant plasmid pET30a-CmFucDH contains the ORF shown in SEQ ID No.2, encoding the protein shown in SEQ ID No.1. The 1st to 344th positions of SEQ ID No.1 are the amino acid sequence of the fucose dehydrogenase in Cyclobacterium marinum, the 345th and 346th amino acids are obtained by translation of the restriction site XhoI (CTCGAG), and the 347th to 352nd positions are 6His tags.

[0060] The recombinant plasmid was transformed into E. coli BL21 to obtain E. coli BL21-CmFucDH. The specific process is as follows:

[0061] (1) The recombinant plasmid pET30a-CmFucDH dry powder returned from gene synthesis was centrifuged and diluted with water to 1 ng / μL;

[0062] (2) Take 1 μL of the diluted recombinant plasmid pET30a-CmFucDH and transfer it into 100 μL of competent cells;

[0063] (3) After standing for 25 minutes, adjust the temperature of the water bath, quickly place the dish in a 42°C water bath for 1 minute, take it out and quickly put it back in ice, and let it stand for 2 minutes to reduce the damage of E. coli.

[0064] (4) Take 200 μL of sterilized LB liquid medium and add it to each tube of competent cells after heat shock and culture at 37°C and 800 rpm for 50 min to allow them to recover;

[0065] (5) Add the shaken cultured bacterial solution evenly and spread it on LB solid medium containing 0.1 mg / mL kanamycin. Place the plate upside down in a 37°C constant temperature incubator and culture for 12-16 hours until the plaque grows into a single colony suitable for picking.

[0066] 2. Heterologous expression of recombinant plasmids in E. coli

[0067] (1) Pick a single colony transferred into E. coli BL21 (i.e., E. coli BL21-CmFucDH containing the recombinant plasmid) and inoculate it into 3 mL of LB liquid medium, add 3 μL of 50 mg / mL kanamycin, and culture it in a constant temperature shaker at 37°C and 250 rpm overnight;

[0068] (2) Take 1 mL of bacterial solution and inoculate it into 400 mL of fresh LB medium and culture it at 37°C and 180 rpm until the OD 600 The concentration of the cell culture medium was 0.5-0.8, and then 400 μL 1 M IPTG (isopropyl-β-D-thiogalactopyranoside) was added, and the culture was continued at 18°C ​​and 180 rpm for 18-20 hours.

[0069] (3) After the culture, the cells were collected by centrifugation (4000 rpm, 15 min, 4°C), resuspended in 10 mL of lysis buffer (formula: 100 mM NaCl, 50 mM Tris, 1% Triton X-100, 1 mM phenyl-methylsulfonylfluoride, pH 8.0), and ultrasonically lysed in an ice bath for 20 min (each time with an amplitude of 20 μm, lasting 15 s, repeated 40 times in total), and then centrifuged (20000 g, 20 min, 4°C) to remove cell debris and collect the cell lysate.

[0070] 3. Nickel affinity chromatography column purification of recombinant vector

[0071] The cell lysate was loaded onto a nickel column (Ni 2+ -nitrilotriacetate agarose affinity chromatography column, column bed volume 2 mL) (Qiagen), after loading, use 50 mL of washing buffer (formula: 50 mM Tris-HCl, 50 mM NaCl, pH 8.0) to fully wash the unadsorbed protein, and the recombinant protein is eluted with elution buffer (formula: 300 mM imidazole, 50 mM Tris-HCl, 50 mM NaCl, pH 8.0).

[0072] The results of SDS-PAGE electrophoresis showed that the CmFucDH recombinant expression vector (i.e., pET30a-CmFucDH) could express a large amount of soluble protein in BL21(DE3) cells, and a relatively pure target protein could be obtained after purification ( Figure 2 ), which can be used for subsequent enzyme activity determination. The volume of the eluate containing the target protein is about 4 mL, which can be directly used for subsequent enzyme activity analysis.

[0073] Various reagents were prepared as follows: (1) Washing 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 make up 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, make up to 500 mL, and store at room temperature.

[0074] 4. Enzyme activity analysis of recombinant plasmid expression products

[0075] The purified recombinant enzyme fucose dehydrogenase CmFucDH could detect fucosidase activity when using L-fucose as substrate.

[0076] Fucose dehydrogenase activity assay: The reaction system is 50 μL, containing 0.4 mM L-fucose, 2 mM NAD + , 50mMTris·HCl buffer (pH8.0) and 0.72mg / mL CmFucDH enzyme solution (Table 1). An equal volume of water was added to the reaction mixture instead of L-fucose solution as a negative control. All samples were immediately placed on the microplate reader for reading. The parameters of the microplate reader were set as follows: temperature was 37°C, absorbance was 340nm, the value was read every 30s, and the monitoring time was 30min. Compared with the negative control, the ultraviolet absorption intensity of the reaction mixture containing L-fucose at a wavelength of 340nm gradually increased, and remained basically stable after 30 minutes of incubation ( Figure 3 ), which fully proved that fucose dehydrogenase CmFucDH showed good catalytic activity towards L-fucose.

[0077] Table 1. Fucose dehydrogenase activity assay reaction system

[0078] Reagent name Volume (μL) L-Fucose (2mM) 10 <![CDATA[NAD + (50mM)]]> 2 Tris·HCl (1M, pH 8.0) 2.5 CmFucDH (7.2 mg / mL) 5 <![CDATA[ddH2O]]> 30.5

[0079] Example 2: Analysis of substrate specificity of fucose dehydrogenase

[0080] 50 μL of reaction mixture was prepared according to the reaction system shown in Table 2, and the activity of the fucose dehydrogenase CmFucDH prepared in Example 1 on different sugar substrates was measured. The reaction mixture contained different sugar substrates with a final concentration of 1 mM, 2 mM NAD +, 50mM Tris·HCl buffer and 0.47mg / mL CmFucDH enzyme solution. In this experiment, an equal volume of water was added to the reaction mixture instead of the sugar solution as a negative control. All samples were immediately placed on the microplate reader for reading. The parameters of the microplate reader were set as follows: temperature was 37°C, absorbance was 340nm, the value was read every 30s, and the monitoring time was 30min. After 30 minutes of incubation, only the reaction mixture containing L-fucose gradually increased and remained stable in terms of ultraviolet absorption intensity at a wavelength of 340nm. The results showed that CmFucDH had no activity on sugars such as D-glucose, maltose, D-lactose, D-galactose, D-xylose, ND-acetylglucosamine and D-mannose, but specifically acted on L-fucose ( Figure 4 ). Therefore, the enzyme can be used for the determination of L-fucose in actual samples.

[0081] Table 2. Fucose dehydrogenase substrate specificity determination reaction system

[0082] Reagent name Volume (μL) Various sugar solutions (5mM) 10 <![CDATA[NAD + (50mM)]]> 2 Tris·HCl (1M, pH 8.0) 2.5 CmFucDH (4.7 mg / mL) 5 <![CDATA[ddH2O]]> 30.5

[0083] Example 3: Establishment of the enzymatic method for detecting fucose

[0084] 50 μL of reaction mixture was prepared according to the reaction system shown in Table 3, and the activity of fucose dehydrogenase CmFucDH prepared in Example 1 on L-fucose was measured. The reaction mixture contained different final concentrations of L-fucose (0, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2 and 0.5 mM), 2 mM NAD + , 50mM Tris·HCl buffer, 50mM glucose and 0.72mg / mL CmFucDH enzyme solution. Glucose was added to the reaction system because the actual sample may contain a large amount of glucose, so as to simulate whether the presence of glucose would interfere with the detection of fucose by this method. All samples were immediately placed on the microplate reader for reading. The parameters of the microplate reader were set as follows: temperature was 37℃, absorbance was 340nm, reading was performed every 30s, and monitoring time was 30min.

[0085] When the incubation time reaches 30 minutes, the absorbance of the reaction system tends to be stable ( Figure 5 A standard curve was drawn with different concentrations of L-fucose as the horizontal axis and the absorbance value at 30 minutes as the vertical axis ( Figure 5 In B and C). In the range of L-fucose concentration of 0-0.5 mM, there is a good linear relationship between the absorbance of the reaction system and the L-fucose concentration, and the fitting coefficient R 2In order to further evaluate the linear relationship between L-fucose and absorbance in the low concentration range, a concentration gradient of 0-0.1mM was specially selected to establish the standard curve. The results showed that a good linear relationship was still maintained in this concentration range, and the fitting coefficient was 0.9971.

[0086] In UV spectrophotometry, the concentration corresponding to 0.01 absorbance after deducting the blank value is usually used as the limit of detection (LOD), and the limit of quantification (LOQ) is usually set at 3.3 times the limit of detection. Based on this principle, we calculated that the LOD of this method for L-fucose is 0.005mM (i.e., since the total volume of the measured sample is 50μL, 0.005mM×50μL=0.25nmol), and the LOQ is 0.0165mM (i.e., since the total volume of the measured sample is 50μL, 0.0165mM×50μL=0.825nmol). These data show that this method has high sensitivity and accuracy and is suitable for the quantitative detection of L-fucose in actual samples.

[0087] Table 3. Fucose dehydrogenase activity assay reaction system

[0088] Reagent name Volume (μL) L-fucose (0-5mM) 5 <![CDATA[NAD + (50mM)]]> 2 Tris·HCl (1M, pH 8.0) 2.5 CmFucDH (7.2 mg / mL) 5 D-Glucose (1M) 2.5 <![CDATA[ddH2O]]> 33

[0089] Example 4: Application of fucose dehydrogenase in detecting fucose in glycoprotein samples

[0090] Take 5 mg of horseradish peroxidase (HRP), 5 mg of lactoferrin and 5 mg of amylase to determine the content of fucose in its glycosylation structure. The specific steps are as follows:

[0091] 1) All samples were mixed evenly with 100 μL of 6 M hydrochloric acid solution and incubated at 80 °C for 12 hours;

[0092] 2) adjusting the pH of the reaction mixture to 7.0-8.0 with about 100 μL of 6M NaOH solution, and adjusting the total volume of the sample to 500 μL with water to obtain a sample stock solution to be tested (i.e., the sample after acid hydrolysis);

[0093] 3) Centrifuge at 12000 rpm, 4°C and remove the supernatant;

[0094] 4) After acid hydrolysis, all samples turned brown. In order to avoid interference of impurities in the samples with subsequent sample testing, ultrafiltration centrifugation (ultrafiltration tube membrane pore size 10 kDa, centrifugal speed 8000 rpm) was used to obtain the filtrate containing free fucose for later use;

[0095] 5) Prepare 50 μL of reaction mixture according to the reaction system shown in Table 4 (CmFucDH is the fucose dehydrogenase prepared in Example 1), put it into a microplate reader, monitor the absorbance of the reaction mixture at 37°C and 340 nm ultraviolet, calculate the difference between the absorbance value and the negative control after it stabilizes, and thus quantify the L-fucose in the sample.

[0096] Note: After acid hydrolysis, all samples turned brown. To avoid the influence of color background, an equal volume of water (or inactivated enzyme solution) was used as a negative control instead of the enzyme solution. Compared with the negative control, horseradish peroxidase showed a higher absorbance value, while lactoferrin and amylase showed relatively small changes in absorbance values.

[0097] The specific method for quantifying L-fucose in the sample is as follows: For small "differences" (less than 0.2), substitute the 0-0.1 mM calibration curve (y = 2.1165x + 0.0036, R 2 =0.9971, see Figure 5 In the middle B), the “difference” is larger (greater than 0.2), and the 0-0.5mM standard curve (y = 2.071x + 0.006, R 2 =0.9996, see Figure 5 C), calculate the L-fucose concentration. Then, on the one hand, calculate the fucose mass in the 50μl reaction system according to the formula (fucose mass in 50μl reaction system = fucose concentration × volume × molar mass of fucose); on the other hand, according to the dilution multiple after sample pretreatment, convert the 50μl reaction system to contain 200μg original solid sample and 16.2μl original liquid sample. Then use the ratio of the fucose mass obtained above to the original sample to get the fucose content in the original sample.

[0098] The results are as follows: amylase contains 0.43 mg / g of L-fucose; lactoferrin is 0.94 mg / g; horseradish peroxidase is 21.08 mg / g. Figure 6 As shown in A, B, C and H.

[0099] Table 4. Reaction system for determination of fucose in solid powder samples

[0100] Reagent name Volume (μL) Sample after acid hydrolysis (10mg / mL) 20 <![CDATA[NAD + (50mM)]]> 2 Tris·HCl (1M, pH 8.0) 2.5 <![CDATA[ddH2O]]> 20.5 CmFucDH (7.2 mg / mL) 5

[0101] Example 5: Application of fucose dehydrogenase in detecting fucose in milk powder

[0102] Take 5 mg of a commercially available milk powder and determine the fucose content in the glycosylation structure of its whey protein. The specific steps are as follows:

[0103] 1) After defatting and removing casein from the sample, the precipitate was dried, mixed evenly with 100 μL of 6 M hydrochloric acid solution, and incubated at 80°C for 12 hours;

[0104] 2) adjusting the pH of the reaction mixture to 7.0-8.0 with about 100 μL of 6M NaOH solution, and adjusting the total volume of the sample to 500 μL with water to obtain a sample stock solution to be tested (i.e., the sample after acid hydrolysis);

[0105] 3) Centrifuge at 12000 rpm, 4°C and remove the supernatant;

[0106] 4) After acid hydrolysis, all samples turned brown. In order to avoid interference of impurities in the samples with subsequent sample testing, ultrafiltration centrifugation (ultrafiltration tube membrane pore size 10 kDa, centrifugal speed 8000 rpm) was used to obtain the filtrate containing free fucose for later use;

[0107] 5) Prepare 50 μL of reaction mixture according to the reaction system shown in Table 4 (CmFucDH is the fucose dehydrogenase prepared in Example 1), put it into a microplate reader, monitor the absorbance of the reaction mixture at 37°C and 340 nm UV, calculate the difference between the absorbance value and the negative control after it stabilizes, and thus quantify the L-fucose in the sample (see Example 4 for specific operations).

[0108] Note: After acid hydrolysis, all samples turned brown. To avoid color background effects, an equal volume of water (or inactivated enzyme solution) was used as a negative control instead of the enzyme solution in the reaction mixture.

[0109] The results showed that compared with the negative control, the fucose content in the whey protein of a commercially available milk powder was 1.32 mg / g. Figure 6 As shown in D and H.

[0110] Example 6: Application of fucose dehydrogenase in detecting fucose in liquid milk samples

[0111] Take 200 μL of skimmed and casein-free human milk, cow milk, and goat milk. The specific steps are as follows:

[0112] 1) All samples were mixed evenly with 100 μL of 6 M hydrochloric acid solution and incubated at 80 °C for 12 hours;

[0113] 2) adjusting the pH of the reaction mixture to 7.0-8.0 with about 100 μL of 6M NaOH solution, and adjusting the total volume of the sample to 500 μL with water to obtain a sample stock solution to be tested (i.e., the sample after acid hydrolysis);

[0114] 3) Centrifuge at 12000 rpm, 4°C and remove the supernatant;

[0115] 4) After acid hydrolysis, all samples turned brown. In order to avoid interference of impurities in the samples with subsequent sample testing, ultrafiltration centrifugation (ultrafiltration tube membrane pore size 10 kDa, centrifugal speed 8000 rpm) was used to obtain the filtrate containing free fucose for later use;

[0116] 5) Prepare 50 μL of reaction mixture according to the reaction system shown in Table 5 (CmFucDH is the fucose dehydrogenase prepared in Example 1), put it into a microplate reader, monitor the absorbance of the reaction mixture at 37°C and 340 nm UV, calculate the difference between the absorbance value and the negative control after it stabilizes, and thus quantify the L-fucose in the sample (for specific operations, see Example 4).

[0117] Note: After acid hydrolysis, all samples turned brown. To avoid color background effects, an equal volume of water (or inactivated enzyme solution) was used as a negative control instead of the enzyme solution in the reaction mixture.

[0118] Compared with the negative control, the absorbance values ​​of liquid human milk and goat milk changed greatly, while the absorbance values ​​of liquid bovine milk and cow milk powder changed less.

[0119] The results are as follows: human milk is 183.63 mg / L; goat milk is 38.54 mg / L; cow milk is 11.13 mg / L. Figure 6 As shown in E, F, G and I.

[0120] Table 5. Reaction system for determination of fucose in liquid dairy products

[0121] Reagent name Volume (μL) Sample after acid hydrolysis (500μL) 40.5 <![CDATA[NAD + (50mM)]]> 2 Tris·HCl (1M, pH 8.0) 2.5 CmFucDH (7.2 mg / mL) 5

[0122] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for detecting fucose content, comprising the following steps: in the presence of fucose dehydrogenase and coenzyme NAD + Under the action of NAD, it catalyzes the dehydrogenation reaction of free fucose. + Reduced to NADH; the fucose content is detected by ultraviolet absorption of NADH at a wavelength of 340nm; The fucose dehydrogenase is a protein having an amino acid sequence as shown in SEQ ID No.1 or positions 1 to 344 of SEQ ID No.1 or positions 1 to 346 of SEQ ID No.

1.

2. The method according to claim 1, characterized in that: The method comprises the following steps: (A1) Drawing a standard curve: Add the fucose dehydrogenase and coenzyme NAD into a series of free fucose standard solutions with known concentrations. + and reaction buffer to obtain a series of reaction systems; the series of reaction systems were incubated at 37° C. for 30 min and then OD was detected 340nm value; then according to the concentration of free fucose standard and its corresponding OD 340nm The values ​​were plotted to obtain the standard curve; (A2) After replacing the free fucose standard solution in (A1) with the sample to be tested containing free fucose, proceed according to (A1), and then convert the OD 340nm The value is substituted into the standard curve to calculate the content of free fucose in the sample to be tested.

3. The method according to claim 2, characterized in that: The test sample containing free fucose is obtained by subjecting a fucosylated glycoconjugate sample to acid hydrolysis.

4. The method according to claim 3, characterized in that: The acid hydrolysis is hydrochloric acid hydrolysis.

5. The method according to claim 3, characterized in that: The acid hydrolysis condition is incubation at 80° C. for 12-16 hours.

6. The method according to claim 3, characterized in that: The fucosylated glycoconjugate sample is glycoprotein, oligosaccharide, milk powder or liquid milk.

7. The method according to claim 1, characterized in that: The fucose dehydrogenase is a protein shown in SEQ ID No. 1 expressed by an Escherichia coli expression system.

8. The method according to any one of claims 2 to 7, characterized in that: The pH of the reaction buffer was 8.

9. The method according to claim 8, characterized in that: The reaction buffer is Tris·HCl with a pH of 8.

10. The method according to any one of claims 1 to 7, characterized in that: In the reaction system, the final concentration of the fucose dehydrogenase is 0.47-0.72 mg / mL.

11. The method according to any one of claims 2 to 7, characterized in that: In the reaction system, the coenzyme NAD + The final concentration is 2 mM.

12. The method according to claim 9, characterized in that: In the reaction system, the final concentration of Tris·HCl is 50 mM.

13. Use of fucose dehydrogenase or a kit of reagents in any of the following: (B1) Detection of fucose content; (B2) preparing a product for detecting fucose content; (B3) detecting the free fucose content in the sample to be tested; (B4) preparing a product for detecting the free fucose content in a sample to be tested; (B5) detecting the fucose content in the fucosylated glycoconjugate sample; (B6) preparing a product for detecting the fucose content in a fucosylated glycoconjugate sample; The fucose dehydrogenase is a protein with an amino acid sequence as shown in SEQ ID No.1 or positions 1 to 344 of SEQ ID No.1 or positions 1 to 346 of SEQ ID No.1; The kit includes: The fucose dehydrogenase and the coenzyme NAD + .

14. The use according to claim 13, characterized in that: The fucose dehydrogenase is a protein shown in SEQ ID No. 1 expressed by an Escherichia coli expression system.

15. The use according to claim 13, characterized in that: The reagent set also includes all or part of the following: Tris·HCl and hydrochloric acid.

Images