Application of fucosidase

Abstract

The invention discloses application of fucosidase, the amino acid sequence of the fucosidase is shown as SEQ ID NO.1, the fucosidase specifically acts on alpha-1, 2 glucosidic bonds, alpha-1, 3 glucosidic bonds and alpha-1, 4 glycosides in fucosyl disaccharide, and the fucosidase can be used for specifically catalytically degrading the alpha-1, 2 glucosidic bonds, alpha-1, 3 glucosidic bonds and alpha-1, 4 glycosides containing alpha-1, 2 glucosidic bonds, alpha-1, 3 glucosidic bonds and alpha-1, 4 glycosides in fucosyl disaccharide. The fucosyl disaccharide containing the 2, 4 glycosidic bonds can be used for carbohydrate analysis and detection.

Description

Technical Field

[0001] The present invention relates to the technical field of biotechnology, and in particular to an application of fucosidase, in particular to an application of fucosidase specifically acting on α-1,2 glycosidic bonds, α-1,3 glycosidic bonds and α-1,4 glycosidic bonds. Background Art

[0002] α-fucose is widely present in HMO, glycoproteins and glycolipids, and plays an important physiological function in different biological processes such as immune recognition, genetic and neural development, and host-intestinal flora interaction. α-fucosidase can specifically hydrolyze and release α-fucose, thereby affecting physiological functions.

[0003] Fucosidase has important applications in carbohydrate analysis. In the glycomics analysis of disease sugar metabolism, fucosidase can diagnose MODY diabetes from the sugar spectrum, solving the defects of diagnosis at the genetic level. In allergic reactions caused by plant and animal glycoproteins, fucosidase can specifically recognize and hydrolyze the N-glycans of allergens, thereby assisting diagnosis and treatment. Since fucosyl exists in different ways in different biomacromolecules, different fucose modifications have characteristic effects or influences. Therefore, α-fucosidase that is specific to specific fucose modifications has potential molecular diagnostic value. The specificity of fucosidase is related to its protein structure, and it is difficult to predict its specificity by amino acid sequence. The α-fucosidases identified so far include α1,2-fucosidase for blood group antigen H, α1,3 / 4-fucosidase for Lewis oligosaccharide antigens, α1,3 / 4-fucosidase for sialic acid-modified Lewis oligosaccharide antigens, and α1,3 or α1,4-fucosidase for disaccharides. Discovering fucosidases with new specificity is of great significance. Summary of the invention

[0004] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the present invention aims to provide the application of fucosidase.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] The first aspect of the present invention provides the use of fucosidase PgoldGH29C in the preparation of a detection probe, wherein the detection probe is used to detect the fucosyl disaccharide in a sample, the amino acid sequence of the fucosidase is as shown in SEQ ID NO.1, the fucosyl disaccharide comprises a first monosaccharide group and a second monosaccharide group connected by a glycosidic bond, the first monosaccharide group is a fucose group; the glycosidic bond comprises at least one of an α-1,2 glycosidic bond, an α-1,3 glycosidic bond, and an α-1,4 glycosidic bond.

[0007] Wherein, SEQ ID NO.1 is:

[0008] MKYSFVTILFLALSLQLGYGQDLILPVPSNQPSSAQQKQIARKYGMFIHFGINTFHDQEWTDGSKPASSYRPTAIDADQWIKTAKDAGMKYVILVAKHHEGFCLWDSKYTEYDVANSGNKTNVIEEVAKACKKYDIGLGLYYSLWDRKVNADVKDKTLDAAYNEYMIKQLNELI DIVQPYTHIVEFWFDGGWEKEHERWPAREIYQTIKSREPECQIGINWTIGLPENPDAHPVLPENQKEGYPIRYFPSDFRLGDPYLPADNDPKLFSHDGKLYYMPWESTICISERWFYNTTDKKYKTVEELTGLYHQCTKNDNILILNCPPNREGKIRDADVTLLKELRKKIQM.

[0009] The present invention provides the application of fucosidase PgoldGH29C in the preparation of a fucosyl disaccharide detection probe, wherein the fucosyl disaccharide is a fucosyl disaccharide containing an α-1,2 glycosidic bond, an α-1,3 glycosidic bond, or an α-1,4 glycosidic bond. The fucosidase has a stronger catalytic hydrolysis effect on the fucosyl disaccharide containing an α-1,2 glycosidic bond, an α-1,3 glycosidic bond, or an α-1,4 glycosidic bond, and can more effectively cut the fucose group connected by the α-1,2 glycosidic bond, the α-1,3 glycosidic bond, or the α-1,4 glycosidic bond. The detection probe can be used to detect whether there is a fucosyl disaccharide containing an α-1,2 glycosidic bond, an α-1,3 glycosidic bond, or an α-1,4 glycosidic bond in the sample to be tested.

[0010] In one or more embodiments of the present invention, the second monosaccharide group is selected from at least one of a galactose group and an acetylglucose group.

[0011] In one or more embodiments of the present invention, the sugars in the sample include one or more disaccharides, wherein disaccharides refer to disaccharides generally recognized by those skilled in the art, i.e., sugars formed by two molecules of monosaccharides through glycosidic bonds, and there is no particular limitation on monosaccharides and glycosidic bonds.

[0012] The second aspect of the present invention provides the use of fucosidase PgoldGH29C in the preparation of a detection kit, wherein the detection kit is used to detect the fucosyl disaccharide in a sample, the amino acid sequence of the fucosidase is as shown in SEQ ID NO.1, the fucosyl disaccharide comprises a first monosaccharide group and a second monosaccharide group connected by a glycosidic bond, the first monosaccharide group is a fucose group; the glycosidic bond comprises at least one of an α-1,2 glycosidic bond, an α-1,3 glycosidic bond, and an α-1,4 glycosidic bond.

[0013] Wherein, SEQ ID NO.1 is:

[0014] MKYSFVTILFLALSLQLGYGQDLILPVPSNQPSSAQQKQIARKYGMFIHFGINTFHDQEWTDGSKPASSYRPTAIDADQWIKTAKDAGMKYVILVAKHHEGFCLWDSKYTEYDVANSGNKTNVIEEVAKACKKYDIGLGLYYSLWDRKVNADVKDKTLDAAYNEYMIKQLNELI DIVQPYTHIVEFWFDGGWEKEHERWPAREIYQTIKSREPECQIGINWTIGLPENPDAHPVLPENQKEGYPIRYFPSDFRLGDPYLPADNDPKLFSHDGKLYYMPWESTICISERWFYNTTDKKYKTVEELTGLYHQCTKNDNILILNCPPNREGKIRDADVTLLKELRKKIQM.

[0015] In one or more embodiments of the present invention, the second monosaccharide group is selected from at least one of a galactose group and an acetylglucose group.

[0016] In one or more embodiments of the present invention, the sugars in the sample include one or more disaccharides, wherein disaccharides refer to disaccharides generally recognized by those skilled in the art, i.e., sugars formed by two molecules of monosaccharides through glycosidic bonds, and there is no particular limitation on monosaccharides and glycosidic bonds.

[0017] The third aspect of the present invention provides a detection method for fucosyl disaccharide in a sample, the method comprising:

[0018] Fucosidase PgoldGH29C was used to perform enzymatic hydrolysis reaction with the sample;

[0019] Analyze the products of enzymatic hydrolysis reactions;

[0020] The amino acid sequence of the fucosidase is shown in SEQ ID NO.1, the fucosyl disaccharide comprises a first monosaccharide and a second monosaccharide connected by a glycosidic bond, the first monosaccharide is a fucose group; the glycosidic bond comprises at least one of an α-1,2 glycosidic bond, an α-1,3 glycosidic bond, and an α-1,4 glycosidic bond.

[0021] Wherein, SEQ ID NO.1 is:

[0022] MKYSFVTILFLALSLQLGYGQDLILPVPSNQPSSAQQKQIARKYGMFIHFGINTFHDQEWTDGSKPASSYRPTAIDADQWIKTAKDAGMKYVILVAKHHEGFCLWDSKYTEYDVANSGNKTNVIEEVAKACKKYDIGLGLYYSLWDRKVNADVKDKTLDAAYNEYMIKQLNELI DIVQPYTHIVEFWFDGGWEKEHERWPAREIYQTIKSREPECQIGINWTIGLPENPDAHPVLPENQKEGYPIRYFPSDFRLGDPYLPADNDPKLFSHDGKLYYMPWESTICISERWFYNTTDKKYKTVEELTGLYHQCTKNDNILILNCPPNREGKIRDADVTLLKELRKKIQM.

[0023] In one or more embodiments of the present invention, the second monosaccharide group is selected from at least one of a galactose group and an acetylglucose group.

[0024] In one or more embodiments of the present invention, the enzymatic hydrolysis reaction is carried out at a pH of 4-6.

[0025] In one or more embodiments of the present invention, the enzymatic hydrolysis reaction is carried out at 35-40°C.

[0026] In one or more embodiments of the present invention, the enzymatic hydrolysis reaction time is 3-6 hours.

[0027] In one or more embodiments of the present invention, the concentration of the fucosidase in the enzymatic reaction system is 0.025-10 μM.

[0028] In one or more embodiments of the present invention, the concentration of the sample is 1-10 mM.

[0029] In one or more embodiments of the present invention, the analyzing the product of the enzymatic hydrolysis reaction comprises analyzing the product of the enzymatic hydrolysis reaction by at least one of silica gel thin layer chromatography, high performance liquid chromatography, ion chromatography, and mass spectrometry.

[0030] In one or more embodiments of the present invention, the sugars in the sample include one or more disaccharides, wherein disaccharides refer to disaccharides generally recognized by those skilled in the art, i.e., sugars formed by two molecules of monosaccharides through glycosidic bonds, and there is no particular limitation on monosaccharides and glycosidic bonds.

[0031] The fourth aspect of the present invention provides the use of fucosidase PgoldGH29C in the degradation of fucosyl disaccharides, wherein the degradation of fucosyl disaccharides includes using the fucosidase as a catalyst to catalyze an enzymatic hydrolysis reaction of a substrate, the amino acid sequence of the fucosidase is shown in SEQ ID NO.1, the fucosyl disaccharide comprises a first monosaccharide group and a second monosaccharide group connected by a glycosidic bond, the first monosaccharide group is a fucose group, and the glycosidic bond comprises at least one of an α-1,2 glycosidic bond, an α-1,3 glycosidic bond, and an α-1,4 glycosidic bond.

[0032] Wherein, SEQ ID NO.1 is:

[0033] MKYSFVTILFLALSLQLGYGQDLILPVPSNQPSSAQQKQIARKYGMFIHFGINTFHDQEWTDGSKPASSYRPTAIDADQWIKTAKDAGMKYVILVAKHHEGFCLWDSKYTEYDVANSGNKTNVIEEVAKACKKYDIGLGLYYSLWDRKVNADVKDKTLDAAYNEYMIKQLNELI DIVQPYTHIVEFWFDGGWEKEHERWPAREIYQTIKSREPECQIGINWTIGLPENPDAHPVLPENQKEGYPIRYFPSDFRLGDPYLPADNDPKLFSHDGKLYYMPWESTICISERWFYNTTDKKYKTVEELTGLYHQCTKNDNILILNCPPNREGKIRDADVTLLKELRKKIQM.

[0034] In one or more embodiments of the present invention, the second monosaccharide group is selected from at least one of a galactose group and an acetylglucose group.

[0035] In one or more embodiments of the present invention, the enzymatic hydrolysis reaction is carried out at pH 4-6.

[0036] In one or more embodiments of the present invention, the enzymatic hydrolysis reaction is carried out at 35-40°C.

[0037] In one or more embodiments of the present invention, the enzymatic hydrolysis reaction time is 3-6 hours.

[0038] In one or more embodiments of the present invention, the concentration of the fucosidase in the enzymatic reaction system is 0.025-10 μM.

[0039] In one or more embodiments of the present invention, the concentration of the substrate is 1-10 mM.

[0040] In one or more embodiments of the present invention, the substrate contains the fucosyl disaccharide and at least one other disaccharide. The other disaccharide refers to a disaccharide generally known to those skilled in the art, that is, a sugar formed by two molecules of monosaccharide through a glycosidic bond, and there is no particular limitation on the monosaccharide and the glycosidic bond.

[0041] The fifth aspect of the present invention provides a method for degrading fucosylated disaccharides, comprising using fucosidase PgoldGH29C as a catalyst to catalyze an enzymatic hydrolysis reaction of a substrate, wherein the amino acid sequence of the fucosidase is as shown in SEQ ID NO.1, and the fucosylated disaccharide comprises a first monosaccharide group and a second monosaccharide group connected by a glycosidic bond, wherein the first monosaccharide group is a fucose group, and the glycosidic bond comprises at least one of an α-1,2 glycosidic bond, an α-1,3 glycosidic bond, and an α-1,4 glycosidic bond.

[0042] Wherein, SEQ ID NO.1 is:

[0043] MKYSFVTILFLALSLQLGYGQDLILPVPSNQPSSAQQKQIARKYGMFIHFGINTFHDQEWTDGSKPASSYRPTAIDADQWIKTAKDAGMKYVILVAKHHEGFCLWDSKYTEYDVANSGNKTNVIEEVAKACKKYDIGLGLYYSLWDRKVNADVKDKTLDAAYNEYMIKQLNELI DIVQPYTHIVEFWFDGGWEKEHERWPAREIYQTIKSREPECQIGINWTIGLPENPDAHPVLPENQKEGYPIRYFPSDFRLGDPYLPADNDPKLFSHDGKLYYMPWESTICISERWFYNTTDKKYKTVEELTGLYHQCTKNDNILILNCPPNREGKIRDADVTLLKELRKKIQM.

[0044] In one or more embodiments of the present invention, the second monosaccharide group is selected from at least one of a galactose group and an acetylglucose group.

[0045] In one or more embodiments of the present invention, the substrate contains the fucosyl disaccharide and at least one other disaccharide. The other disaccharide refers to a disaccharide generally known to those skilled in the art, that is, a sugar formed by two molecules of monosaccharide through a glycosidic bond, and there is no particular limitation on the monosaccharide and the glycosidic bond.

[0046] In one or more embodiments of the present invention, the enzymatic hydrolysis reaction is carried out at pH 4-6.

[0047] In one or more embodiments of the present invention, the enzymatic hydrolysis reaction is carried out at 35-40°C.

[0048] In one or more embodiments of the present invention, the enzymatic hydrolysis reaction time is 3-6 hours.

[0049] In one or more embodiments of the present invention, the concentration of the fucosidase in the enzymatic reaction system is 0.025-10 μM.

[0050] In one or more embodiments of the present invention, the concentration of the substrate in the enzymatic reaction system is 1-10 mM.

[0051] The beneficial effect of the present invention is that the present invention provides a fucosidase PgoldGH29C which specifically acts on α-1,2 glycosidic bonds, α-1,3 glycosidic bonds and α-1,4 glycosidic bonds in fucosyl disaccharides. The enzyme can be used to specifically catalyze the degradation of fucosyl disaccharides containing α-1,2 glycosidic bonds, α-1,3 glycosidic bonds and α-1,4 glycosidic bonds, and can be used for carbohydrate analysis and detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The figure shows the SDS-PAGE result of purified PgoldGH29C.

[0053] Figure 2 is the activity of PgoldGH29C under different pH conditions.

[0054] Figure 3 The TLC results of PgoldGH29C acting on different fucoidan oligosaccharides. DETAILED DESCRIPTION

[0055] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0056] The first aspect of the present invention provides the use of fucosidase PgoldGH29C in the preparation of a fucosyl disaccharide detection probe, wherein the detection probe is used to detect the fucosyl disaccharide in a sample, the amino acid sequence of the fucosidase is shown in SEQ ID NO.1, the fucosyl disaccharide comprises a first monosaccharide group and a second monosaccharide group connected by a glycosidic bond, the first monosaccharide group is a fucose group; the glycosidic bond comprises at least one of an α-1,2 glycosidic bond, an α-1,3 glycosidic bond, and an α-1,4 glycosidic bond.

[0057] Wherein, SEQ ID NO.1 is:

[0058] MKYSFVTILFLALSLQLGYGQDLILPVPSNQPSSAQQKQIARKYGMFIHFGINTFHDQEWTDGSKPASSYRPTAIDADQWIKTAKDAGMKYVILVAKHHEGFCLWDSKYTEYDVANSGNKTNVIEEVAKACKKYDIGLGLYYSLWDRKVNADVKDKTLDAAYNEYMIKQLNELI DIVQPYTHIVEFWFDGGWEKEHERWPAREIYQTIKSREPECQIGINWTIGLPENPDAHPVLPENQKEGYPIRYFPSDFRLGDPYLPADNDPKLFSHDGKLYYMPWESTICISERWFYNTTDKKYKTVEELTGLYHQCTKNDNILILNCPPNREGKIRDADVTLLKELRKKIQM.

[0059] In one or more embodiments of the present invention, the second monosaccharide group is selected from at least one of a galactose group and an acetylglucose group.

[0060] In one or more embodiments of the present invention, the sugars in the sample include one or more disaccharides, wherein disaccharides refer to disaccharides generally recognized by those skilled in the art, i.e., sugars formed by two molecules of monosaccharides through glycosidic bonds, and there is no particular limitation on monosaccharides and glycosidic bonds.

[0061] The second aspect of the present invention provides the use of fucosidase PgoldGH29C in the preparation of a fucosyl disaccharide detection kit, wherein the detection kit is used to detect the fucosyl disaccharide in a sample, the amino acid sequence of the fucosidase is as shown in SEQ ID NO.1, the fucosyl disaccharide comprises a first monosaccharide group and a second monosaccharide group connected by a glycosidic bond, the first monosaccharide group is a fucose group; the glycosidic bond comprises at least one of an α-1,2 glycosidic bond, an α-1,3 glycosidic bond, and an α-1,4 glycosidic bond.

[0062] Wherein, SEQ ID NO.1 is:

[0063] MKYSFVTILFLALSLQLGYGQDLILPVPSNQPSSAQQKQIARKYGMFIHFGINTFHDQEWTDGSKPASSYRPTAIDADQWIKTAKDAGMKYVILVAKHHEGFCLWDSKYTEYDVANSGNKTNVIEEVAKACKKYDIGLGLYYSLWDRKVNADVKDKTLDAAYNEYMIKQLNELI DIVQPYTHIVEFWFDGGWEKEHERWPAREIYQTIKSREPECQIGINWTIGLPENPDAHPVLPENQKEGYPIRYFPSDFRLGDPYLPADNDPKLFSHDGKLYYMPWESTICISERWFYNTTDKKYKTVEELTGLYHQCTKNDNILILNCPPNREGKIRDADVTLLKELRKKIQM.

[0064] In one or more embodiments of the present invention, the second monosaccharide group is selected from at least one of a galactose group and an acetylglucose group.

[0065] In one or more embodiments of the present invention, the sugars in the sample include one or more disaccharides, wherein disaccharides refer to disaccharides generally recognized by those skilled in the art, i.e., sugars formed by two molecules of monosaccharides through glycosidic bonds, and there is no particular limitation on monosaccharides and glycosidic bonds.

[0066] The third aspect of the present invention provides a detection method for fucosyl disaccharide in a sample, the method comprising:

[0067] Fucosidase PgoldGH29C was used to perform enzymatic hydrolysis reaction with the sample;

[0068] Analyze the products of enzymatic hydrolysis reactions;

[0069] The amino acid sequence of the fucosidase is shown in SEQ ID NO.1, the fucosyl disaccharide comprises a first monosaccharide and a second monosaccharide connected by a glycosidic bond, the first monosaccharide is a fucose group; the glycosidic bond comprises at least one of an α-1,2 glycosidic bond, an α-1,3 glycosidic bond, and an α-1,4 glycosidic bond.

[0070] Wherein, SEQ ID NO.1 is:

[0071] MKYSFVTILFLALSLQLGYGQDLILPVPSNQPSSAQQKQIARKYGMFIHFGINTFHDQEWTDGSKPASSYRPTAIDADQWIKTAKDAGMKYVILVAKHHEGFCLWDSKYTEYDVANSGNKTNVIEEVAKACKKYDIGLGLYYSLWDRKVNADVKDKTLDAAYNEYMIKQLNELI DIVQPYTHIVEFWFDGGWEKEHERWPAREIYQTIKSREPECQIGINWTIGLPENPDAHPVLPENQKEGYPIRYFPSDFRLGDPYLPADNDPKLFSHDGKLYYMPWESTICISERWFYNTTDKKYKTVEELTGLYHQCTKNDNILILNCPPNREGKIRDADVTLLKELRKKIQM.

[0072] In one or more embodiments of the present invention, the second monosaccharide group is selected from at least one of a galactose group and an acetylglucose group.

[0073] In one or more embodiments of the present invention, the enzymatic hydrolysis reaction is carried out at a pH of 4-6.

[0074] In one or more embodiments of the present invention, the enzymatic hydrolysis reaction is carried out at 35-40°C.

[0075] In one or more embodiments of the present invention, the enzymatic hydrolysis reaction time is 3-6 hours.

[0076] In one or more embodiments of the present invention, the concentration of the fucosidase in the enzymatic reaction system is 0.025-10 μM.

[0077] In one or more embodiments of the present invention, the concentration of the sample is 1-10 mM.

[0078] In one or more embodiments of the present invention, the analyzing the product of the enzymatic hydrolysis reaction comprises analyzing the product of the enzymatic hydrolysis reaction by at least one of silica gel thin layer chromatography, high performance liquid chromatography, ion chromatography, and mass spectrometry.

[0079] In one or more embodiments of the present invention, the sugars in the sample include one or more disaccharides, wherein disaccharides refer to disaccharides generally recognized by those skilled in the art, i.e., sugars formed by two molecules of monosaccharides through glycosidic bonds, and there is no particular limitation on monosaccharides and glycosidic bonds.

[0080] The fourth aspect of the present invention provides the use of fucosidase PgoldGH29C in the degradation of fucosyl disaccharides, wherein the degradation of fucosyl disaccharides includes using the fucosidase as a catalyst to catalyze an enzymatic hydrolysis reaction of a substrate, the amino acid sequence of the fucosidase is shown in SEQ ID NO.1, the fucosyl disaccharide comprises a first monosaccharide group and a second monosaccharide group connected by a glycosidic bond, the first monosaccharide group is a fucose group, and the glycosidic bond comprises at least one of an α-1,2 glycosidic bond, an α-1,3 glycosidic bond, and an α-1,4 glycosidic bond.

[0081] Wherein, SEQ ID NO.1 is:

[0082] MKYSFVTILFLALSLQLGYGQDLILPVPSNQPSSAQQKQIARKYGMFIHFGINTFHDQEWTDGSKPASSYRPTAIDADQWIKTAKDAGMKYVILVAKHHEGFCLWDSKYTEYDVANSGNKTNVIEEVAKACKKYDIGLGLYYSLWDRKVNADVKDKTLDAAYNEYMIKQLNELI DIVQPYTHIVEFWFDGGWEKEHERWPAREIYQTIKSREPECQIGINWTIGLPENPDAHPVLPENQKEGYPIRYFPSDFRLGDPYLPADNDPKLFSHDGKLYYMPWESTICISERWFYNTTDKKYKTVEELTGLYHQCTKNDNILILNCPPNREGKIRDADVTLLKELRKKIQM.

[0083] In one or more embodiments of the present invention, the second monosaccharide group is selected from at least one of a galactose group and an acetylglucose group.

[0084] In one or more embodiments of the present invention, the enzymatic hydrolysis reaction is carried out at pH 4-6.

[0085] In one or more embodiments of the present invention, the enzymatic hydrolysis reaction is carried out at 35-40°C.

[0086] In one or more embodiments of the present invention, the enzymatic hydrolysis reaction time is 3-6 hours.

[0087] In one or more embodiments of the present invention, the concentration of the fucosidase in the enzymatic reaction system is 0.025-10 μM.

[0088] In one or more embodiments of the present invention, the concentration of the substrate is 1-10 mM.

[0089] In one or more embodiments of the present invention, the substrate contains the fucosyl disaccharide and at least one other disaccharide. The other disaccharide refers to a disaccharide generally known to those skilled in the art, that is, a sugar formed by two molecules of monosaccharide through a glycosidic bond, and there is no particular limitation on the monosaccharide and the glycosidic bond.

[0090] The fifth aspect of the present invention provides a method for degrading fucosylated disaccharides, comprising using fucosidase PgoldGH29C as a catalyst to catalyze an enzymatic hydrolysis reaction of a substrate, wherein the amino acid sequence of the fucosidase is as shown in SEQ ID NO.1, and the fucosylated disaccharide comprises a first monosaccharide group and a second monosaccharide group connected by a glycosidic bond, wherein the first monosaccharide group is a fucose group, and the glycosidic bond comprises at least one of an α-1,2 glycosidic bond, an α-1,3 glycosidic bond, and an α-1,4 glycosidic bond.

[0091] Wherein, SEQ ID NO.1 is:

[0092] MKYSFVTILFLALSLQLGYGQDLILPVPSNQPSSAQQKQIARKYGMFIHFGINTFHDQEWTDGSKPASSYRPTAIDADQWIKTAKDAGMKYVILVAKHHEGFCLWDSKYTEYDVANSGNKTNVIEEVAKACKKYDIGLGLYYSLWDRKVNADVKDKTLDAAYNEYMIKQLNELI DIVQPYTHIVEFWFDGGWEKEHERWPAREIYQTIKSREPECQIGINWTIGLPENPDAHPVLPENQKEGYPIRYFPSDFRLGDPYLPADNDPKLFSHDGKLYYMPWESTICISERWFYNTTDKKYKTVEELTGLYHQCTKNDNILILNCPPNREGKIRDADVTLLKELRKKIQM.

[0093] In one or more embodiments of the present invention, the second monosaccharide group is selected from at least one of a galactose group and an acetylglucose group;.

[0094] In one or more embodiments of the present invention, the substrate contains the fucosyl disaccharide and at least one other disaccharide. The other disaccharide refers to a disaccharide generally known to those skilled in the art, that is, a sugar formed by two molecules of monosaccharide through a glycosidic bond, and there is no particular limitation on the monosaccharide and the glycosidic bond.

[0095] In one or more embodiments of the present invention, the enzymatic hydrolysis reaction is carried out at pH 4-6.

[0096] In one or more embodiments of the present invention, the enzymatic hydrolysis reaction is carried out at 35-40°C.

[0097] In one or more embodiments of the present invention, the enzymatic hydrolysis reaction time is 3-6 hours.

[0098] In one or more embodiments of the present invention, the concentration of the fucosidase in the enzymatic reaction system is 0.025-10 μM.

[0099] In one or more embodiments of the present invention, the concentration of the substrate in the enzymatic reaction system is 1-10 mM.

[0100] Example 1 Heterologous Expression of PgoldGH29C in E. coli

[0101] 1. Experimental Materials

[0102] Escherichia coli TunerDE3, pET28a, purchased from Shanghai Angyu Biotechnology Co., Ltd. 2 mL disposable cobalt ion resin, purchased from Takara Co., Ltd. K-fucose kit was purchased from Megazyme Co., Ltd.

[0103] 2. Cloning, expression and purification methods

[0104] The DNA sequence of PgoldGH29C was removed from the signal peptide and the sequence (SEQID NO.2) after codon optimization in E. coli was cloned into the NdeI and XhoI sites of pET28a to obtain the plasmid pET28a-PgoldGH29C. The plasmid was transformed into competent cells TunerDE3 and cultured at 37°C overnight. The next day, a single clone was picked from the bacterial plate and inoculated into 10mL liquid LB medium, and cultured at 37°C and 180r / min for 16 hours to obtain seed liquid. 10mL of seed liquid was drawn into 1L LB medium, cultured at 140r / min and 37°C until OD600 was about 0.4, IPTG was added to a final concentration of 0.2mM, and then cultured at 140r / min and 16°C for 16h. The bacterial liquid was collected by centrifugation, the supernatant was discarded, the bacterial cells were resuspended with 20mL Tris Buffer (pH=8.0), and ultrasonically disrupted on ice at 220W for 1min. The supernatant was centrifuged to obtain CFE (Cell free extract) fraction, which was passed through a column according to the instructions. The purified fraction was dialyzed in Tris Buffer (pH=8.0) to remove imidazole.

[0105] SEQ ID NO.2:

[0106] CTGATCTTGCCGGTTCCATCTAACCAGCCGTCTTCTGCTCAGCAGAAACAGATCGCGCGT

[0107] AAATACGGTATGTTCATCCACTTCGGCATCAACACCTTCCACGACCAAGAATGGACCGAT

[0108] GGCTCTAAACCGGCGTCTTCTTACCGTCCGACCGCTATCGACGCTGACCAGTGGATCAA

[0109] GACTGCGAAAGACGCGGGTATGAAATACGTGATCTTGGTTGCGAAACACCACGAAGGT

[0110] TTCTGCCTGTGGGACTCTAAATACACCGAATACGACGTTGCGAACTCTGGTAACAAGAC

[0111] CAACGTTATCGAAGAAGTTGCGAAAGCGTGCAAAAAATACGATATCGGTCTGGGTCTGT

[0112] ACTACTCTCTGTGGGATCGTAAAGTTAACGCTGACGTTAAAGACAAGACTCTGGACGCA

[0113] GCATACAACGAATACATGATCAAACAGCTGAACGAACTGATCGACATCGTTCAGCCGTA

[0114] CACTCACATCGTTGAATTCTGGTTCGACGGTGGTTGGGAGAAAGAACACGAACGTTGG

[0115] CCAGCTCGTGAAATCTACCAGACCATCAAGTCTCGTGAACCGGAATGCCAGATCGGTAT

[0116] CAACTGGACCATCGGTCTGCCGGAGAATCCAGACGCTCATCCGGTACTTCCGGAGAACC

[0117] AGAAAGAAGGTTATCCGATCCGTTACTTTCCGTCTGACTTCCGTCTGGGTGATCCATACC

[0118] TGCCGGCTGACAACGACCCGAAACTGTTCAGCCACGATGGTAAACTGTACTACATGCCA

[0119] TGGGAATCTACCATCTGCATCTCTGAACGTTGGTTCTACAACACCACTGACAAGAAGTA

[0120] CAAGACCGTTGAAGAACTGACCGGTCTGTACCACCAGTGCACCAAGAACGACAACATT

[0121] CTGATTCTGAACTGTCCGCCGAACCGTGAAGGTAAGATCCGTGATGCGGACGTAACTCTGCTGAAAGAACTGCGTAAGAAGATCCAGATGTAA.

[0122] Liquid LB medium: by mass fraction (g / 100mL), including 0.5% yeast powder, 1% tryptone, 1% sodium chloride, pH 7.0, the solvent is water, and the preparation method is to mix the components evenly and sterilize at 121°C for 20 minutes.

[0123] Solid LB medium: calculated by mass fraction (g / 100mL), including 0.5% yeast powder, 1% tryptone, 1% sodium chloride, 1.5% agar, pH 7.0, solvent is water, and the preparation method is to mix the components evenly and sterilize at 121°C for 20 minutes.

[0124] The purified PgoldGH29C was subjected to SDS-PAGE detection, and the results were as follows Figure 1 As shown, NB is the unbound fraction, W1 is the washing fraction of Tris Buffer (pH=8.0), W2 is the washing fraction of Tris Buffer (pH=8.0) containing 10mM imidazole, E1 is the elution fraction of Tris Buffer (pH=8.0) containing 20mM imidazole, E2 and E3 are the elution fractions of Tris Buffer (pH=8.0) containing 100mM imidazole, and E4 is the elution fraction of Tris Buffer (pH=8.0) containing 200mM imidazole. The rightmost lane is the insoluble fraction obtained by centrifugation and taking the supernatant. Most of the purified protein is located in the E2 fraction, and its band position is consistent with its theoretical molecular weight of 40.2kDa.

[0125] Example 2 PgoldGH29C acts on 2-chloro-4-nitrobenzene-alpha-L-fucoside

[0126] Enzyme reaction conditions: The enzyme was incubated with 1mM 2-chloro-4-nitrobenzene-alpha-L-fucoside (CNP-FUC) in citric acid-sodium phosphate buffer (pH=3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8) at 37°C for 4h, and the final enzyme concentration was 0.025μM. The concentration of 2-chloro-4-nitrobenzene released by the enzyme reaction was linearly related to the absorbance at 405nm. The product concentration in the enzyme reaction was obtained from the standard curve, and the initial reaction rate was calculated. Figure 2 It was found that the optimum pH of PgoldGH29C is 4.5.

[0127] Example 3 PgoldGH29C acts on different fucoidan oligosaccharides

[0128] In order to test the specificity of PgoldGH29C to different glycosidic bonds, different fucoidan oligosaccharides were used as substrates for enzymatic hydrolysis. The fucoidan oligosaccharides used in this example are shown in the following table:

[0129]

[0130]

[0131] Enzyme reaction conditions: The enzyme and substrate were incubated in citric acid-sodium phosphate buffer (pH = 4.5) at 37°C for 4 h, the final enzyme concentration was 10 μM, the final concentration of disaccharide substrate was 6.88 mM, and the final concentration of trisaccharide and above substrates was 2 mM. Deionized water was used instead of enzyme in the negative control.

[0132] Take 8 μg of fucose standard and 4 μL of reaction and spot on silica gel thin layer chromatography plate (Merck, catalog number 105729). The developing agent is n-butanol: acetic acid: water with a volume ratio of 2:1:1. The developing time is about 1 hour. The color development method is to blow dry, spray the color developer, and heat at 85℃ for 10 minutes. The formula of the color developer is: 200mL acetone, 4mL aniline, 4g diphenylamine, 20mL 85% phosphoric acid.

[0133] The results of silica gel thin layer chromatography are as follows Figure 3 As shown, each TLC is from left to right: fucose standard, enzyme reaction, negative control. The results show that the enzyme has a significant effect on disaccharides connected by α-1,2 glycosidic bonds, α-1,3 glycosidic bonds, and α-1,4 glycosidic bonds, has no α-1,6 fucosidase activity, and has a significantly reduced effect on trisaccharides and above.

[0134] The enzymatic hydrolysis effect was quantitatively analyzed as follows:

[0135] Quantitative analysis of fucose released by hydrolysis: According to the instructions of Megazyme's k-fucose kit, 10 μL of fucose standard and enzymatic hydrolysis products were mixed with 250 μL of buffer reagent and NADP+, and the absorbance A1 value was recorded after A340 nm stabilized. Then 5 μL of L-fucose dehydrogenase was added, and the absorbance A2 value was recorded after A340 nm stabilized. The standard curve was drawn using the absorbance difference A2-A1 and the fucose concentration, and the fucose concentration in the enzymatic hydrolysis reaction was calculated based on this. Among them, the enzymatic hydrolysis percentage = fucose concentration in the enzymatic hydrolysis reaction / substrate concentration.

[0136] The quantitative analysis of the enzymatic hydrolysis effect is shown in the following table. The enzymatic hydrolysis percentages of disaccharides linked by α-1,2 glycosidic bonds, α-1,3 glycosidic bonds, and α-1,4 glycosidic bonds are all above 60%, and the enzymatic hydrolysis percentage of disaccharides linked by α-1,6 glycosidic bonds is 3%. The effect on trisaccharides and above is significantly reduced, which corresponds to the results of silica gel thin layer chromatography.

[0137]

[0138] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of fucosidase in preparing a detection probe, wherein the detection probe is used to detect fucosyl disaccharide in a sample, wherein the amino acid sequence of the fucosidase is shown in SEQ ID NO.1, wherein the fucosyl disaccharide comprises a first monosaccharide group and a second monosaccharide group connected by a glycosidic bond, wherein the first monosaccharide group is a fucose group; and the glycosidic bond comprises at least one of an α-1,2 glycosidic bond, an α-1,3 glycosidic bond, and an α-1,4 glycosidic bond.

2. The use according to claim 1, characterized in that: The second monosaccharide group is selected from at least one of a galactose group and an acetyl glucose group; Preferably, the sugars in the sample comprise one or more disaccharides.

3. Use of fucosidase in the preparation of a detection kit, wherein the detection kit is used to detect fucosyl disaccharide in a sample, wherein the amino acid sequence of the fucosidase is as shown in SEQ ID NO.1, and the fucosyl disaccharide comprises a first monosaccharide group and a second monosaccharide group connected by a glycosidic bond, wherein the first monosaccharide group is a fucose group; and the glycosidic bond comprises at least one of an α-1,2 glycosidic bond, an α-1,3 glycosidic bond, and an α-1,4 glycosidic bond.

4. The use according to claim 3, characterized in that: The second monosaccharide group is selected from at least one of a galactose group and an acetyl glucose group; Preferably, the sugars in the sample comprise one or more disaccharides.

5. A detection method for fucosyl disaccharide in a sample, the method comprising: Perform enzymatic hydrolysis reaction with the sample using fucosidase; Analyze the products of enzymatic hydrolysis reactions; The amino acid sequence of the fucosidase is shown in SEQ ID NO.1, the fucosyl disaccharide comprises a first monosaccharide and a second monosaccharide connected by a glycosidic bond, the first monosaccharide is a fucose group; the glycosidic bond comprises at least one of an α-1,2 glycosidic bond, an α-1,3 glycosidic bond, and an α-1,4 glycosidic bond.

6. The detection according to claim 5, characterized in that The second monosaccharide group is selected from at least one of a galactose group and an acetyl glucose group.

7. The detection according to claim 5, characterized in that The enzymatic hydrolysis reaction is carried out at a pH of 4-6; Preferably, the enzymatic hydrolysis reaction is carried out at 35-40°C; Preferably, the enzymatic reaction time is 3-6h; Preferably, the concentration of the fucosidase in the enzymatic reaction system is 0.025-10 μM; preferably, The concentration of the sample is 1-10 mM; Preferably, the analyzing the product of the enzymatic hydrolysis reaction comprises analyzing the product of the enzymatic hydrolysis reaction by at least one of silica gel thin layer chromatography, high performance liquid chromatography, ion chromatography, and mass spectrometry.

8. The detection according to claim 5, characterized in that The sugars in the sample include one or more disaccharides.

9. Use of a fucosidase in degrading fucosyl disaccharide, wherein the degradation of the fucosyl disaccharide comprises using the fucosidase as a catalyst to catalyze an enzymatic hydrolysis reaction of a substrate, wherein the amino acid sequence of the fucosidase is as shown in SEQ ID NO.1, wherein the fucosyl disaccharide comprises a first monosaccharide group and a second monosaccharide group connected by a glycosidic bond, wherein the first monosaccharide group is a fucose group, and the glycosidic bond comprises at least one of an α-1,2 glycosidic bond, an α-1,3 glycosidic bond, and an α-1,4 glycosidic bond; Preferably, the second monosaccharide group is selected from at least one of a galactose group and an acetyl glucose group; Preferably, the enzymatic hydrolysis reaction is carried out at pH 4-6; Preferably, the enzymatic hydrolysis reaction is carried out at 35-40°C; Preferably, the enzymatic reaction time is 3-6h; Preferably, the concentration of the fucosidase in the enzymatic reaction system is 0.025-10 μM; Preferably, the concentration of the substrate is 1-10 mM; Preferably, the substrate contains the fucosyl disaccharide and at least one other disaccharide.

10. A method for degrading fucosyl disaccharide, comprising using fucosidase as a catalyst to catalyze an enzymatic hydrolysis reaction of a substrate, wherein the amino acid sequence of the fucosidase is as shown in SEQ ID NO.1, and the fucosyl disaccharide comprises a first monosaccharide group and a second monosaccharide group connected by a glycosidic bond, wherein the first monosaccharide group is a fucose group, and the glycosidic bond comprises at least one of an α-1,2 glycosidic bond, an α-1,3 glycosidic bond, and an α-1,4 glycosidic bond; Preferably, the second monosaccharide group is selected from at least one of a galactose group and an acetyl glucose group; Preferably, the substrate contains the fucosyl disaccharide and at least one other disaccharide; Preferably, the enzymatic hydrolysis reaction is carried out at pH 4-6; Preferably, the enzymatic hydrolysis reaction is carried out at 35-40°C; Preferably, the enzymatic reaction time is 3-6h; Preferably, the concentration of the fucosidase in the enzymatic reaction system is 0.025-10 μM; Preferably, the concentration of the substrate in the enzymatic reaction system is 1-10 mM.

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