Fucoidin degrading enzyme OUC-MgFucD1-H359D as well as coding gene and application thereof

Abstract

The invention discloses a fucoidin degrading enzyme OUC-MgFucD1-H359D as well as a coding gene and application thereof, and belongs to the technical field of hydrolase. The amino acid sequence of the fucoidin degrading enzyme OUC-MgFucD1-H359D is as shown in SEQ ID NO. 31, and the nucleotide sequence of the coding gene of the fucoidin degrading enzyme OUC-MgFucD1-H359D is as shown in SEQ ID NO. 32. The invention further discloses the application of the fucoidin degrading enzyme OUC-MgFucD1-H359D in the degradation of fucoidin. The fucoidin degrading enzyme OUC-MgFucD1-H359D which is higher in stability and better in pH tolerance is constructed through a calculation driving design strategy, the fucoidin degrading enzyme OUC-MgFucD1-H359D still keeps more than 80% of activity after being incubated for 84 hours at the temperature of 30 DEG C, and the pH tolerance range is widened to 7.0-9.0. The invention provides an efficient and stable enzymolysis tool for large-scale preparation of the fucoidan oligosaccharide.

Description

Technical Field

[0001] The present invention relates to a fucoidan-degrading enzyme OUC-MgFucD1-H359D, its encoding gene and applications, and belongs to the technical field of hydrolases. Background Art

[0002] CN 119913226 A discloses the application of fucoidan-degrading enzyme OUC-MgFucD1 in the preparation of fucosylligosaccharides. By using the unique substrate recognition mechanism of fucoidan-degrading enzyme OUC-MgFucD1, fucoidan is degraded into fucose with one sulfate group, fucobiose with two sulfate groups, and fucotriose with one sulfate group. However, the fucoidan-degrading enzyme OUC-MgFucD1 has the following two deficiencies, which limit its industrial application: one is insufficient thermal stability: its residual activity rate is only 20% after incubation at 30 °C for 72 hours, and it is almost completely inactivated after 84 hours. When applied, enzyme preparations need to be frequently supplemented, increasing production costs; the other is a narrow pH application range: it only maintains high activity near pH 7.5 and is sensitive to fluctuations in the pH of the reaction system. Therefore, there is an urgent need for a fucoidan-degrading enzyme with stronger thermal stability and better pH tolerance. Summary of the Invention

[0003] In view of the above-mentioned prior art, the present invention provides a fucoidan-degrading enzyme OUC-MgFucD1-H359D, its encoding gene and applications, and belongs to the technical field of hydrolases.

[0004] The present invention is achieved by the following technical solutions: A fucoidan-degrading enzyme OUC-MgFucD1-H359D, whose amino acid sequence is as shown in SEQ ID NO.31.

[0005] The encoding gene of the fucoidan-degrading enzyme OUC-MgFucD1-H359D, whose nucleotide sequence is as shown in SEQ ID NO.32.

[0006] The application of the fucoidan-degrading enzyme OUC-MgFucD1-H359D in degrading fucoidan.

[0007] Further, the degradation products are fucose (FucS), fucobiose (Fuc2S2), and fucotriose (Fuc3S); the molecular formula of fucose is C6H 12 O8S, the molecular formula of fucobiose is C 12 H 22 O 15 S2, and the molecular formula of fucotriose is C 18 H 32 O 16 S.

[0008] In view of the industrial application bottlenecks of the wild-type fucoidan-degrading enzyme OUC-MgFucD1, such as poor thermal stability and narrow pH application range, through a computational-driven design strategy, a fucoidan-degrading enzyme OUC-MgFucD1-H359D with stronger stability and better pH tolerance was successfully constructed. On the premise of maintaining the specific catalytic function of the wild enzyme, it still retains more than 80% of its activity after incubation at 30 °C for 84 hours, and the pH tolerance range is broadened to 7.0 - 9.0; the present invention also explains the mechanism of its enhanced stability at the molecular level. The fucoidan-degrading enzyme OUC-MgFucD1-H359D of the present invention is suitable for industrial production of high-value fucosaccharides. The present invention provides an efficient and stable enzymatic hydrolysis tool for large-scale preparation of high-purity fucosaccharides, which can accelerate the industrial application process in fields such as targeted drug delivery systems and functional food additives.

[0009] All terms and phrases used in the present invention have the general meanings well-known to those skilled in the art. Description of the Drawings

[0010] Figure 1 : Mutation sites predicted by three online tools.

[0011] Figure 2 : Agarose gel electrophoresis detection results of 14 mutant plasmids. Among them, M represents the standard DNA Marker, "bp" represents base pairs, and lanes 1 - 14 represent the mutant plasmids corresponding to E19Y, D39E, R57F, S59H, L98F, K181A, K213N, E220D, A255G, W256Q, N275E, S300V, L344I, H359D in sequence.

[0012] Figure 3 : SDS-PAGE detection results of crude enzyme solutions of the wild enzyme and 14 mutant enzymes. Among them, M represents the standard protein Marker, "kDa" represents molecular weight, lane 1 represents the wild enzyme, and lanes 2 - 15 represent MgFucD1 (E19Y), MgFucD1 (D39E), MgFucD1 (R57F), MgFucD1 (S59H), MgFucD1 (L98F), MgFucD1 (K181A), MgFucD1 (K213N), MgFucD1 (E220D), MgFucD1 (A255G), MgFucD1 (W256Q), MgFucD1 (N275E), MgFucD1 (S300V), MgFucD1 (L344I), MgFucD1 (H359D) in sequence.

[0013] Figure 4: Relative enzyme activities of wild enzyme and 14 mutant enzymes, where M represents the wild enzyme, and 1 - 14 represent MgFucD1(E19Y), MgFucD1(D39E), MgFucD1(R57F), MgFucD1(S59H), MgFucD1(L98F), MgFucD1(K181A), MgFucD1(K213N), MgFucD1(E220D), MgFucD1(A255G), MgFucD1(W256Q), MgFucD1(N275E), MgFucD1(S300V), MgFucD1(L344I), MgFucD1(H359D) in sequence.

[0014] Figure 5 : Relative enzyme activities of wild enzyme and 14 mutant enzymes after heat treatment, where M represents the wild enzyme, and 1 - 14 represent MgFucD1(E19Y), MgFucD1(D39E), MgFucD1(R57F), MgFucD1(S59H), MgFucD1(L98F), MgFucD1(K181A), MgFucD1(K213N), MgFucD1(E220D), MgFucD1(A255G), MgFucD1(W256Q), MgFucD1(N275E), MgFucD1(S300V), MgFucD1(L344I), MgFucD1(H359D) in sequence.

[0015] Figure 6 : SDS - PAGE electrophoresis detection results of pure enzyme solutions of wild enzyme and 4 mutant enzymes, where M represents the standard protein Marker, "kDa" represents the molecular weight, lane 1 represents the wild enzyme, and lanes 2 - 5 represent MgFucD1(K181A), MgFucD1(K213N), MgFucD1(N275E) and MgFucD1(H359D) in sequence.

[0016] Figure 7 : Relative enzyme activities of wild enzyme and 4 mutant enzymes after heat treatment for different times, where "MgFucD1" represents the wild enzyme, the same below.

[0017] Figure 8 : Schematic diagram of the effect of reaction temperature on relative enzyme activity.

[0018] Figure 9 : Schematic diagram of the effect of reaction pH on relative enzyme activity.

[0019] Figure 10 : Schematic diagram of the comparison of Tm values between wild enzyme and MgFucD1(H359D).

[0020] Figure 11: Schematic diagram of the product composition of wild enzyme and MgFucD1 (H359D).

[0021] Figure 12 : Schematic diagram of the comparison of molecular dynamics RMSD between wild enzyme and MgFucD1 (H359D) at 303 K.

[0022] Figure 13 : Schematic diagram of the comparison of molecular dynamics RMSD between wild enzyme and MgFucD1 (H359D) at 333 K.

[0023] Figure 14 : Schematic diagram of the comparison of molecular dynamics RMSF between wild enzyme and MgFucD1 (H359D) at 303 K.

[0024] Figure 15 : Schematic diagram of the comparison of molecular dynamics RMSF between wild enzyme and MgFucD1 (H359D) at 333 K.

[0025] Figure 16 : Schematic diagram of the comparison of molecular dynamics Rg between wild enzyme and MgFucD1 (H359D) at 303 K.

[0026] Figure 17 : Schematic diagram of the comparison of molecular dynamics Rg between wild enzyme and MgFucD1 (H359D) at 333 K.

[0027] Figure 18 : Schematic diagram of the comparison of molecular dynamics SASA between wild enzyme and MgFucD1 (H359D) at 303 K.

[0028] Figure 19 : Schematic diagram of the comparison of molecular dynamics SASA between wild enzyme and MgFucD1 (H359D) at 333 K. Detailed implementation manners

[0029] The present invention will be further described below in conjunction with embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.

[0030] The instruments, reagents, and materials involved in the following embodiments, unless otherwise specified, are all conventional instruments, reagents, and materials existing in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, are all conventional experimental methods and detection methods existing in the prior art.

[0031] The wild enzyme plasmid pET28a(+)-OUC-MgFucD1 used in the present invention was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and preserved in the laboratory where the inventors are located.

[0032] The Escherichia coli DH5α competent cells used in the present invention were purchased from Beijing Tsingke Biotechnology Co., Ltd.

[0033] The Escherichia coli BL21(DE3) competent cells used in the present invention were purchased from Beijing Tsingke Biotechnology Co., Ltd.

[0034] The composition of the LB medium used in the present invention is as follows: sodium chloride 10 g / L; tryptone 10 g / L; yeast extract 5 g / L; agar powder 15 g / L (added when preparing solid medium); the balance is water; sterilization conditions: 115°C, 30 min.

[0035] The pHBH powder used in the present invention was purchased from Sigma-aldrich Co., Ltd.

[0036] The rapid plasmid miniprep kit used in the present invention was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0037] The fucoidan used in the present invention was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0038] Example 1 Prediction of potential thermostable sites Fucoidan is a sulfated polysaccharide of marine origin, with unique antioxidant, anticoagulant, anti-inflammatory and immune activation functions, and has attracted extensive attention in the fields of biomedicine, functional foods, etc. in recent years. However, the high molecular weight characteristics of such polysaccharides (usually 50-100 kDa) lead to poor solubility and low bioavailability, seriously restricting their in vivo targeting delivery efficiency. Research shows that enzymatically hydrolyzing fucoidan into fucose or fuco-oligosaccharides with a degree of polymerization of 2-6 (molecular weight < 3 kDa) can expose more active sulfate groups, thereby significantly enhancing its binding ability to cell surface receptors and amplifying anti-tumor, anti-viral, anti-hypoxia and other targeting activities.

[0039] According to the record of CN 119913226 A, the scientific research team where the inventors of the present application are located screened a fucoidan-degrading enzyme OUC-MgFucD1 with a unique substrate recognition mechanism. Its amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2; it can degrade fucoidan into fucose, fucobiose and fucotriose. However, its thermal stability is insufficient and the applicable pH range is narrow. The present invention attempts to mutate and transform it in order to obtain mutants with stronger thermal stability and better pH tolerance to meet the requirements of long-term stability of enzyme preparations for industrial production.

[0040] The amino acid sequence of the fucoidan-degrading enzyme OUC-MgFucD1 is shown below, as shown in SEQ ID NO.1: NTSESEWLLGSWGVRLIVEGGVELDKASKSSDWVKGAQDIVDNLPTVGHVFTNFNHRASGYWFTLRDNPYVDIAKEIHPDFVPSLENEQIILDVIDVLKKAGKKVILYIATDGPSARSGTKDNAEYKTAWENYYNEKFNGDEGLAYRTLCRGFIERFKGLADGYWLDHTSGIAGELPDFIKMIKEVDPTVIIASNGIVNNENTSPHNYFKDEKGEFLLVESDGVDDQDDRHYKIRSFNTDDLYTDFTSGHPTPLAWGAPPNSWAYEEFTFPEIANAMTNYEVTKDNIKHAWMPMRMKWTSPKAELMFDTEQAYRFVRTLTDAGCAITWGNTNTYGFITEDEMVLMKEIDKRLQIKPMPHYVPYKRPEGAKLVGEDKEN.

[0041] The nucleotide sequence of the encoding gene of the fucoidan-degrading enzyme OUC-MgFucD1 is shown below (direction 5'-3'), as shown in SEQ ID NO.2:

[0042] First, the three-dimensional structure of the wild enzyme, fucoidan-degrading enzyme OUC-MgFucD1, was predicted using AlphaFold3 with a pTM of 0.94 for subsequent computational design.

[0043] Then, potential stabilizing mutations of the fucoidan-degrading enzyme OUC-MgFucD1 were predicted using three online tools, PROSS, HotSpot Wizard 3, and FireProt 2.0, and 43, 22, and 36 mutation sites were identified respectively.

[0044] The website of PROSS is: https: / / pross.weizmann.ac.il / step / pross-terms / .

[0045] The website of HotSpot Wizard 3 is: https: / / loschmidt.chemi.muni.cz / hotspotwizard / .

[0046] The website of FireProt 2.0 is: https: / / loschmidt.chemi.muni.cz / fireprotweb / .

[0047] Subsequently, all single mutations predicted by the above three online tools were compared, and 14 mutation sites recommended by at least two tools were selected, namely E19Y, D39E, R57F, S59H, L98F, K181A, K213N, E220D, A255G, W256Q, N275E, S300V, L344I, H359D. The mutation sites predicted by the three online tools are as Figure 1 shown.

[0048] Example 2 Construction and Induced Expression of Mutant Enzymes According to the prediction in Example 1, the mutant enzymes corresponding to each mutation site were constructed and induced for expression. The 14 mutant enzymes were temporarily named in sequence as: MgFucD1(E19Y), MgFucD1(D39E), MgFucD1(R57F), MgFucD1(S59H), MgFucD1(L98F), MgFucD1(K181A), MgFucD1(K213N), MgFucD1(E220D), MgFucD1(A255G), MgFucD1(W256Q), MgFucD1(N275E), MgFucD1(S300V), MgFucD1(L344I), MgFucD1(H359D); The steps are as follows: (1) The mutants were obtained by one-step PCR amplification with the wild enzyme plasmid pET28a(+)-OUC-MgFucD1 as the template and specific primers for each mutation site. The nucleotide sequences of the specific primers for 14 mutation sites are shown in Table 1, successively as SEQ ID NO.3 - 30, and the specific primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The PCR reaction system was: 20 μL of water, 25 μL of P525 enzyme, 2 μL of each specific primer, 1 μL of template, and the total system was 50 μL. The PCR reaction conditions were: pre-denaturation at 95°C for 5 min, denaturation at 98°C for 10 s, annealing at 55°C for 15 s, extension at 72°C for 4 min, and 30 cycles of reaction. The PCR products were verified by agarose gel electrophoresis. The agarose gel electrophoresis detection results of 14 mutant plasmids are as Figure 2 shown. The base pairs of the mutant plasmids were approximately 6500 bp, which was consistent with the theoretical value, indicating that 14 mutant plasmids were successfully constructed.

[0049] Table 1 Nucleotide sequences of specific primers for 14 mutation sites

[0050]

[0051] (2) The wild enzyme plasmid and each mutant plasmid were separately transformed into Escherichia coli DH5α competent cells, spread on solid LB medium plates containing 50 μg / mL kanamycin sulfate, and cultured in an incubator at 37°C for 16 h. Single colonies were picked and transferred into liquid LB medium containing 50 μg / mL kanamycin sulfate, and cultured on a shaker at 37°C and 220 rpm for 8 h. Plasmids were extracted using a rapid plasmid mini-prep kit and verified by sequencing.

[0052] (3) The plasmids with correct sequencing results were transformed into Escherichia coli BL21(DE3) competent cells, and the constructed recombinant engineering bacteria grew on the kanamycin sulfate resistance plates.

[0053] (4) Single colonies were picked and inoculated into 5 ml of liquid LB medium containing 50 μg / mL kanamycin sulfate, and cultured on a shaker at 37°C and 220 rpm for 12 h for activation. Then, they were inoculated into 50 mL of liquid LB medium containing 50 μg / mL kanamycin sulfate at an inoculation amount of 1% (volume percentage), and cultured on a shaker at 37°C and 220 rpm until the OD 600 value of the bacterial liquid reached 0.6. 0.5‰ Isopropyl-β-D-thiogalactoside (IPTG) (100 mM) was added, and induced expression was carried out on a shaker at 20°C and 220 rpm for 18 h.

[0054] (5) After the cultivation was completed, the culture medium was taken, centrifuged at 8000 rpm for 10 min at 4°C, the cells were collected, resuspended in ultrapure water, sonicated for 15 min, and centrifuged at 9000 rpm for 10 min. The supernatant was the crude enzyme solution. SDS-PAGE electrophoresis was used to detect the crude enzyme solutions of the wild enzyme and 14 mutant enzymes. The SDS-PAGE detection results of the crude enzyme solutions of the wild enzyme and 14 mutant enzymes are as Figure 3 shown. It can be seen that all mutant enzymes were correctly expressed (43 kDa).

[0055] Example 3 Preliminary Screening by Heat Treatment and Determination of Enzyme Activity The crude enzyme solutions of the wild enzyme and 14 mutant enzymes obtained in Example 2 were assayed for enzyme activity. The assay method was as follows: 10 μL of the crude enzyme solution was added to 65 μL of the substrate solution (prepared from fucoidan, CaCl2, NaCl, and Tris-HCl buffer with a pH of 7.5, where the concentration of fucoidan was 2 mg / mL, the concentration of CaCl2 was 20 mM, the concentration of NaCl was 25 mM, and the concentration of Tris-HCl was 20 mM). After mixing evenly, the reaction was carried out at 30°C for 1 h, and the reaction was terminated by boiling water bath for 5 min. 225 μL of water was added to dilute the reaction solution, and then 100 μL of 4-hydroxybenzoyl hydrazide (pHBH) solution was added, followed by boiling water bath for 5 min. The reaction solution was cooled to room temperature, centrifuged, and the supernatant was taken to measure OD 415 . The control group was replaced with an inactivated enzyme solution.

[0056] The preparation method of the pHBH solution was as follows: Weigh the pHBH powder and dissolve it in 2 M hydrochloric acid to prepare a pHBH stock solution with a concentration of 0.2 g / mL. The pHBH stock solution was mixed with 2 M NaOH solution in a volume ratio of 1:9 to obtain the pHBH solution. This solution should be prepared and used immediately.

[0057] The enzyme activity (U) was defined as: The amount of enzyme required to produce 1 μg of reducing sugar per minute under standard conditions was 1 U.

[0058] Taking the enzyme activity of the wild enzyme as 100%, the relative enzyme activities of the 14 mutant enzymes were calculated. The relative enzyme activities of the wild enzyme and 14 mutant enzymes are as Figure 4 shown. Compared with the wild enzyme, there were 7 mutant enzymes with relative enzyme activities greater than 80%, which were: MgFucD1 (S59H), MgFucD1 (K181A), MgFucD1 (K213N), MgFucD1 (N275E), MgFucD1 (S300V), MgFucD1 (L344I), MgFucD1 (H359D).

[0059] Then, the crude enzyme solutions of the wild-type enzyme and 14 mutant enzymes were heat-treated: incubated at 30 °C for 12 hours. The residual enzyme activities of the wild-type enzyme and 14 mutant enzymes after heat treatment were measured using the same method as above. Taking their respective initial enzyme activities as 100%, the relative enzyme activities were calculated.

[0060] The relative enzyme activities of the wild-type enzyme and 14 mutant enzymes after heat treatment are as Figure 5 shown. There are 8 mutant enzymes with residual enzyme activities greater than 80%, which are, in order: MgFucD1 (D39E), MgFucD1 (L98F), MgFucD1 (K181A), MgFucD1 (K213N), MgFucD1 (E220D), MgFucD1 (W256Q), MgFucD1 (N275E), MgFucD1 (H359D).

[0061] There are 4 mutant enzymes that meet the above two conditions, which are, in order: MgFucD1 (K181A), MgFucD1 (K213N), MgFucD1 (N275E), and MgFucD1 (H359D). These 4 mutant enzymes exhibit good thermal stability without sacrificing enzyme activity. Their crude enzyme solutions were purified for further study.

[0062] Example 4 Study on the Thermal Stability of Mutant Enzymes The crude enzyme solutions of the 4 mutant enzymes MgFucD1 (K181A), MgFucD1 (K213N), MgFucD1 (N275E), and MgFucD1 (H359D) were purified by affinity chromatography using a Ni 2+ -NTA column: First, the column was equilibrated with a 10 mM imidazole solution (10 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl, pH 7.5). The crude enzyme solution was added to bind to the Ni 2+ -NTA column. Then, weakly bound impurity proteins were eluted with a 40 mM imidazole solution (40 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl). Then, the target protein was eluted with a 200 mM imidazole solution (200 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl), and the eluate was collected. Ultrafiltration concentration was performed using an ultrafiltration centrifugal tube with a molecular weight cut-off of 10 kDa at 4 °C and 4000 rpm to obtain a pure enzyme solution. SDS-PAGE electrophoresis was used to detect the pure enzyme solutions of the wild-type enzyme and 4 mutant enzymes. The SDS-PAGE electrophoresis results of the pure enzyme solutions of the wild-type enzyme and 4 mutant enzymes are as Figure 6 shown. It can be seen that the molecular weights of the mutant enzymes are consistent with the predicted values, which is 43 kDa. The protein concentrations of the pure enzyme solutions were measured and uniformly diluted with water to 1 mg / mL for standby.

[0063] The pure enzyme solutions of the wild enzyme and 4 mutant enzymes were incubated at 30 °C for 0 h, 2 h, 4 h, 10 h, 16 h, 24 h, 36 h, 48 h, 60 h, 72 h, and 84 h respectively, and the enzyme activities were measured according to the method of Example 3.

[0064] It was determined that before heat treatment (incubation for 0 h), the enzyme activities of the wild enzyme, MgFucD1 (K181A), MgFucD1 (K213N), MgFucD1 (N275E), and MgFucD1 (H359D) were 112.87 U / mg, 110.23 U / mg, 119.18 U / mg, 111.25 U / mg, and 136.79 U / mg respectively. It can be seen that MgFucD1 (H359D) has the highest enzyme activity.

[0065] Taking the enzyme activity before heat treatment of each enzyme as 100%, the relative enzyme activities of the wild enzyme and 4 mutant enzymes after heat treatment for different times were calculated. The relative enzyme activities of the wild enzyme and 4 mutant enzymes after heat treatment for different times are as Figure 7 shown. It can be seen that after incubation at 30 °C for 84 h, MgFucD1 (H359D) still retains more than 80% of its activity, while the activity of the wild enzyme is almost reduced to 0; although the stabilities of the 3 mutant enzymes MgFucD1 (K181A), MgFucD1 (K213N), and MgFucD1 (N275E) are improved at each time period, the trend is almost the same as that of the wild enzyme. Therefore, MgFucD1 (H359D) has the best thermal stability. This mutant enzyme was officially named fucoidan-degrading enzyme OUC-MgFucD1-H359D. Its amino acid sequence is as shown in SEQ ID NO.31, and the nucleotide sequence of its encoding gene is as shown in SEQ ID NO.32.

[0066] The amino acid sequence of the fucoidan-degrading enzyme OUC-MgFucD1-H359D is as follows, as shown in SEQ ID NO.31: NTSESEWLLGSWGVRLIVEGGVELDKASKSSDWVKGAQDIVDNLPTVGHVFTNFNHRASGYWFTLRDNPYVDIAKEIHPDFVPSLENEQIILDVIDVLKKAGKKVILYIATDGPSARSGTKDNAEYKTAWENYYNEKFNGDEGLAYRTLCRGFIERFKGLADGYWLDHTSGIAGELPDFIKMIKEVDPTVIIASNGIVNNENTSPHNYFKDEKGEFLLVESDGVDDQDDRHYKIRSFNTDDLYTDFTSGHPTPLAWGAPPNSWAYEEFTFPEIANAMTNYEVTKDNIKHAWMPMRMKWTSPKAELMFDTEQAYRFVRTLTDAGCAITWGNTNTYGFITEDEMVLMKEIDKRLQIKPMPDYVPYKRPEGAKLVGEDKEN。

[0067] The nucleotide sequence of the encoding gene of fucoidan-degrading enzyme OUC-MgFucD1-H359D is shown below (direction 5'-3'), as shown in SEQ ID NO.32:

[0068] Example 5 Characterization of the Enzymatic Properties of Fucoidan-Degrading Enzyme OUC-MgFucD1-H359D The enzymatic properties of the fucoidan-degrading enzyme OUC-MgFucD1-H359D were studied, with the wild-type enzyme as a control, as described below.

[0069] (1) Effect of reaction temperature on relative enzyme activity Take 65 μL of the substrate solution (prepared in Example 3), add 10 μL of the pure enzyme solution (prepared in Example 4), and react at 4 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, and 60 °C for 1 h, and terminate the reaction by boiling water bath for 5 min. Determine the enzyme activity according to the method of Example 3. All experiments were performed in triplicate. Taking the highest enzyme activity as 100%, calculate the relative enzyme activities of the wild-type enzyme and MgFucD1 (H359D) under each temperature condition.

[0070] The schematic diagram of the effect of reaction temperature on relative enzyme activity is as Figure 8 shown. It can be seen that the optimal reaction temperature of MgFucD1 (H359D) is the same as that of the wild-type enzyme, both are 30 °C. However, in the non-optimal temperature range (especially the high-temperature region), the enzyme activity decay rate of MgFucD1 (H359D) is significantly lower than that of the wild-type enzyme, and it can still retain more than 60% of its activity at 60 °C. This characteristic improves its applicability in industrial scenarios with fluctuating environmental temperatures.

[0071] (2) Effect of reaction pH on relative enzyme activity Using buffers with different pH values as solvents, prepare fucoidan buffer solutions with different pH values (containing 2 mg / mL of fucoidan, 20 mM of CaCl2, and 25 mM of NaCl). The buffers with different pH values used are: 20 mM citrate-sodium citrate buffer (pH values are 3.0, 4.0, 5.0, 6.0), 20 mM phosphate buffer (pH values are 6.0, 7.0), 20 mM Tris-HCl buffer (pH values are 7.0, 7.5, 8.0, 8.5, 9.0), and 20 mM glycine-sodium hydroxide buffer (pH values are 9.0, 10.0, 11.0).

[0072] Take 65 μL of each fucoidan buffer solution with different pH values, add 10 μL of the pure enzyme solution (prepared in Example 4) to each, react at 30 °C for 1 h, terminate the reaction by boiling water bath for 5 min, and determine the enzyme activity according to the method of Example 3. All experiments were performed in triplicate. Taking the highest enzyme activity as 100%, calculate the relative enzyme activities of the wild-type enzyme and MgFucD1 (H359D) under each pH condition.

[0073] The schematic diagram of the effect of reaction pH on relative enzyme activity is as Figure 9 shown. It can be seen that the optimal reaction pH of the wild enzyme is 7.5; the optimal reaction pH of MgFucD1 (H359D) migrates to 8.0, and maintains more than 80% enzyme activity in the range of pH 7.0 - 9.0 (while the wild enzyme only maintains the same activity in the range of pH 7.0 - 7.5). This indicates that the mutation significantly broadens the pH adaptation ability of the enzyme, which is beneficial to reducing the pH regulation cost in industrial production.

[0074] (3) Thermodynamic stability analysis The thermodynamic stability of proteins can be evaluated by their melting temperature. The thermal denaturation temperature (Tm value) of the wild enzyme and MgFucD1 (H359D) was measured by differential scanning calorimetry (DSC): 8 mL of pure enzyme solution (prepared in Example 4) was taken and freeze-dried in a freeze dryer for 3 days; the protease powder was put into a sealed aluminum crucible, the test temperature range was set at 100 - 260 °C, and the heating and cooling rate was controlled at 2 °C / min to measure the Tm value. The schematic diagram of the comparison of the Tm values of the wild enzyme and MgFucD1 (H359D) is as Figure 10 shown. The Tm value of MgFucD1 (H359D) is 214.5 °C, which is 4.5 °C higher than that of the wild enzyme.

[0075] Example 6 Analysis of the products of the fucoidan-degrading enzyme OUC-MgFucD1-H359D Take 133 μL of the substrate solution, add 227 μL of ultrapure water, add 40 μL of pure enzyme solution (prepared in Example 4), mix well. The concentration of fucoidan in the reaction system is 2 mg / mL, the concentration of CaCl2 is 20 mM, the concentration of NaCl is 25 mM, the concentration of Tris-HCl is 20 mM, and the pH is 8. React at 30 °C for 24 h, terminate the reaction by boiling water bath for 10 min; centrifuge at 12000 rpm for 10 min to remove impurities such as proteins in the system, collect the supernatant, filter through a 0.22 μm filter membrane, and perform HPLC detection. The HPLC detection conditions are as follows: The chromatographic column uses a Superdex 30 Increase 10 / 300 GL column (10×300 - 310 mm) (GE Healthcare, USA), the mobile phase is 0.2 M NH4HCO3 solution, the flow rate is 0.4 mL / min, and the detector is a differential detector.

[0076] The substrate solution is prepared from fucoidan, CaCl2, NaCl, and Tris-HCl buffer with a pH of 8, wherein the concentration of fucoidan is 6 mg / mL, the concentration of CaCl2 is 60 mM, the concentration of NaCl is 75 mM, and the concentration of Tris-HCl is 60 mM.

[0077] Results: The schematic diagram of the product composition of the wild enzyme and MgFucD1 (H359D) is as follows Figure 11 shown. MgFucD1 (H359D) can degrade fucoidan into fucosooligosaccharides with different molecular weights and degrees of polymerization, and its product composition is exactly the same as that of the wild enzyme; however, through peak area calculation, the yields of each product have increased. Specifically, product I is fucotriose sulfate Fuc3S (molecular formula C 18 H 32 O 16 S) with one sulfate group. The peak area of the wild enzyme is 31592.611, and the peak area of MgFucD1 (H359D) is 36067.909; product II is fucobiose disulfate Fuc2S2 (molecular formula C 12 H 22 O 15 S2) with two sulfate groups. The peak area of the wild enzyme is 38127.513, and the peak area of MgFucD1 (H359D) is 40563.884; product III is fucose sulfate FucS (molecular formula C6H 12 O8S) with one sulfate group. The peak area of the wild enzyme is 12310.594, and the peak area of MgFucD1 (H359D) is 14017.120. It shows that the improved stability enables MgFucD1 (H359D) to maintain the active conformation in the reaction system for a longer time, and the measured product accumulation may be higher due to continuous catalysis.

[0078] Example 7 Molecular Dynamics Simulation of Wild Enzyme and MgFucD1 (H359D) Based on the GROMACS 2023 molecular dynamics platform, the kinetic characteristics of the wild enzyme and MgFucD1 (H359D) were analyzed using an all-atom model. The AMBER14SB_PARMBSC1 force field and the TIP3P water model were selected. The simulation system was constructed through a cubic solvent box, and the periodic boundary condition was set to 1 nm. In the initial equilibrium stage, the system first underwent a 500-ps NVT ensemble equilibration stage (canonical ensemble), followed by a 1000-ps NPT ensemble relaxation process (isothermal-isobaric ensemble). Temperature control was achieved using the Nose-Hoover thermostat algorithm to stabilize the system temperature at 303 K (the optimal temperature of the enzyme) and 333 K. Pressure control was realized through the Parrinello-Rahman pressure coupling method to maintain a 1-bar standard atmospheric pressure environment. Finally, 100-ns kinetic simulations were performed on the two proteases respectively.

[0079] (1) RMSD (Root Mean Square Deviation) The RMSD can measure the deviation of a protein molecule from its initial conformation during the simulation process. If the RMSD value reaches a stable value, it is considered that the system has reached equilibrium. The schematic diagram of the comparison of the molecular dynamics RMSD of the wild-type enzyme and MgFucD1 (H359D) at 303 K is as Figure 12 shown. The schematic diagram of the comparison of the molecular dynamics RMSD of the wild-type enzyme and MgFucD1 (H359D) at 333 K is as Figure 13 shown. At 303 K, both the wild-type enzyme and MgFucD1 (H359D) tend to be stable after 40 ns, and at 333 K, they tend to be stable after 60 ns, indicating that the subsequent trajectories can be used for the next analysis; the difference between the two may be due to the conformational adaptive adjustment of the mutant enzyme, forming a new equilibrium state.

[0080] (2) RMSF (Root Mean Square Fluctuation) The RMSF reflects the fluctuation degree of each amino acid during the molecular dynamics simulation. The larger the RMSF value, the stronger the thermal motion ability and the greater the flexibility of this region. The schematic diagram of the comparison of the molecular dynamics RMSF of the wild-type enzyme and MgFucD1 (H359D) at 303 K is as Figure 14 shown. The schematic diagram of the comparison of the molecular dynamics RMSF of the wild-type enzyme and MgFucD1 (H359D) at 333 K is as Figure 15 shown; the analysis shows that at 303 K, the average RMSF value of the wild-type enzyme is 0.1230 nm, while the average RMSF value of MgFucD1 (H359D) is 0.1098 nm, a decrease of 10.8%. And in 80.7% of the regions, the RMSF value of the mutant enzyme is lower, indicating that the overall fluctuation of the mutant enzyme is smaller; in addition, at 117 - 122, 165 - 171 (key catalytic sites), 272 - 280, and 297 - 302, huge changes before and after the mutation can be observed, and the RMSF values decrease by 22.8%, 33.0%, 44.2%, and 56.9% respectively compared with the wild-type enzyme; when the temperature reaches 333 K, the RMSF value at 250 - 256 (another key catalytic site) of the mutant enzyme decreases by 47.1% compared with the wild-type enzyme, meaning that the introduction of the mutation increases the rigidity of the local conformation and is more conducive to the improvement of protein stability.

[0081] (3) Rg (Radius of Gyration) During the MD simulation, Rg represents the compactness of protein molecules. A smaller Rg value indicates a more compact structure, while a larger value may imply an unfolded or loose conformation. The schematic diagram of the molecular dynamics Rg comparison between the wild-type enzyme and MgFucD1 (H359D) at 303 K is as follows Figure 16 shown. The schematic diagram of the molecular dynamics Rg comparison between the wild-type enzyme and MgFucD1 (H359D) at 333 K is as follows Figure 17 shown; at 303 K, the Rg value of the wild-type enzyme is stable in the range of about 2.15 nm, while that of MgFucD1 (H359D) is stable at about 2.13 nm; at 333 K, the Rg value of the wild-type enzyme is stable in the range of about 2.15 nm, while that of MgFucD1 (H359D) is stable at about 2.12 nm. Regardless of the temperature, the Rg value of the mutant enzyme is lower, indicating that the introduction of the H359D mutation site makes the protein molecular structure more compact and the conformation more stable.

[0082] (4) SASA (Solvent Accessible Surface Area) SASA is an important indicator to describe the exposure degree of the hydrophobic core of proteins. An increase in SASA means a larger contact area between the molecule and water, and the molecular structure is looser. The schematic diagram of the molecular dynamics SASA comparison between the wild-type enzyme and MgFucD1 (H359D) at 303 K is as follows Figure 18 shown. The schematic diagram of the molecular dynamics SASA comparison between the wild-type enzyme and MgFucD1 (H359D) at 333 K is as follows Figure 19 shown; similar to Rg, the SASA value of MgFucD1 (H359D) is approximately 10 nm lower than that of the wild-type enzyme, indicating that fewer surface residues of the mutant enzyme are exposed to the solvent, and the hydrophobic interaction is enhanced, further supporting the improvement of stability.

[0083] Conclusion: In the present invention, the amino acid at position 359 of the wild-type enzyme is mutated from histidine to aspartic acid. Through such computational design, the synchronous optimization of thermal stability and pH adaptability is achieved, effectively overcoming the bottlenecks of frequent enzyme supplementation and acid-base regulation in industrial applications. The fucoidan-degrading enzyme OUC-MgFucD1-H359D has efficient and stable catalytic characteristics, providing a new tool for the development of targeted drug delivery, anti-tumor adjuvant therapy, and functional foods.

[0084] The above embodiments are provided to those skilled in the art to fully disclose and describe how to implement and use the claimed embodiments, rather than to limit the scope disclosed herein. Modifications that are obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A fucoidan-degrading enzyme OUC-MgFucD1-H359D, characterized in that: The amino acid sequence is as shown in SEQ ID NO.

31.

2. The coding gene of the fucoidan-degrading enzyme OUC-MgFucD1-H359D according to claim 1, characterized in that: The nucleotide sequence is as shown in SEQ ID NO.

32.

3. Use of the fucoidan-degrading enzyme OUC-MgFucD1-H359D according to claim 1 in degrading fucoidan.

4. Use of the fucoidan-degrading enzyme OUC-MgFucD1-H359D according to claim 3 in degrading fucoidan, characterized in that: The degradation products are fucose, fucobiose and fucotriose; the molecular formula of fucose is C6H 12 O8S, the molecular formula of fucobiose is C 12 H 22 O 15 S2, the molecular formula of fucotriose is C 18 H 32 O 16 S.

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