Fucoidan-degrading enzyme OUC-MgFucD1-H359D and its encoding gene and application

Abstract

The present invention discloses a fucoidan degrading enzyme OUC‑MgFucD1‑H359D and its encoding gene and application, which belong to the field of hydrolase technology. The amino acid sequence of the fucoidan degrading enzyme OUC‑MgFucD1‑H359D is shown in SEQ ID NO.31, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.32. Application of the fucoidan degrading enzyme OUC‑MgFucD1‑H359D in degrading fucoidan. The present invention constructs a fucoidan degrading enzyme OUC‑MgFucD1‑H359D with stronger stability and better pH tolerance through a computationally driven design strategy. It still retains more than 80% of its activity after incubation at 30°C for 84 hours, and the pH tolerance range is widened to 7.0-9.0. The present invention provides an efficient and stable enzymatic hydrolysis tool for the large-scale preparation of fucoidan oligosaccharides.

Description

Technical Field

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

[0002] CN 119913226 A discloses the use of the fucoidan-degrading enzyme OUC-MgFucD1 in the preparation of fucoidan oligosaccharides. This enzyme utilizes the unique substrate recognition mechanism of the fucoidan-degrading enzyme OUC-MgFucD1 to degrade fucoidan into fucose containing one sulfate group, fucobiose containing two sulfate groups, and fucotriose containing one sulfate group. However, the fucoidan-degrading enzyme OUC-MgFucD1 has two deficiencies that limit its industrial application: first, insufficient thermal stability: after incubation at 30°C for 72 hours, its residual activity is only 20%, and after 84 hours, it is almost completely inactivated, requiring frequent enzyme replenishment during use, increasing production costs; second, a narrow pH range of application: it maintains high activity only around pH 7.5 and is sensitive to fluctuations in the pH of the reaction system. Therefore, there is an urgent need for fucoidan-degrading enzymes with greater thermal stability and improved pH tolerance. Summary of the Invention

[0003] In response to the above-mentioned prior art, the present invention provides a fucoidan-degrading enzyme OUC-MgFucD1-H359D and its encoding gene and application, belonging to the field of hydrolase technology.

[0004] The present invention is achieved through the following technical solutions:

[0005] A fucoidan-degrading enzyme OUC-MgFucD1-H359D, whose amino acid sequence is shown in SEQ ID NO.31.

[0006] The nucleotide sequence of the gene encoding the fucoidan degrading enzyme OUC-MgFucD1-H359D is shown in SEQ ID NO.32.

[0007] Application of the fucoidan degrading enzyme OUC-MgFucD1-H359D in degrading fucoidan.

[0008] Furthermore, the degradation products are fucose (FucS), fucobiose (Fuc2S2) and fucotriose (Fuc3S); the molecular formula of fucose is C6H 12 O8S, the molecular formula of fucoidan is C 12 H 22 O 15 S2, the molecular formula of fucotriose is C 18 H 32 O 16 S.

[0009] This invention addresses the industrial application bottlenecks of the wild-type fucoidan-degrading enzyme OUC-MgFucD1, namely poor thermal stability and a narrow pH range. Through a computationally driven design strategy, the author successfully constructed a more stable and pH-tolerant fucoidan-degrading enzyme, OUC-MgFucD1-H359D. While maintaining the specific catalytic function of the wild-type enzyme, OUC-MgFucD1-H359D retains over 80% of its activity after 84 hours of incubation at 30°C, and its pH tolerance is extended to 7.0-9.0. The 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 the industrial production of high-value-added fucoidan oligosaccharides. This invention provides an efficient and stable enzymatic hydrolysis tool for the large-scale preparation of high-purity fucoidan oligosaccharides, which can accelerate their industrial application in areas such as targeted drug delivery systems and functional food additives.

[0010] Various terms and phrases used herein have the general meanings that are well known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 : Mutation sites predicted by three online tools.

[0012] Figure 2 : Agarose gel electrophoresis detection results of 14 mutant plasmids, where M represents standard DNA marker, "bp" represents base pairs, and lanes 1 to 14 represent the mutant plasmids corresponding to E19Y, D39E, R57F, S59H, L98F, K181A, K213N, E220D, A255G, W256Q, N275E, S300V, L344I, and H359D, respectively.

[0013] Figure 3 : SDS-PAGE detection results of crude enzyme solutions of wild-type enzyme and 14 mutant enzymes, where M represents standard protein marker and kDa represents molecular weight. Lane 1 represents wild-type enzyme, and lanes 2 to 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), and MgFucD1(H359D), respectively.

[0014] Figure 4: Relative enzymatic activities of the wild-type enzyme and 14 mutant enzymes, where M represents the wild-type enzyme and 1 to 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), and MgFucD1(H359D), respectively.

[0015] Figure 5 : Relative enzyme activities of the wild type enzyme and 14 mutant enzymes after heat treatment, where M represents the wild type enzyme, and 1 to 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), and MgFucD1 (H359D), respectively.

[0016] Figure 6 : SDS-PAGE electrophoresis results of pure enzyme solutions of the wild-type enzyme and four mutant enzymes, where M represents a standard protein marker and “kDa” represents the molecular weight. Lane 1 represents the wild-type enzyme, and lanes 2 to 5 represent MgFucD1 (K181A), MgFucD1 (K213N), MgFucD1 (N275E), and MgFucD1 (H359D), respectively.

[0017] Figure 7 : Relative enzyme activities of the wild-type enzyme and four mutant enzymes after heat treatment for different time periods, where “MgFucD1” represents the wild-type enzyme, the same below.

[0018] Figure 8 : Schematic diagram of the effect of reaction temperature on relative enzyme activity.

[0019] Figure 9 : Schematic diagram of the effect of reaction pH on relative enzyme activity.

[0020] Figure 10 : Schematic diagram comparing the Tm values ​​of wild-type enzyme and MgFucD1 (H359D).

[0021] Figure 11: Schematic diagram of the product composition of the wild-type enzyme and MgFucD1 (H359D).

[0022] Figure 12 : Schematic diagram of molecular dynamics RMSD comparison between wild-type enzyme and MgFucD1 (H359D) at 303 K.

[0023] Figure 13 : Schematic diagram of molecular dynamics RMSD comparison between wild-type enzyme and MgFucD1 (H359D) at 333 K.

[0024] Figure 14 : Schematic diagram of molecular dynamics RMSF comparison between wild-type enzyme and MgFucD1 (H359D) under 303 K conditions.

[0025] Figure 15 : Schematic diagram of molecular dynamics RMSF comparison between wild-type enzyme and MgFucD1 (H359D) at 333 K.

[0026] Figure 16 : Schematic diagram of molecular dynamics Rg comparison between wild-type enzyme and MgFucD1 (H359D) at 303 K.

[0027] Figure 17 : Schematic diagram of molecular dynamics Rg comparison between wild-type enzyme and MgFucD1 (H359D) at 333 K.

[0028] Figure 18 : Schematic diagram of the molecular dynamics SASA comparison of the wild-type enzyme and MgFucD1 (H359D) at 303 K.

[0029] Figure 19 : Schematic diagram of the molecular dynamics SASA comparison of the wild-type enzyme and MgFucD1 (H359D) at 333 K. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will appreciate that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0031] Unless otherwise specified, the instruments, reagents, and materials used in the following examples are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods and detection methods used in the following examples are all conventional experimental methods and detection methods available in the prior art, unless otherwise specified.

[0032] 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 of the inventor.

[0033] The Escherichia coli DH5α competent cells used in the present invention were purchased from Beijing Qingke Biotechnology Co., Ltd.

[0034] The Escherichia coli BL21 (DE3) competent cells used in the present invention were purchased from Beijing Qingke Biotechnology Co., Ltd.

[0035] The LB culture medium used in the present invention comprises the following components: 10 g / L sodium chloride; 10 g / L tryptone; 5 g / L yeast extract; 15 g / L agar powder (added when preparing the solid culture medium); the balance being water; and sterilization conditions: 115°C for 30 min.

[0036] The pHBH powder used in the present invention was purchased from Sigma-Aldrich Co., Ltd.

[0037] The rapid plasmid mini-extraction kit used in the present invention was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0038] The fucoidan used in the present invention was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0039] Example 1 Prediction of potential thermostability sites

[0040] Fucoidan is a marine-derived sulfated polysaccharide with unique antioxidant, anticoagulant, anti-inflammatory, and immune-stimulating properties. In recent years, it has attracted widespread attention in the fields of biopharmaceuticals and functional foods. However, the high molecular weight of this polysaccharide (typically 50-100 kDa) results in poor solubility and low bioavailability, which severely limits its targeted delivery efficiency in vivo. Studies have shown that enzymatic hydrolysis of fucoidan into fucose or fucoidan 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 its targeted activities such as anti-tumor, anti-viral, and anti-hypoxia.

[0041] According to CN 119913226 A, the research team of the inventors of this application identified OUC-MgFucD1, a fucoidan-degrading enzyme 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. OUC-MgFucD1 can degrade fucoidan into fucose, fucobiose, and fucotriose. However, its thermal stability is insufficient and its pH range of application is narrow. The present invention attempts to modify it through mutations to obtain mutants with enhanced thermal stability and pH tolerance, thereby meeting the long-term stability requirements of enzyme preparations for industrial production.

[0042] The amino acid sequence of fucoidan degrading enzyme OUC-MgFucD1 is shown below, as shown in SEQ ID NO.1:

[0043] NTSESEWLLGSWGVRLIVEGGVELDKASKSSDWVKGAQDIVDNLPTVGHVFTNFNHRASGYWFTLRDNPYVDIAKEIHPDFVPSLENEQIILDVIDVLKKAGKKVILYIATDGPSRSGTKDNAEYKTAWENYYNEKFNGDEGLAYRTLCRGFIERFKGLADGYWLDHTSGIAGELPDFIKMIKEVDPT VIIASNGIVNNENTSPHNYFKDEKGEFLLVESDGVDDQDDRHYKIRSFNTDDLYTDFTSGHPTPLAWGAPPNSWAYEEFTFPEIANAMTNYEVTKDNIKHAWMPMRMKWTSPKAELMFDTEQAYRFVRTLTDAGCAITWGNTNTYGFITEDEMVLMKEIDKRLQIKPMPHYVPYKRPEGAKLVGEDKEN.

[0044] The nucleotide sequence of the gene encoding the fucoidan degrading enzyme OUC-MgFucD1 is as follows (5'-3' direction), as shown in SEQ ID NO. 2:

[0045]

[0046] First, AlphaFold3 was used to predict the three-dimensional structure of the wild enzyme, fucoidan-degrading enzyme OUC-MgFucD1, with pTM=0.94, for subsequent computational design.

[0047] Then, three online tools, PROSS, HotSpot Wizard 3, and FireProt 2.0, were used to predict potential stabilizing mutations of the fucoidan-degrading enzyme OUC-MgFucD1, and 43, 22, and 36 mutation sites were identified, respectively.

[0048] The website of PROSS is: https: / / pross.weizmann.ac.il / step / pross-terms / .

[0049] The website for HotSpot Wizard 3 is: https: / / loschmidt.chemi.muni.cz / hotspotwizard / .

[0050] The website for FireProt 2.0 is: https: / / loschmidt.chemi.muni.cz / fireprotweb / .

[0051] Then, all the single mutations predicted by the three online tools were compared, and 14 mutation sites that were recommended by at least two tools were screened out, namely E19Y, D39E, R57F, S59H, L98F, K181A, K213N, E220D, A255G, W256Q, N275E, S300V, L344I, and H359D. Figure 1 shown.

[0052] Example 2 Construction and induced expression of mutant enzyme

[0053] According to the prediction of Example 1, the mutant enzymes corresponding to the mutation sites were constructed and induced to express. The 14 mutant enzymes were temporarily named: 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), and MgFucD1 (H359D). The steps are as follows:

[0054] (1) The mutation was amplified by one-step PCR using the wild enzyme plasmid pET28a(+)-OUC-MgFucD1 as a template and specific primers for each mutation site; the nucleotide sequences of the specific primers for the 14 mutation sites are shown in Table 1, and are shown in SEQ ID NO.3 to 30, respectively. 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 a total system of 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 the 14 mutant plasmids are shown in Figure 1. Figure 2 As shown, the base pair length of the mutant plasmid was approximately 6500 bp, which was consistent with its theoretical value, indicating that all 14 mutant plasmids were successfully constructed.

[0055] Table 1 Nucleotide sequences of specific primers for 14 mutation sites

[0056]

[0057] (2) The wild enzyme plasmid and each mutant plasmid were transformed into Escherichia coli DH5α competent cells, spread on solid LB medium plates containing 50 μg / mL kanamycin sulfate, and cultured in a 37°C incubator for 16 h. Single colonies were picked and transferred to liquid LB medium containing 50 μg / mL kanamycin sulfate, and cultured in a shaking incubator at 37°C and 220 rpm for 8 h. The plasmids were extracted using a rapid plasmid extraction kit and verified by sequencing.

[0058] (3) The plasmid with the correct sequencing results was transformed into Escherichia coli BL21 (DE3) competent cells, and the constructed recombinant engineered bacteria grew on kanamycin sulfate resistance plates.

[0059] (4) Pick a single colony and inoculate it into 5 ml of liquid LB medium containing 50 μg / mL kanamycin sulfate. Incubate it at 37°C and 220 rpm for 12 h. Then, inoculate it into 50 mL of liquid LB medium containing 50 μg / mL kanamycin sulfate at a volume of 1% (volume percentage). Incubate it at 37°C and 220 rpm until the bacterial liquid OD reaches 0. 600 The value reached 0.6. 0.5‰ isopropyl-β-D-thiogalactoside (IPTG) (100 mM) was added and expression was induced for 18 h at 20°C and 220 rpm in a shaking incubator.

[0060] (5) After the culture is completed, the culture medium is taken and centrifuged at 4°C and 8000 rpm for 10 min. The bacteria are collected and resuspended in ultrapure water. Ultrasonic disruption is performed for 15 min and centrifuged at 9000 rpm for 10 min. The supernatant is the crude enzyme solution. The crude enzyme solutions of the wild enzyme and 14 mutant enzymes are subjected to SDS-PAGE electrophoresis. The SDS-PAGE detection results of the crude enzyme solutions of the wild enzyme and 14 mutant enzymes are as follows: Figure 3 As shown, all mutant enzymes were correctly expressed (43 kDa).

[0061] Example 3 Preliminary screening of heat treatment and determination of enzyme activity

[0062] The crude enzyme solutions of the wild enzyme and 14 mutant enzymes obtained in Example 2 were subjected to enzyme activity assay. The assay method was as follows: 10 μL of the crude enzyme solution was added to 65 μL of a substrate solution (prepared with fucoidan, CaCl2, NaCl, and a Tris-HCl buffer solution with a pH of 7.5, wherein 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), mixed evenly, reacted at 30°C for 1 h, and then incubated in a boiling water bath for 5 min to terminate the reaction process; 225 μL of water was added to dilute the reaction solution, and then 100 μL of 4-hydroxybenzoylhydrazide (pHBH) solution was added and incubated in a boiling water bath for 5 min; the reaction solution was cooled to room temperature, centrifuged, and the supernatant was collected to determine the OD 415 The control group was replaced with inactivated enzyme solution.

[0063] The pHBH solution is prepared by weighing pHBH powder and dissolving it in 2 M hydrochloric acid to prepare a pHBH stock solution with a concentration of 0.2 g / mL. The pHBH stock solution is then mixed with a 2 M NaOH solution in a volume ratio of 1:9 to obtain the pHBH solution. This solution must be prepared immediately before use.

[0064] Enzyme activity (U) is defined as the amount of enzyme required to produce 1 μg of reducing sugar per minute under standard conditions.

[0065] The relative activity of the wild enzyme and the 14 mutant enzymes was calculated with the wild enzyme activity as 100%. Figure 4 As shown, compared with the wild-type enzyme, there are seven mutant enzymes with relative enzyme activities greater than 80%, namely: MgFucD1 (S59H), MgFucD1 (K181A), MgFucD1 (K213N), MgFucD1 (N275E), MgFucD1 (S300V), MgFucD1 (L344I), and MgFucD1 (H359D).

[0066] The crude enzyme solutions of the wild-type enzyme and the 14 mutant enzymes were then heat-treated by incubation at 30°C for 12 hours. The residual enzyme activities of the wild-type enzyme and the 14 mutant enzymes after heat treatment were determined using the same method as above. Relative enzyme activities were calculated, with the initial enzyme activity set as 100%.

[0067] The relative enzyme activities of wild-type enzyme and 14 mutant enzymes after heat treatment are as follows Figure 5 As shown, there are 8 mutant enzymes with residual enzyme activity greater than 80%, namely: MgFucD1 (D39E), MgFucD1 (L98F), MgFucD1 (K181A), MgFucD1 (K213N), MgFucD1 (E220D), MgFucD1 (W256Q), MgFucD1 (N275E), and MgFucD1 (H359D).

[0068] Four mutant enzymes met both of these conditions: MgFucD1 (K181A), MgFucD1 (K213N), MgFucD1 (N275E), and MgFucD1 (H359D). These four mutant enzymes exhibited excellent thermostability without sacrificing enzymatic activity, and their crude enzymes were purified for further study.

[0069] Example 4 Study on the thermal stability of mutant enzymes

[0070] Ni was used to treat the crude enzyme solutions of the four mutant enzymes MgFucD1 (K181A), MgFucD1 (K213N), MgFucD1 (N275E), and MgFucD1 (H359D). 2+ -NTA column affinity chromatography purification: First, equilibrate the column with 10 mM imidazole solution (10 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl, pH 7.5), add the crude enzyme solution to allow it to bind to Ni 2+ -NTA column binding, then eluted with 40mM imidazole solution (40mM imidazole, 500mM NaCl, 50mM Tris-HCl) the weakly binding impurities, and then eluted with 200mM imidazole solution (200mM imidazole, 500mM NaCl, 50mM Tris-HCl) the target protein, and collected the eluate. Use an ultrafiltration centrifuge tube with a molecular weight cutoff of 10kDa to perform ultrafiltration and concentration at 4℃ and 4000rpm to obtain pure enzyme solution. The pure enzyme solutions of the wild enzyme and the four mutant enzymes were subjected to SDS-PAGE electrophoresis detection. The results of SDS-PAGE electrophoresis detection of the pure enzyme solutions of the wild enzyme and the four mutant enzymes are shown as follows. Figure 6 As shown, the molecular weight of each mutant enzyme is consistent with the predicted value of 43 kDa. The protein concentration of the pure enzyme solution was determined and diluted with water to 1 mg / mL for later use.

[0071] The pure enzyme solutions of the wild enzyme and the four 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 activity was determined according to the method of Example 3.

[0072] Before heat treatment (0 h incubation), the enzyme activities of the wild-type 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. MgFucD1(H359D) exhibited the highest enzyme activity.

[0073] The enzyme activity before heat treatment was taken as 100%, and the relative enzyme activities of the wild enzyme and the four mutant enzymes after heat treatment for different times were calculated. Figure 7 As shown in the figure, after incubation at 30°C for 84 hours, MgFucD1(H359D) still retained over 80% of its activity, while the activity of the wild-type enzyme dropped to almost zero. Although the stability of the three mutant enzymes, MgFucD1(K181A), MgFucD1(K213N), and MgFucD1(N275E), improved slightly at various time points, the trends were almost identical to those of the wild-type enzyme. Therefore, MgFucD1(H359D) exhibited the best thermal stability, and this mutant enzyme was formally named fucoidan-degrading enzyme OUC-MgFucD1-H359D. Its amino acid sequence is shown in SEQ ID NO. 31, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO. 32.

[0074] The amino acid sequence of fucoidan degrading enzyme OUC-MgFucD1-H359D is shown below, as shown in SEQ ID NO.31:

[0075] NTSESEWLLGSWGVRLIVEGGVELDKASKSSDWVKGAQDIVDNLPTVGHVFTNFNHRASGYWFTLRDNPYVDIAKEIHPDFVPSLENEQIILDVIDVLKKAGKKVILYIATDGPSRSGTKDNAEYKTAWENYYNEKFNGDEGLAYRTLCRGFIERFKGLADGYWLDHTSGIAGELPDFIKMIKEVDPT VIIASNGIVNNENTSPHNYFKDEKGEFLLVESDGVDDQDDRHYKIRSFNTDDLYTDFTSGHPTPLAWGAPPNSWAYEEFTFPEIANAMTNYEVTKDNIKHAWMPMRMKWTSPKAELMFDTEQAYRFVRTLTDAGCAITWGNTNTYGFITEDEMVLMKEIDKRLQIKPMPDYVPYKRPEGAKLVGEDKEN.

[0076] The nucleotide sequence of the gene encoding the fucoidan degrading enzyme OUC-MgFucD1-H359D is as follows (5'-3' direction), as shown in SEQ ID NO.32:

[0077]

[0078] Example 5 Characterization of the Enzymatic Properties of Fucoidan-Degrading Enzyme OUC-MgFucD1-H359D

[0079] The enzymatic properties of fucoidan-degrading enzyme OUC-MgFucD1-H359D were studied, with the wild enzyme as a control, as shown below.

[0080] (1) Effect of reaction temperature on relative enzyme activity

[0081] To 65 μL of substrate solution (prepared in Example 3), add 10 μL of pure enzyme solution (prepared in Example 4). Incubate 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. Terminate the reaction in a boiling water bath for 5 min. Determine enzyme activity according to the method in Example 3. Each experiment was performed in triplicate. The relative enzyme activity of the wild-type enzyme and MgFucD1 (H359D) at each temperature was calculated, with the highest enzyme activity defined as 100%.

[0082] Schematic diagram of the effect of reaction temperature on relative enzyme activity Figure 8 As shown in the figure, the optimal reaction temperature of MgFucD1 (H359D) is the same as that of the wild enzyme, both at 30°C. However, in the non-optimal temperature range (especially the high temperature zone), the enzyme activity decay rate of MgFucD1 (H359D) is significantly lower than that of the wild enzyme, and it can still retain more than 60% of its activity at 60°C. This feature improves its applicability in industrial scenarios with fluctuating ambient temperatures.

[0083] (2) Effect of reaction pH on relative enzyme activity

[0084] Fucoidan buffer solutions (containing 2 mg / mL fucoidan, 20 mM CaCl2, and 25 mM NaCl) of different pH values ​​were prepared using buffer solutions of different pH values ​​as solvents. The buffer solutions used were: 20 mM citric acid-sodium citrate buffer (pH 3.0, 4.0, 5.0, and 6.0), 20 mM phosphate buffer (pH 6.0 and 7.0), 20 mM Tris-HCl buffer (pH 7.0, 7.5, 8.0, 8.5, and 9.0), and 20 mM glycine-sodium hydroxide buffer (pH 9.0, 10.0, and 11.0).

[0085] To each solution, 10 μL of pure enzyme solution (prepared in Example 4) was added to 65 μL of each fucoidan buffer solution at different pH values. The reaction was incubated at 30°C for 1 h. The reaction was terminated in a boiling water bath for 5 min. Enzyme activity was determined according to the method in Example 3. Each experiment was performed in triplicate. The relative enzyme activity of the wild-type enzyme and MgFucD1 (H359D) at each pH value was calculated, with the highest enzyme activity defined as 100%.

[0086] Schematic diagram of the effect of reaction pH on relative enzyme activity Figure 9 As shown in the figure, the optimal reaction pH of the wild-type enzyme is 7.5; the optimal reaction pH of MgFucD1 (H359D) shifts to 8.0, and the enzyme activity is maintained at more than 80% in the pH range of 7.0-9.0 (while the wild-type enzyme only maintains the same activity in the pH range of 7.0-7.5). This indicates that the mutation significantly broadens the enzyme's pH adaptability, which is beneficial for reducing the cost of pH control in industrial production.

[0087] (3) Thermodynamic stability analysis

[0088] The thermodynamic stability of a protein can be evaluated by its melting temperature. Differential scanning calorimetry (DSC) was used to determine the thermal denaturation temperature (Tm value) of the wild-type enzyme and MgFucD1 (H359D): 8 mL of pure enzyme solution (prepared in Example 4) was taken and placed in a freeze dryer for 3 days; the protease powder was placed in a sealed aluminum crucible, the test temperature range was set to 100-260°C, the heating and cooling rate was controlled to 2°C / min, and the Tm value was determined. A schematic diagram comparing the Tm values ​​of the wild-type enzyme and MgFucD1 (H359D) is shown in FIG. Figure 10 As shown, the Tm value of MgFucD1 (H359D) was 214.5°C, which was 4.5°C higher than that of the wild-type enzyme.

[0089] Example 6 Analysis of the Action Products of Fucoidan Degrading Enzyme OUC-MgFucD1-H359D

[0090] To 133 μL of substrate solution, add 227 μL of ultrapure water and 40 μL of pure enzyme solution (prepared in Example 4) and mix thoroughly. The reaction system contains 2 mg / mL fucoidan, 20 mM CaCl₂, 25 mM NaCl, 20 mM Tris-HCl, and a pH of 8. The reaction was incubated at 30°C for 24 h and terminated in a boiling water bath for 10 min. The supernatant was collected, filtered through a 0.22 μm filter, and analyzed by HPLC. HPLC conditions were as follows: a Superdex 30 Increase 10 / 300 GL column (10 × 300-310 mm) (GE Healthcare, USA), a 0.2 M NH₄HCO₃ solution as the mobile phase, a flow rate of 0.4 mL / min, and a differential detection detector.

[0091] 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.

[0092] Results: The product composition diagram of wild enzyme and MgFucD1 (H359D) is shown in Figure 11 As shown in the figure, MgFucD1 (H359D) can degrade fucoidan into fucoidan oligosaccharides of different molecular weights and polymerization degrees. The product composition is exactly the same as that of the wild enzyme; however, the yield of each product is increased according to the peak area calculation. Specifically, product I is a fucotriose Fuc3S (molecular formula C 18 H 32 O 16 S), the peak area of ​​the wild enzyme was 31592.611, and the peak area of ​​MgFucD1 (H359D) was 36067.909; product II was fucose Fuc2S2 containing two sulfate groups (molecular formula C 12 H 22 O 15 S2), the peak area of ​​the wild enzyme was 38127.513, and the peak area of ​​MgFucD1 (H359D) was 40563.884; product III was fucose FucS containing a sulfate group (molecular formula C6H 12The peak area of ​​the wild-type enzyme was 12310.594, while that of MgFucD1 (H359D) was 14017.120. This indicates that the improved stability allows MgFucD1 (H359D) to maintain its active conformation longer in the reaction system, possibly leading to the higher measured product accumulation due to continued catalysis.

[0093] Example 7 Molecular dynamics simulation of wild-type enzyme and MgFucD1 (H359D)

[0094] The dynamics of the wild-type enzyme and MgFucD1 (H359D) were analyzed using an all-atom model using the GROMACS 2023 molecular dynamics platform. The AMBER14SB_PARMBSC1 force field and the TIP3P water model were used, and the simulation system was constructed using a cubic solvation box with a periodic boundary condition of 1 nm. During the initial equilibrium phase, the system first underwent a 500 ps NVT ensemble equilibration phase (canonical ensemble), followed by a 1000 ps NPT ensemble relaxation phase (isothermal and isobaric ensemble). Temperature control was achieved using the Nose-Hoover thermal bath algorithm, maintaining the system temperatures at 303 K (the enzyme's optimal temperature) and 333 K. Pressure control was achieved using the Parrinello-Rahman pressure coupling method, maintaining a standard atmospheric pressure of 1 bar. Finally, 100 ns dynamics simulations were performed for each of the two proteases.

[0095] (1) RMSD (Root Mean Square Deviation)

[0096] RMSD can measure the degree of deviation of protein molecules from the initial conformation during the simulation. If the RMSD value reaches a stable value, the system is considered to have reached equilibrium. The molecular dynamics RMSD comparison diagram of the wild-type enzyme and MgFucD1 (H359D) at 303 K is shown in the figure. Figure 12 The molecular dynamics RMSD comparison diagram of the wild-type enzyme and MgFucD1 (H359D) at 333 K is shown in Figure 2. Figure 13 As shown in the figure, at 303 K, both the wild enzyme and MgFucD1 (H359D) stabilized after 40 ns, and at 333 K after 60 ns, indicating that the subsequent trajectories can be used for the next step of analysis; the difference between the two may be due to the conformational adaptive adjustment of the mutant enzyme, forming a new equilibrium state.

[0097] (2) RMSF (Root Mean Square Fluctuation)

[0098] RMSF reflects the degree of fluctuation 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 the region. The molecular dynamics RMSF comparison diagram of the wild type enzyme and MgFucD1 (H359D) at 303 K is shown in the figure. Figure 14 The molecular dynamics RMSF comparison diagram of the wild-type enzyme and MgFucD1 (H359D) at 333 K is shown in Figure 2. Figure 15 As shown; analysis showed that at 303 K, the average RMSF of the wild enzyme was 0.1230 nm, while the average RMSF of MgFucD1 (H359D) was 0.1098 nm, a decrease of 10.8%, and 80.7% of the regions of the mutant enzyme had lower RMSF values, indicating that the mutant enzyme had smaller overall fluctuations; in addition, huge changes before and after the mutation were observed at 117-122, 165-171 (key catalytic sites), 272-280 and 297-302, with RMSF values ​​decreasing by 22.8%, 33.0%, 44.2% and 56.9% compared with the wild enzyme, respectively; and when the temperature reached 333 K, the RMSF value of the mutant enzyme at 250-256 (another key catalytic site) decreased by 47.1% compared with the wild enzyme, which means that the introduction of the mutation increased the rigidity of the local conformation and was more conducive to improving the stability of the protein.

[0099] (3) Rg (Radius of Gyration)

[0100] During MD simulation, Rg represents the compactness of protein molecules. Smaller Rg values ​​indicate a tighter structure, while larger values ​​may indicate an unfolded or loose conformation. A schematic diagram comparing the molecular dynamics Rg of the wild-type enzyme and MgFucD1 (H359D) at 303 K is shown in Figure 2. Figure 16 The molecular dynamics Rg comparison diagram of wild-type enzyme and MgFucD1 (H359D) at 333 K is shown in the figure. Figure 17 As shown in the figure, at 303 K, the Rg value of the wild-type enzyme stabilized around 2.15 nm, while that of MgFucD1 (H359D) stabilized around 2.13 nm. At 333 K, the Rg value of the wild-type enzyme stabilized around 2.15 nm, while that of MgFucD1 (H359D) stabilized around 2.12 nm. Regardless of the temperature, the Rg value of the mutant enzyme was lower, indicating that the introduction of the H359D mutation site made the protein molecular structure more compact and the conformation more stable.

[0101] (4) SASA (Solvent Accessible Surface Area)

[0102] SASA is an important indicator for describing the degree of exposure of the hydrophobic core of a protein. An increase in SASA means a larger contact area between the molecule and water and a looser molecular structure. A schematic diagram of the molecular dynamics SASA comparison of the wild-type enzyme and MgFucD1 (H359D) at 303 K is shown in the figure. Figure 18 The molecular dynamics SASA comparison diagram of the wild-type enzyme and MgFucD1 (H359D) at 333 K is shown in Figure 19 As shown; similar to Rg, the SASA value of MgFucD1 (H359D) is approximately 10 nm lower than that of the wild-type enzyme, indicating that the surface residues of the mutant enzyme are less exposed to the solvent and the hydrophobic interactions are enhanced, further supporting the improved stability.

[0103] Conclusion: This computational design, which mutates the amino acid at position 359 of the wild-type enzyme from histidine to aspartic acid, achieves simultaneous optimization of thermal stability and pH adaptability, effectively overcoming the bottlenecks of frequent enzyme replenishment and acid-base regulation in industrial applications. The fucoidan-degrading enzyme OUC-MgFucD1-H359D exhibits efficient and stable catalytic properties, providing a new tool for the development of targeted drug delivery, adjuvant anti-tumor therapy, and functional foods.

[0104] The above examples 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 of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

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

31.

2. The gene encoding the fucoidan-degrading enzyme OUC-MgFucD1-H359D according to claim 1, characterized in that: The nucleotide sequence is 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 in degrading fucoidan according to claim 3, characterized in that: The degradation products are fucose, fucobiose and fucotriose; the molecular formula of fucose is C6H 12 O8S, the molecular formula of fucoidan 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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