A recombinant mannuronic acid c-5 epimerase mc5en3 and its use

Abstract

The application discloses a kind of recombinant mannuronic acid C-5 epimerase MC5EN3 and its application, belong to enzyme engineering technical field.The amino acid sequence of the recombinant mannuronic acid C-5 epimerase MC5EN3 of the present application is as shown in SEQ ID NO.1.The present application further provides a kind of preparation method of recombinant mannuronic acid C-5 epimerase MC5EN3.The recombinant enzyme MC5EN3 prepared by the method can catalyze β-D-mannuronic acid (M) in the content of 31.32% G of alginate raw material into α-L-guluronate (G), and the alginate with the content of 53.35% G is prepared.Further, the alginate composite hydrogel wound dressing is prepared, the dressing can effectively promote wound healing, and the effect of promoting wound healing is better than that of the alginate composite hydrogel with the content of 31.32%, which greatly expands the application of alginate in food, biological medicine and biomaterials.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and in particular to a recombinant mannouronic acid C-5 epimerase MC5EN3 and its applications. Background Technology

[0002] Skin trauma is very common in clinical practice and daily life, including external injuries, burns, surgical incisions, and difficult-to-heal wounds associated with chronic diseases such as diabetes. Traditional dry dressings such as gauze and cotton pads mainly serve to mechanically cover and simply absorb exudate, making it difficult to maintain a moist environment for the wound. Furthermore, they tend to adhere to newly formed tissue during dressing changes, causing secondary damage and significant pain, which is detrimental to wound healing quality and patient comfort. In recent years, hydrogel dressings, characterized by high water content, good flexibility, and suitable microenvironment regulation capabilities, have gradually become an important development direction for wound care materials.

[0003] Alginate is a natural anionic polysaccharide extracted from brown algae. Its main chain consists of β-D-mannuronic acid (M) and α-L-guluronic acid (G) arranged in different sequences. Alginate possesses excellent biocompatibility, biodegradability, and the ability to easily form gels in the presence of divalent metal ions, making it widely used in food thickeners, drug carriers, and tissue engineering scaffolds. In wound repair, alginate is often combined with polymers such as gelatin, trehalose, and chitosan to prepare composite hydrogel wound dressings that absorb exudate, have moderate adhesion, and possess a certain level of mechanical strength.

[0004] Existing research and applications have shown that the M / G ratio on alginate chains has a significant impact on its gel properties, mechanical strength, degradation behavior, and interactions with cells and tissues. Generally, as the G content increases, the M / G ratio of alginate increases with the calcium content. 2+ The "egg-box" structure formed by divalent ions is more regular, and the gel network is denser, resulting in improved strength and stability of the hydrogel, which is more conducive to forming a complete and continuous protective barrier at the wound site. In contrast, alginate with a low G content or a high M ratio has a relatively loose gel structure. The composite hydrogel is prone to deformation or structural collapse when subjected to body fluid flushing and mechanical stretching, which limits the effectiveness of the dressing in complex wound environments.

[0005] Currently, industrially used alginate is mostly derived directly from different algal species and origins. The M / G ratio primarily depends on the natural source and extraction process, resulting in significant batch-to-batch variations and a relatively limited range of selectable G content. To improve alginate performance, existing technologies have proposed various chemical modification or physical blending strategies, such as oxidation, grafting, crosslinking, and compounding with other polymer materials. These methods can alter the rheological and gel properties of materials to some extent, but they often suffer from complex reaction conditions, the need for organic solvents or chemical crosslinking agents, and imprecise modification sites, potentially introducing safety hazards and hindering their widespread use in wound dressings where biosafety is paramount. Furthermore, simply changing the amount of alginate in the formulation or simply mixing alginate from different sources makes it difficult to achieve precise and predictable control of the M / G ratio.

[0006] Mannuronic acid C-5 epimerases are a class of enzymes capable of epimerizing β-D-mannuronic acid residues in alginate chains to α-L-guluronic acid under mild conditions. Biocatalytic modification of alginate using these enzymes can progressively increase the glycogen content without cleaving the polysaccharide backbone, thus obtaining a series of alginates with varying glycogen contents. This provides a new approach for constructing tunable composite hydrogel dressings. While existing literature includes mannuronic acid C-5 epimerases cloned from certain natural strains, these natural enzymes typically suffer from limited catalytic efficiency, insufficient substrate adaptability and transformation depth, and unsatisfactory thermal and operational stability. These limitations restrict their application in modifying complex raw material alginates, making it difficult to balance transformation efficiency, cost, and industrial scale-up. To improve enzyme performance, directed evolution techniques are gaining increasing attention.

[0007] In existing technologies, alginate composite hydrogel wound dressings still have significant shortcomings in terms of controllable adjustment of the glucose content of raw alginate, enzyme modification efficiency and stability, and compatibility with industrial expression systems. There is an urgent need to provide a recombinant mannuronic acid C-5 epimerase obtained through molecular modification and its suitable heterologous expression system to increase the glucose ratio of alginate with moderate glucose content under mild conditions. This can be further used to construct alginate composite hydrogel wound dressings with excellent gelation properties and effective wound healing promotion, thereby enhancing the application of alginate and its composite hydrogels in food, biomedicine, and biomaterials fields. Summary of the Invention

[0008] In view of this, the purpose of this invention is to provide a recombinant mannouronic acid C-5 epimerase MC5EN3 and its application in the preparation of alginate composite hydrogel wound dressings. This invention also provides a method for preparing a method of error-prone PCR mutation of the recombinant mannouronic acid C-5 epimerase MC5EN3 and its heterologous expression in Pichia pastoris. The recombinant mannouronic acid C-5 epimerase MC5EN3 improves the efficiency of catalyzing the conversion of M to G, enabling the conversion of M to G in alginate raw material with a G content of 31.32% to obtain alginate with a G content of 53.35%. Further preparation of alginate composite hydrogel wound dressings using this method can effectively promote wound healing, and the wound healing effect is superior to that of 31.32% alginate-based hydrogels, greatly expanding the application of alginate and its composite hydrogels in the fields of food, biomedicine, and biomaterials.

[0009] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a recombinant mannuronic acid C-5 epimerase MC5EN3, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0010] In a second aspect, the present invention provides a biomaterial related to the recombinant mannuronic acid C-5 epimerase MC5EN3 described above, wherein the biomaterial is any one of the following: 1) A nucleic acid molecule encoding the recombinant mannuronic acid C-5 epimerase MC5EN3 as described in claim 1; 2) An expression cassette containing the nucleic acid molecule described in 1); 3) A recombinant vector containing the nucleic acid molecule described in 1); 4) A recombinant vector containing the expression cassette described in 2); 5) Recombinant microorganisms containing the nucleic acid molecules described in 1); 6) Recombinant microorganisms containing the expression cassette described in 2); 7) Recombinant microorganisms containing the recombinant vector described in 3); 8) Recombinant microorganisms containing the recombinant vector described in 4).

[0011] Furthermore, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO: 2; and / or the recombinant microorganism is a recombinant yeast.

[0012] In this invention, SEQ ID NO. 2 is a mutant obtained by translating the mannuronic acid C-5 epimerase of the original amino acid sequence SEQ ID NO. 3 into an encoded nucleotide sequence, and then artificially synthesizing the modified nucleotide fragment, and performing error-prone PCR on the artificially synthesized fragment.

[0013] Furthermore, the recombinant yeast is Pichia pastoris X33.

[0014] In a third aspect, the present invention provides a method for preparing recombinant mannuronic acid C-5 epimerase MC5EN3, wherein the gene encoding the aforementioned recombinant mannuronic acid C-5 epimerase MC5EN3 is cloned into a recombinant expression vector, the recombinant expression vector is introduced into a host cell, a positive recombinant host is screened, the positive recombinant host is cultured, expression is induced, and recombinant mannuronic acid C-5 epimerase MC5EN3 is obtained.

[0015] Further, the recombinant expression vector is one or more of the following: Escherichia coli expression vector, yeast expression vector, Bacillus subtilis expression vector, lactic acid bacteria expression vector, Streptomyces expression vector, bacteriophage vector, filamentous fungus expression vector, plant expression vector, insect expression vector, or mammalian cell expression vector; and / or the host cell includes Escherichia coli host cell, yeast host cell, Bacillus subtilis host cell, lactic acid bacteria host cell, actinomycete host cell, filamentous fungus host cell, insect cell, or mammalian cell.

[0016] Specifically, in this invention, the gene fragment of the original mannouronic acid C-5 epimerase is amplified by error-prone PCR, the amplified error-prone PCR fragment is detected by agarose gel electrophoresis, and inserted into the expression vector of Pichia pastoris X33 to construct a recombinant expression vector of recombinant mannouronic acid C-5 epimerase; after linearization by enzyme digestion, it is transformed into Pichia pastoris X33, positive transformants are screened and verified, and recombinant mannouronic acid C-5 epimerase MC5EN3 is obtained by fermentation expression.

[0017] In a fourth aspect, the present invention provides the application of the recombinant mannuronic acid C-5 epimerase MC5EN3 or the biomaterials described above in catalyzing the conversion of β-D-mannuronic acid (M) to α-L-guluronic acid (G).

[0018] In a fifth aspect, the present invention provides a method for catalyzing the conversion of β-D-mannuronic acid to α-L-guluronic acid, comprising adding the above-described recombinant mannuronic acid C-5 epimerase MC5EN3 to a substrate for catalytic reaction to obtain a product with increased α-L-guluronic acid.

[0019] Furthermore, the catalytic reaction temperature is 35-39°C, and the pH value is 6-8; and / or The catalytic reaction time is 8-15 h; and / or The substrate includes at least one of alginate and alginate oligosaccharides.

[0020] Specifically, the recombinant mannouronic acid C-5 epimerase MC5EN3 obtained in preparation was used as the catalytic enzyme, and alginate raw material with a 1% G content of 31.32% was used as the substrate. The reaction was carried out at 37°C for 12 h to increase the G content of the substrate from 31.32% to 53.35%, thereby enabling the preparation and provision of alginate with a stable G content of 53.35%.

[0021] In a sixth aspect, the present invention provides a brown alginate composite hydrogel wound dressing, comprising the product prepared by the method described above.

[0022] Specifically, a 1% alginate solution with a G content of 52.35% was prepared using a calcium carbonate solution with a calcium ion to carboxyl group ratio of 0.25. After the solution was dissolved evenly, 0.06 g of calcium carbonate and 0.075 g of citric acid aqueous solution, as well as 10% small molecule hyaluronic acid solution, were added. The solution was poured into a 12-well plate, left to stand for one day, and then a portion was frozen and lyophilized to prepare a composite hydrogel sample.

[0023] In a seventh aspect, the present invention provides the application of the above-described alginate composite hydrogel wound dressing in promoting wound healing.

[0024] The beneficial effects of this invention include at least the following: (1) The present invention uses error-prone PCR directed evolution technology to modify and obtain recombinant mannuronic acid C-5 epimerase MC5EN3, which has a significantly better efficiency than the original enzyme in catalyzing the conversion of β-D-mannuronic acid (M) to α-L-guluronic acid (G): for 1% polyM substrate, the G content can be increased from 13.32% to 34.08%, which is much higher than the conversion limit of 23.35% of the original enzyme; for commercial alginate substrate, it can achieve an isomerization conversion rate of 22.03% and increase the G content from 31.32% to 53.35%, thus solving the problems of limited catalytic efficiency and insufficient conversion depth of naturally derived enzymes.

[0025] The recombinase was successfully expressed heterologously and efficiently in Pichia pastoris X33. The expression system is adapted to the industrial fermentation process, and the fermentation process does not require complex conditions. The active enzyme can be obtained through simple induction. After purification, it has good stability, which provides feasibility for large-scale industrial production and reduces production costs.

[0026] (2) The alginate is modified by biocatalysis. The reaction conditions are mild and there is no need to use potentially harmful reagents such as organic solvents and chemical crosslinking agents. This avoids the safety hazards caused by chemical modification and meets the stringent requirements for biosafety of biomedical materials such as wound dressings.

[0027] (3) The composite hydrogel prepared based on high-G content alginate has a more regular "egg-box" structure due to the increased G content, the gel network is denser, the degree of cross-linking is higher, and the adsorbed Ca 2+ With the increased total volume, the mechanical strength and stability are significantly improved, effectively resisting the erosion of bodily fluids and mechanical stretching, preventing structural deformation or collapse, and making it suitable for complex wound environments. This composite hydrogel, as a dressing, effectively promotes wound healing, and its wound-healing effect is superior to that of 31.32% alginate-based hydrogels; it greatly expands the application of alginate and its composite hydrogels in food, biomedicine, and biomaterials. Attached Figure Description

[0028] Figure 1 Nucleic acid electrophoresis for gene amplification of recombinant mannouronic acid C-5 epimerase MC5EN3.

[0029] Figure 2 Electrophoresis of the protein expression product of recombinant mannuronic acid C-5 epimerase MC5EN3.

[0030] Figure 3 The catalytic efficiency of the recombinant mannouronic acid C-5 epimerase MC5EN3 is given.

[0031] Figure 4 HPLC analysis of the M and G content after catalysis by recombinant mannouronic acid C-5 epimerase MC5EN3.

[0032] Figure 5 This is a SEM image of the composite hydrogel dressing.

[0033] Figure 6 The image shows the FTIR spectrum of the composite hydrogel dressing.

[0034] Figure 7 The effect of composite hydrogel dressing on wound healing. Detailed Implementation

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0037] The following specific embodiments illustrate the solution proposed in this invention: Example 1: Error-prone PCR of recombinant mannuronic acid C-5 epimerase MC5EN3 Using the amino acid sequence of the mannuronic acid C-5 epimerase KAA8560786.1 as the original amino acid sequence, reverse translation of this amino acid sequence was performed using Snap gene software to obtain the nucleotide sequence encoding this amino acid sequence, which was then artificially synthesized. Using this nucleotide sequence as a template, a fallibility-prone PCR reaction was performed. The DNA amplification enzyme used in the reaction was Phantadine. ® The Super-Fidelity DNA Polymerase reaction system consisted of: 10 μL Buffer; 1 μL dNTP; 1 μL Phanta; 2 μL front primer (5'-GATACTGCTCAACCAGTTGATGCTC-3', SEQ ID NO. 4); 2 μL back primer (5'-ATCCTGCATCTCAGTTTGTCTTTCAACT-3', SEQ ID NO. 5); 1 μL genomic template; and ddH2O to a final volume of 50 μL. The annealing temperature was set at 58℃, and the extension time was 72℃ for 3 min. The PCR products were analyzed using agarose gel electrophoresis. The results are shown below. Figure 1 As shown, the DNA amplification band has the correct molecular weight. The verified PCR product was further purified using DpnI enzyme digestion and the Cycle Pure Kit PCR purification kit, following the instructions for use of both the DpnI enzyme and the Cycle Pure Kit PCR purification kit. The purified DNA fragment is the PCR amplification product prepared by recombinant mannuronic acid C-5 epimerase MC5EN3.

[0038] Example 2 Construction of Recombinant Expression Vector The Pichia pastoris X33 expression vector was linearized by digestion with Not I and EcoRI. The error-prone PCR product of the mannouronic acid C-5 epimerase MC5EN3 was then ligated into the linearized expression vector via in vitro homologous recombination. The homologous recombination ligase used was Exnase II, and the ligation system (50 μL) consisted of: CE II Buffer 2 μL; Exnase II 0.5 μL; linearized vector 0.5 μL; PCR product 1 μL; ddH2O 1 μL. The ligation conditions were 37 ℃ for 30 min. The ligation product was then transformed into *E. coli* DH5α using a heat shock method and cultured at 37 ℃ for 12–16 h. Single colony selection was performed, and positive clones were verified, completing the construction of the mannouronic acid C-5 epimerase MC5EN3 recombinant expression vector.

[0039] Example 3: Expression of recombinant mannuronic acid C-5 epimerase MC5EN3 in Pichia pastoris The constructed recombinant expression vector was linearized by Sac I enzyme digestion. The linearized recombinant expression vector was then transformed into Pichia pastoris X33 competent cells via electroporation. The specific transformation method was as follows: the linearized recombinant expression vector was transformed into competent cells, incubated on ice for 30 min, and then electroporated. 1 mL of pre-chilled 1 M sorbitol solution was added, and the cells were incubated at 30°C for 1 h. The cells were then plated onto LB agar plates containing Zeocin antibiotic and incubated at 30°C for 2-3 days. The resulting yeast transformants were screened for positive transformants and inoculated into BMGY liquid medium for shake-flask fermentation at 30°C and 200 rpm. Induction was performed by adding 1% methanol every 24 h, for a total of 3 additions. After expression, the cells were centrifuged, and the supernatant was collected, which was the prepared mannouronic acid C-5 epimerase MC5EN3. Fermentation was analyzed using SDS-PAGE, and the results are shown below. Figure 2 As shown, the fermentation broth contains a band with a molecular weight similar to that of the mannouronic acid C-5 epimerase MC5EN3, indicating that the mannouronic acid C-5 epimerase N2MC5E was successfully expressed in Pichia pastoris X33.

[0040] Example 4: Evaluation of the conversion efficiency of M to G catalyzed by recombinant mannuronic acid C-5 epimerase MC5EN3. The heterologously expressed recombinant mannuronate C-5 epimerase MC5EN3 was compared with the original mannuronate C-5 epimerase using Ni +Affinity chromatography purification was performed, and the protein concentration of the purified enzyme sample was determined using a BCA kit. The contents of M and G in the substrate before and after the enzyme reaction were determined by HPLC to evaluate the conversion efficiency of recombinant mannuronic acid C-5 epimerase MC5EN3 and mannuronic acid C-5 epimerase in the conversion of M to G. The specific reaction system was as follows: 900 μL of 1% polyM solution was added to a 10 mL test tube, followed by 100 μL of appropriately diluted enzyme solution (concentration 200 μg / mL). The mixture was vortexed and incubated at 37°C for 12 h. After the water bath, the reaction system was placed in a boiling water bath for 10 min, immediately cooled, and then subjected to acid hydrolysis and derivatization to determine the M and G contents in the sample.

[0041] When determining the G and M content in alginate using HPLC, an XDB-C18 column was used. The sample injection volume was 20 μL, the absorbance was set to 245 nm, and the column temperature was 25 ℃. Mobile phase A was 0.05 mol / L phosphate buffer (KH₂PO₄, NaOH, pH 6.9), and mobile phase B was acetonitrile. The elution mobile phase composition was 17% acetonitrile + 83% phosphate buffer, and the flow rate was 1 mL / min.

[0042] The M and G contents of the substrate and the product after reaction with recombinant mannouronic acid C-5 epimerase were determined by HPLC, and the results are as follows: Figure 3 The results showed that the glucose (G) content of 1% poly M substrate was 13.32%. After reaction with recombinant mannuronic acid C-5 epimerase MC5EN3, the G content of the product was 34.08%. Comparative analysis of the conversion efficiency with that of the original mannuronic acid C-5 epimerase showed that the G content of the product after reaction with the original mannuronic acid C-5 epimerase was 23.35%. These results indicate that the recombinant mannuronic acid C-5 epimerase MC5EN3 has the ability to catalyze the conversion of M to G, efficiently converting M in alginate poly M to G, and the conversion efficiency is improved compared to the original mannuronic acid C-5 epimerase. The isomerization efficiency of MC5EN3 can increase the G content in poly M substrate from 13.32% to 34.08%, thus enabling the preparation and provision of alginate with a stable increased G content, which is beneficial for its application in the preparation of high-G-content alginate.

[0043] Example 5: Recombinant mannuronic acid C-5 epimerase MC5EN3 catalyzes the conversion of M to G and prepares alginate with increased G content. After confirming that MC5EN3 possesses catalytic activity for the conversion of M to G, commercially available alginate was used as a raw material to prepare G-enhanced alginate catalyzed by MC5EN3. The contents of M and G in the substrate before and after the enzymatic reaction were determined using HPLC to evaluate the conversion efficiency of recombinant mannouronic acid C-5 epimerase MC5EN3 from M to G. The specific reaction system was as follows: 500 μL of 1% commercially available alginate substrate was added to a 10 mL test tube, followed by 500 μL of MC5EN3 enzyme solution at a concentration of 20 μg / mL. The mixture was vortexed and incubated in a 37°C water bath for 12 h. After the water bath, the reaction system was placed in a boiling water bath for 10 min, immediately cooled, and then subjected to acid hydrolysis and derivatization treatments to determine the M and G contents in the samples.

[0044] The contents of G and M in 1% commercial alginate substrate and alginate prepared by MC5EN3 catalysis were determined by HPLC. The results are as follows: Figure 4 The results showed that the substrate had a G content of 31.32% and an M / G ratio of 2.19. After reaction with recombinant mannouronic acid C-5 epimerase MC5EN3, the product had a G content of 53.35% and an M / G ratio of 0.87. These results indicate that MC5EN3 has the ability to catalyze the conversion of M to G, specifically converting M in alginate polysaccharide to G with an isomerization rate of 32.07%, and the prepared alginate had a G content of 53.35%.

[0045] Example 6: Preparation of alginate composite hydrogel A calcium carbonate solution with a calcium ion to carboxyl group ratio of 0.25 was prepared to obtain a 1% alginate solution. After uniform dissolution, approximately 0.06 g of calcium carbonate and 0.075 g of citric acid aqueous solution, along with 10% small molecule hyaluronic acid solution, were added to each sample. The solution was poured into a 12-well plate, allowed to stand for one day, and then a portion was frozen and lyophilized to prepare an alginate composite hydrogel sample.

[0046] The microstructure of the surfaces of the two hydrogel scaffolds was observed and the surface elements were qualitatively analyzed using scanning electron microscopy-X-ray energy dispersive spectroscopy (SEM-EDS). The results are as follows: Figure 5 As shown, the alginate composite hydrogel structure catalyzed by MC5EN3 is more compact, indicating a higher degree of gel cross-linking. Furthermore, the elemental analysis spectrum shows that the cross-linked Ca in this hydrogel sample... 2+ The total amount is greater. The above results indicate that MC5EN3 catalysis leads to an increase in the G content in the alginate composite hydrogel, thereby making its structure stable and dense. It also shows that the hydrogel sample was successfully prepared and has a dense, cross-linked gel characteristic structure.

[0047] Example 7: FTIR analysis of alginate composite hydrogel Infrared spectroscopy was used to characterize the structure of alginate-based composite hydrogels prepared using MC5EN3 catalysis. The results are as follows: Figure 6 As shown, the alginate composite hydrogel sample can be deposited at 3200-3600 cm⁻¹. -1 A broad peak appears at [value missing], which is a characteristic peak of hydrogen bonding between SA molecules or between SA and water. Furthermore, the FTIR spectrum shows asymmetric stretching vibrations of the carboxylate groups in the composite hydrogel sample (1600-1610 cm⁻¹). -1 ) and symmetrical stretching vibration (1400-1410 cm) -1 The particles moved to lower and higher wavenumbers respectively, indicating that they were successfully cross-linked with calcium ions, and the hydrogel sample was successfully prepared.

[0048] Example 8: Evaluation of the wound healing efficacy of brown alginate composite hydrogel dressing Alginate composite hydrogel dressings prepared using MC5EN3 catalysis were sterilized under UV light for 30 min. Scissors, tweezers, a hair removal device, and a skin punch were prepared. Six- to eight-week-old male ICR mice were used in the experiment and divided into three groups: a negative control group (no wound treatment), a positive control group (3M gel dressing), and an experimental group using the alginate composite hydrogel dressing, with six mice in each group. Mice in different groups were first acclimatized and then anesthetized before having their backs shaved. The general wound area was disinfected with iodine-soaked cotton balls using a circular motion. After 1-2 minutes, the iodine was removed with 75% alcohol cotton balls to model the wound. The mice were then placed on their sides, and the skin on their backs was pulled out with tweezers. The punch was then applied to the pulled-out skin, and holes were made by pressing and gently smoothing the skin. The different groups were treated with their respective dressing samples, and the wound healing status was observed daily, with dressings changed as needed. The animal experiment protocol was approved by the Animal Ethics Committee of Ocean University of China (Shandong, China), and the experimental procedures were conducted in accordance with the guidelines of the National Health Commission of the People's Republic of China and the "Guidelines for the Management and Use of Laboratory Animals: Eighth Edition", ISBN-10: 0-309-15396-4.

[0049] To monitor the wound healing process, images of the wounds of mice were taken at 0, 3, 6, 9, and 12 days post-injury, and the wound size was calculated using ImageJ software. The wound healing rate was calculated using the formula: Wound healing rate = (A0 - A...) t The wound healing rate is calculated as A0 / A0, where A0 is the wound area and A t The area of ​​the wound after a fixed time interval is represented. Results showed that after 3 days of treatment, the relative wound area ratio in the alginate composite hydrogel dressing group (56.80%) was significantly lower than that in the control group (75.70%), indicating that the composite hydrogel can more effectively promote wound healing. Figure 7By day 12, the wounds in the alginate composite hydrogel dressing group and the positive control group had basically healed, with the relative wound area ratios decreasing to 8.04% and 6.69%, respectively, significantly better than the blank control group (20.94%). Figure 7 ).

[0050] The above results demonstrate that the alginate with a G content of 53.35% prepared using the mannouronic acid C-5 epimerase MC5EN3 disclosed in this invention can effectively promote wound healing, thereby greatly expanding the application of alginate and its composite hydrogel in the fields of food, biomedicine and biomaterials.

[0051] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0052] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0053] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A recombinant mannuronic acid C-5 epimerase MC5EN3, characterized in that, The amino acid sequence of the recombinant mannuronic acid C-5 epimerase MC5EN3 is shown in SEQ ID NO:

1.

2. A biomaterial related to the recombinant mannuronic acid C-5 epimerase MC5EN3 of claim 1, characterized in that, The biomaterial is any one of the following: 1) A nucleic acid molecule encoding the recombinant mannuronic acid C-5 epimerase MC5EN3 as described in claim 1; 2) An expression cassette containing the nucleic acid molecule described in 1); 3) A recombinant vector containing the nucleic acid molecule described in 1); 4) A recombinant vector containing the expression cassette described in 2); 5) Recombinant microorganisms containing the nucleic acid molecules described in 1); 6) Recombinant microorganisms containing the expression cassette described in 2); 7) Recombinant microorganisms containing the recombinant vector described in 3); 8) Recombinant microorganisms containing the recombinant vector described in 4).

3. The biomaterial according to claim 2, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 2; and / or the recombinant microorganism is a recombinant yeast.

4. A method for preparing recombinant mannuronic acid C-5 epimerase MC5EN3, characterized in that, The gene encoding the recombinant mannuronic acid C-5 epimerase MC5EN3 as described in claim 1 was cloned into a recombinant expression vector, the recombinant expression vector was introduced into host cells, positive recombinant hosts were screened, the positive recombinant hosts were cultured, expression was induced, and recombinant mannuronic acid C-5 epimerase MC5EN3 was obtained.

5. The recombinant expression vector according to claim 4, characterized in that, The recombinant expression vector is one or more of the following: Escherichia coli expression vector, yeast expression vector, Bacillus subtilis expression vector, lactic acid bacteria expression vector, Streptomyces expression vector, bacteriophage vector, filamentous fungus expression vector, plant expression vector, insect expression vector, or mammalian cell expression vector; and / or the host cell includes Escherichia coli host cell, yeast host cell, Bacillus subtilis host cell, lactic acid bacteria host cell, actinomycete host cell, filamentous fungus host cell, insect cell, or mammalian cell.

6. The application of the recombinant mannuronic acid C-5 epimerase MC5EN3 of claim 1 or the biomaterial of claim 2 in catalyzing the conversion of β-D-mannuronic acid to α-L-guluronic acid.

7. A method for catalyzing the conversion of β-D-mannuronic acid to α-L-guluronic acid, characterized in that, The recombinant mannuronic acid C-5 epimerase MC5EN3 of claim 1 was added to the substrate for catalytic reaction to obtain a product with increased α-L-guluronic acid.

8. The method according to claim 7, characterized in that, The catalytic reaction temperature is 35-39°C, and the pH value is 6-8; and / or The catalytic reaction time is 8-15 h; and / or The substrate includes at least one of alginate and alginate oligosaccharides.

9. A brown alginate composite hydrogel wound dressing, characterized in that, Includes the product prepared by the method of claim 7 or 8.

10. The application of the alginate composite hydrogel wound dressing according to claim 9 in promoting wound healing.

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