Mannuronic acid C-5 epimerase MpMC5E as well as preparation method and application thereof

Abstract

The invention relates to mannuronic acid C-5 epimerase MpMC5E as well as a preparation method and application thereof, and the MpMC5E is prepared by the following method: culturing a host cell containing a mannuronic acid C-5 epimerase MpMC5E coding gene, and inducing the expression of the MpMC5E coding gene to obtain the mannuronic acid C-5 epimerase MpMC5E, the nucleotide sequence of the MpMC5E coding gene is as shown in SEQ ID NO: 2. According to the preparation method disclosed by the invention, efficient soluble expression of MpMC5E derived from marine bacteria in escherichia coli is successfully realized, and a foundation is laid for functional research of MpMC5E. MpMC5E is used as a raw material and is proved to have mannuronic acid C-5 epimerase activity, beta-D-mannuronic acid can be efficiently catalyzed to be converted into alpha-L-guluronic acid, and the isomerization conversion rate reaches up to 56.7% within a short time (20-28 h), so that stable algin with high G content can be provided, and the application of the algin in the fields of food, medicine and the like is greatly expanded.

Description

Technical Field

[0001] The invention belongs to the technical fields of genetic engineering and enzyme engineering, and particularly relates to a mannuronic acid C-5 epimerase MpMC5E and a preparation method and application thereof. Background Art

[0002] Algin is a water-soluble acidic polysaccharide that is widely distributed in the cell walls and extracellular matrix of brown algae. In a broad sense, alginate covers alginic acid, alginates and organic derivatives. Algin is composed of β-D-mannuronic acid (M) and α-L-guluronic acid (G) connected by a β-1,4-glycosidic bond. M and G are isomers, and the difference is only reflected in the position of the C-5 hydroxyl group. In alginate, M and G exist in three distribution forms: polymannuronic acid fragments (polyM), polyguluronic acid fragments (polyG), and mannuronic acid-guluronic acid mixed blocks (polyMG).

[0003] In recent years, brown algae have attracted widespread attention due to their rich content of high-value-added bioactive substances such as polysaccharides and oligosaccharides. Among them, alginate polysaccharides and their oligosaccharide derivatives have shown broad application prospects due to their good biocompatibility, non-toxicity, biodegradability, and functional diversity in various matrices and substrates. Alginate oligosaccharides (AOS) are degradation products of alginate, characterized by low molecular weight, high water solubility, and good bioactivity. The multifunctional properties of alginate oligosaccharides are derived from their unique structure, especially the M / G ratio. The ratio and arrangement order of M and G between different alginate molecules vary, resulting in different physical and chemical properties of alginate. The lower the M / G ratio, the higher the gel strength and rigidity, as well as better mechanical properties and higher antimicrobial activity. By regulating the M / G ratio, its application in related fields such as food and medicine can be targeted and designed.

[0004] Compared with physical and chemical treatment methods, bioenzymatic modification of alginate shows unique advantages. This technology achieves precise control of the molecular structure of alginate through enzymatic reactions, which not only breaks through the limitation of the source of brown algae raw materials, but also avoids the problem of secondary pollution. In brown algae, mannuronic acid C-5 diastereomerase can specifically catalyze the conversion of M to G. This property provides the possibility for the targeted modification of alginate structure. At present, brown algae with high G content in nature not only have the problem of low reserves, but also change with various environmental conditions. Therefore, it is impossible to provide stable high-G content alginate, which greatly limits its application in various fields.

[0005] Mannuronate C-5 epimerase, a key alginate-modifying enzyme, increases the G residue content of the substrate, upgrading alginate or brown algal oligosaccharides into products with long polyG or polyMG residues. Depending on the molecular length, M / G residue ratio, and distribution, these products possess varying properties such as gel strength, hydration capacity, viscosity, and bioactivity, thereby expanding their application range. Mannuronate C-5 epimerase is primarily derived from seaweed and bacteria. Bacterial sources include Pseudomonas genera (e.g., Pseudomonas fluorescens, Pseudomonas syringae, Pseudomonas mendocina, and Pseudomonas aeruginosa) and Azotobacter genera (e.g., Azotobacter vinifera and Azotobacter chrysogenum). Currently, few marine bacterial-derived mannuronate C-5 epimerases have been expressed. Different growth environments may give marine bacterial-derived mannuronate C-5 epimerases unique catalytic properties, such as salt tolerance and high catalytic efficiency. However, soluble expression of marine-derived mannuronate C-5 epimerases is difficult, further limiting their development and application. Summary of the Invention

[0006] Based on this, the purpose of the present invention is a mannuronic acid C-5 epimerase MpMC5E and its preparation method and application. The preparation method successfully achieves the soluble expression of MpMC5E derived from marine bacteria, and the prepared MpMC5E can efficiently catalyze the conversion of β-D-mannuronic acid to α-L-guluronic acid.

[0007] The first aspect of the present invention is to provide a method for preparing mannuronic acid C-5 epimerase MpMC5E, comprising the following steps: culturing a host cell containing a gene encoding mannuronic acid C-5 epimerase MpMC5E, inducing expression of the MpMC5E encoding gene, and obtaining the mannuronic acid C-5 epimerase MpMC5E;

[0008] The nucleotide sequence of the MpMC5E encoding gene is shown in SEQ ID NO: 2.

[0009] In some embodiments, the host cell contains a recombinant expression vector into which the MpMC5E encoding gene is inserted.

[0010] In some embodiments, the recombinant expression vector includes an Escherichia coli recombinant expression vector, a yeast recombinant expression vector, or a Bacillus subtilis recombinant expression vector; and / or, the host cell includes an Escherichia coli cell, a yeast cell, or a Bacillus subtilis cell.

[0011] In some embodiments, the recombinant expression vector is an Escherichia coli recombinant expression vector, the host cell is an Escherichia coli cell, and the preparation method comprises the following steps:

[0012] (1) fermenting the Escherichia coli cells to induce expression of the MpMC5E encoding gene after culturing to the logarithmic growth phase;

[0013] (2) Stop the fermentation, centrifuge the fermentation liquid to collect the bacteria, break the bacteria, and centrifuge to obtain the supernatant.

[0014] In some embodiments, IPTG is used in step (1) to induce the expression of the MpMC5E encoding gene; preferably, the induction conditions are: IPTG 0.1 mM to 0.6 mM, and / or temperature 15° C. to 18° C., and / or pH 6.5 to 7.5.

[0015] In some embodiments, the induction conditions are: IPTG 0.1 mM to 0.3 mM, and / or temperature 15° C. to 17° C., and / or pH 6.5 to 7.0.

[0016] In some embodiments, fermentation is stopped 15 to 20 hours after induction of expression.

[0017] In some embodiments, fermentation is stopped 15 to 18 hours after induction of expression.

[0018] In some embodiments, step (2) further comprises purifying the supernatant; preferably, purification is performed using chromatography; more preferably, purification is performed using affinity chromatography.

[0019] The second aspect of the present invention is to provide mannuronate C-5 epimerase MpMC5E prepared by the above-mentioned preparation method.

[0020] The third aspect of the present invention is to provide the use of the mannuronic acid C-5 epimerase MpMC5E described above in catalyzing the conversion of γ-D-mannuronic acid to α-L-guluronic acid.

[0021] The fourth aspect of the present invention is to provide a method for catalyzing the conversion of β-D-mannuronic acid to α-L-guluronic acid, comprising the following steps: adding the mannuronic acid C-5 epimerase MpMC5E described above to a substrate for catalytic reaction.

[0022] In some embodiments, the temperature of the catalytic reaction is 20° C. to 40° C., and the pH value is 6 to 8.

[0023] In some embodiments, the temperature of the catalytic reaction is 25° C. to 35° C., and the pH value is 6.5 to 7.5.

[0024] In some embodiments, the catalytic reaction time is 20 hours to 28 hours, preferably 20 hours to 26 hours, and more preferably 20 hours to 24 hours.

[0025] In some embodiments, the substrate comprises at least one of alginate and algal oligosaccharide.

[0026] The inventors of the present invention predicted a protein derived from marine bacteria that may have high mannuronic acid C-5 epimerase catalytic activity through a comprehensive evaluation of bioinformatics analysis. After research, the inventors obtained the encoding gene by optimizing the codons and deleting the nucleotide sequence corresponding to the 23 amino acids encoding the N-terminus of the protein. They successfully achieved efficient soluble expression of the encoded protein of the gene in Escherichia coli, laying the foundation for its functional research.

[0027] Furthermore, the present invention confirms that the expressed protein has mannuronic acid C-5 epimerase activity and can catalyze the conversion of β-D-mannuronic acid to α-L-guluronic acid, so it is named mannuronic acid C-5 epimerase MpMC5E, and optimizes the method of using MpMC5E to efficiently catalyze the conversion of β-D-mannuronic acid to α-L-guluronic acid in substrates (including alginate and brown algae oligosaccharides). The method uses MpMC5E as a catalytic enzyme under appropriate temperature and pH conditions, and can achieve an isomerization conversion rate of up to 56.7% in a relatively short period of time (20h to 28h), reducing the M / G ratio in brown algae oligosaccharides to 0.25, thereby providing alginate with a stable high G content, greatly expanding the application of alginate in the fields of food, medicine, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the SDS-PAGE diagram of the target protein expression and purification.

[0029] Figure 2 This is the result of MpMC5E gene amplification.

[0030] Figure 3 This is the SDS-PAGE image of the fermentor-induced expression of MpMC5E.

[0031] Figure 4 The SDS-PAGE diagram of MpMC5E induced expression at different induction temperatures.

[0032] Figure 5 This is the test result of the effect of temperature on MpMC5E enzyme activity.

[0033] Figure 6 This is the test result of the effect of pH on MpMC5E enzyme activity.

[0034] Figure 7 The product of MpMC5E's action on brown algal oligosaccharide (polyMG) 1 H NMR spectrum. DETAILED DESCRIPTION

[0035] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the present disclosure more thorough and comprehensive.

[0036] Experimental procedures in the following examples, where specific conditions are not specified, generally followed conventional conditions, such as those in Molecular Cloning: A Laboratory Manual (4th edition, edited by Green and Sambrook, published in 2013), or according to manufacturer recommendations. All commonly used chemical reagents used in the examples were commercially available.

[0037] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as those belonging to the art and

[0038] The terms "and / or" and "and / or" used in the present invention are intended to be used only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" as used in the present invention includes any and all combinations of one or more of the related listed items.

[0039] Furthermore, as used herein, the term "or" is inclusive and equivalent to the term "and / or," unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly dictates otherwise. Furthermore, throughout this specification, the meanings of "a," "an," and "the" include plural referents. The meaning of "in" includes "in" and "on."

[0040] The present invention is further described in detail below with reference to specific embodiments.

[0041] The vector pET28a, expression host Escherichia coli BL21(DE3), and algal oligosaccharide (polyMG, 3000 kDa) used in the following examples were commercially available and stored in the applicant's laboratory. All other materials and reagents were commercially available unless otherwise specified.

[0042] In the following examples, the culture medium used was as follows:

[0043] 1. Activation culture medium: 1% tryptone, 0.5% yeast powder, 1% NaCl, 1.5% agar powder, sterilize at 121°C for 20 min, add kanamycin to a final concentration of 50 μg / mL before pouring the plate.

[0044] 2. Primary seed culture medium LB (L -1 ): 1% tryptone, 0.5% yeast extract, 1% NaCl, 20 mL test tube filled with 5 mL, sterilized at 121℃ for 20 min.

[0045] 3. Secondary seed culture medium LB (L -1 ): 1% tryptone, 0.5% yeast extract, 1% NaCl, 150 mL in a 500 mL Erlenmeyer flask, sterilize at 115°C for 20 min.

[0046] 4. Component 1: Fermentation medium (L -1 ): 11.5 g yeast powder, 19.5 g peptone, 4 g (NH4)2SO4, 0.98 g citric acid monohydrate, 18 g K2HPO4·3H2O, 3 g KH2PO4, 0.5 mL defoaming agent, sterilize at 121°C for 30 min (prepared according to the 3 L recipe, but the final volume is only adjusted to 2.7 L).

[0047] 5. Component 2 (L -1 ): 30g glucose, 7.38g MgSO4·7H2O, dilute to 150ml, place in a 500mL Erlenmeyer flask and sterilize at 115℃ for 20min.

[0048] 6. Component 3: trace elements (L -1 ): 2.8g FeSO4·7H2O, 0.08g NaI, 1.71g MnSO4·H2O, 2.8gCoSO4·7H2O, 1.13g CaCl2, 0.2g CuCl2·2H2O, 0.14g ZnCl2, sterilized at 121°C for 30 minutes.

[0049] 7. Component 4: Glucose feed medium (L -1 ): 480 g glucose, 8 g MgSO4·7H2O, prepared in 800 mL, microwave-assisted dissolution, and sterilized at 115°C for 20 min.

[0050] In the following examples, the detection methods involved are as follows:

[0051] 1. Wet weight determination: Take three 2 mL centrifuge tubes and accurately weigh their total weight M. Pipette 2 mL of fermentation broth into each 2 mL centrifuge tube and centrifuge at 12000 rpm for 3 min. Carefully discard the supernatant and accurately weigh the total weight M. Calculate the cell wet weight (g / L) = (M' - M) / 6 × 1000.

[0052] 2. OD 600Determination: Use pure water as a blank and dilute the fermentation liquid with pure water a certain number of times so that the OD value measured at a wavelength of 600nm is between 0.2 and 0.8. Then multiply this value by the dilution multiple to obtain the cell OD value in the fermentation liquid.

[0053] 3. Protein content determination

[0054] The protein concentration of crude enzyme solution and pure enzyme solution was determined using the Coomassie Brilliant Blue (Bradford) protein concentration kit.

[0055] 4. Isomerization products 1 H NMR detection

[0056] The isomerization reaction 1 The H NMR spectrum clearly shows the peaks corresponding to the uronic acid residues. The NMR spectrum was integrated using the MestReNova software: ∼5.07 ppm (G, H1) = A; ∼4.78 ppm (G G M, H5) = B1; ~ 4.74ppm (M G M, H5) = B2; ~ 4.70ppm ( M G, H1) = B3, ~ 4.67ppm ( M M, H1) = B4; ~ 4.48ppm ( G G, H5)=C, where the underline indicates the uronic acid residue, H indicates the proton generating the signal, and the letters are used as the calculation names.

[0057] The calculation formula is as follows:

[0058] G = 0.5[A + C + 0.5(B1 + B2 + B3)]

[0059] M=B4+0.5(B1+B2+B3)

[0060] F G =G / (M+G)

[0061] F M =M / (M+G)

[0062] F g +F M =1

[0063] Example 1 Preparation of MpMC5E

[0064] Through literature research, we analyzed the amino acid sequences and structural characteristics of reported mannuronate C-5 epimerases. Using PmC5A (GenBank ID: CP000680.1) from Pseudomonas mendocina.sp. DICP-70 as a template, we performed a BLASTP search of homologous gene sequences in the NCBI database. Marine mannuronate C-5 epimerase sequences with 30-60% identity to the template were selected as candidate genes.

[0065] Based on a comprehensive evaluation of bioinformatics analysis, it was finally determined that the protein derived from Marinobacter pelagius with the amino acid sequence shown in SEQ ID NO: 4 would be selected as the target protein for subsequent gene cloning, expression, and research.

[0066] SEQ ID NO: 4:

[0067]

[0068]

[0069] In order to improve the expression efficiency of the gene in the host system Escherichia coli, the gene sequence encoding the target protein with an amino acid sequence as shown in SEQ ID NO: 4 was codon optimized to obtain the coding gene with a nucleotide sequence as shown in SEQ ID NO: 3. The whole gene synthesis technology was commissioned to Sangon Biotech (Shanghai) Co., Ltd.

[0070] SEQ ID NO: 3:

[0071]

[0072]

[0073] The coding gene fragment with the nucleotide sequence shown in SEQ ID NO: 3 was cloned into the prokaryotic expression vector pET28a to complete the construction of the recombinant plasmid.

[0074] Subsequently, the resulting plasmids were transformed into TOP10 competent E. coli cells. Furthermore, the competent cells were plated onto solid LB plates containing kanamycin at a final concentration of 50 μg / mL and cultured at 37°C for 12–16 hours. A single colony was picked and transferred to 5 mL of liquid LB medium. After incubation at 37°C and 220 rpm for 12–16 hours, the plasmids were extracted and the recombinant plasmids were verified by sequencing.

[0075] After sequencing verification, the recombinant plasmid was transformed into competent E. coli BL21(DE3) cells using the heat shock method. Furthermore, the competent cells were plated onto solid LB plates containing kanamycin at a final concentration of 50 μg / mL and incubated at 37°C for 12–16 hours. A single colony was transferred to 5 mL of liquid LB medium and incubated at 37°C and 220 rpm for 12–16 hours. The bacterial suspension was then stored in glycerol to obtain the recombinant strain.

[0076] Fermentation expression and purification of recombinant strains

[0077] The specific steps include:

[0078] (1) Activation of bacterial strains: Streak the glycerol bacteria on a solid LB plate containing 50 μg / mL kanamycin and culture in a 37°C incubator overnight.

[0079] (2) Preparation of primary seed solution: A single colony was selected from the cultured plate and inoculated into 5 mL LB liquid test tube culture medium containing 50 μg / mL kanamycin under the sterile environment of a UV clean bench. The culture was carried out at 37°C and 220 rpm for 10 to 12 hours.

[0080] (3) Preparation of secondary seed liquid: Take 1 mL of the primary seed liquid and inoculate it into 150 mL of LB liquid shake flask culture medium, and add kanamycin to make the final concentration 50 μg / mL. Continue to culture at 37 ° C and 220 rpm for 12 hours to ensure that the bacteria grow fully and reach the appropriate inoculation concentration. This step provides a sufficient number of activated bacteria for the subsequent fermentation process by expanding the culture scale, while ensuring the activity and consistency of the strain. The entire operation process strictly follows the aseptic operation specifications to avoid the influence of bacterial contamination on the experimental results.

[0081] (4) Control of high-density fermentation: Component 1 (fermentation medium) was poured into a 7L fermenter. Before sterilizing the fermenter, the pH electrode needed to be calibrated using standard solutions of pH 6.86 and pH 4.00. The dissolved oxygen electrode was installed and the ventilation filter and other interfaces were sealed with tin foil. The mixture was sterilized at 121°C for 30 minutes. Ammonia feeding bottles, defoaming agent feeding bottles, and glucose feeding bottles were prepared. The ammonia feeding bottles were sterilized and then filled with ammonia in a fume hood. After the fermenter was sterilized, the ventilation line and condensate line were opened, the speed was adjusted to 300 rpm, the ventilation volume was adjusted to 3 L / min, and the temperature was automatically controlled to 37°C. During inoculation, component 2, component 3, and seed solution were poured in sequence. To calibrate the dissolved oxygen electrode, the ventilation volume was increased to 10 L / min, the speed was increased to 800 rpm, and the dissolved oxygen was calibrated to 100%.

[0082] In the initial stage of fermentation, the ventilation volume is set to 3L / min, the stirring speed is maintained at 100rpm, and the fermentation temperature is controlled at 37°C. The dissolved oxygen level is maintained above 30% by adjusting the rotation speed and dissolved oxygen in a linked manner. The ammonia water is transferred and added to the sterilized feeding bottle, the feeding needle is connected to the fermentation tank, and the pH value of the fermentation liquid is stabilized at around 7.0 using an automatic control system. After fermentation for about 4 hours, the stirring speed will be increased to 800rpm. At this time, the linkage control of the rotation speed and dissolved oxygen will be cancelled, and the ventilation volume will be increased to 6L / min. When the dissolved oxygen drops to the lowest value and rises sharply, the glucose feeding program is started. When OD 600 When the temperature reached about 30°C (about 7 h), the temperature was lowered to 16°C and 0.1 mM IPTG was added for induction.

[0083] After 15 minutes of induction, fermentation was terminated and the fermentation broth was centrifuged in a high-speed refrigerated centrifuge at 4°C and 7000 rpm for 20 minutes. The supernatant culture medium was discarded and the precipitated bacteria were collected. The bacteria were resuspended in Buffer A (50 mM phosphate buffer, pH 7.4) at a ratio of 1:10 (w / v) and ultrasonically disrupted for 20 minutes on ice (duty cycle: 2s on / 2s off, amplitude 40%) until the bacteria were clear and homogenous. After disruption, the supernatant was centrifuged at 4°C and 12000 rpm for 30 minutes. The supernatant was collected and filtered through a 0.45 μm filter to obtain a crude enzyme solution, which was stored on ice until further use. The disrupted bacterial lysate (total bacteria), supernatant, and precipitate were subjected to SDS-PAGE protein electrophoresis.

[0084] Using the AKTApure protein purification system, a Ni-NTA affinity chromatography column (pre-packed with Ni Sepharose 6FF) was connected to the instrument and rinsed sequentially with filtered water and Buffer A (50 mM phosphate buffer, pH 7.4) until the UV absorption peak and conductivity reached equilibrium. The crude enzyme solution was then loaded at a flow rate of 4 mL / min, and the sample that passed through the nickel column was collected. Following loading, the column was rinsed with Buffer C (50 mM phosphate buffer, pH 7.4, containing 20 mM imidazole) to remove unadsorbed proteins until the UV absorption peak and conductivity reached equilibrium again. Finally, the target protein adsorbed to the column was eluted with Buffer B (50 mM phosphate buffer, pH 7.4, containing 500 mM imidazole), and the eluate was collected in aliquots. The solution was concentrated using an ultrafiltration tube at 4°C and 4,000 rpm, and then ultrafiltered using Buffer A (50 mM phosphate buffer, pH 7.4) to exchange the salt. The flow-through and pure enzyme solution were subjected to SDS-PAGE electrophoresis. The SDS-PAGE results of expression and purification are shown in Figure 1As shown (before removing the relevant amino acids), according to the protein electrophoresis diagram, it can be seen that the target protein easily forms a large number of inclusion bodies when expressed in E. coli, and it is difficult to obtain a large amount of recombinant protein in the supernatant, and the soluble expression of the target protein cannot be effectively achieved.

[0085] To successfully achieve soluble expression of the target protein, we conducted extensive research and evaluation, ultimately determining that recombinant expression should be performed after removing the nucleotide sequence encoding the N-terminal 23 amino acids of the target protein. To this end, we designed specific primers (as shown in Table 1) and amplified the target protein by PCR to obtain the coding gene fragment after removing the nucleotide sequence corresponding to the N-terminal 23 amino acids of the target protein. The nucleotide sequence is shown in SEQ ID NO: 2.

[0086] Table 1

[0087]

[0088] SEQ ID NO: 2:

[0089]

[0090] The target protein after removing the 23 relevant amino acids at the N-terminus was named MpMC5E, and its amino acid sequence is shown in SEQ ID NO: 1.

[0091] SEQ ID NO: 1:

[0092]

[0093] The gene fragment (MpMC5E gene) with the nucleotide sequence shown in SEQ ID NO: 2 was cloned into the prokaryotic expression vector pET28a to complete the construction of the recombinant plasmid pET28a-MpMC5E. The results of MpMC5E gene amplification were as follows: Figure 2 shown.

[0094] The recombinant strain containing the recombinant plasmid pET28a-MpMC5E was prepared according to the same recombinant strain preparation process, and the fermentation expression and purification of the recombinant strain were carried out according to the same method. 600 , wet weight and residual glucose content to monitor the fermentation process and protein expression status, and collect E. coli fermentation broth at each time point to prepare total bacteria, supernatant and precipitate for SDS-PAGE electrophoresis analysis. The protein expression at 0, 3, 6, 9, 12 and 15h of induction is shown in Figure 2. Figure 3 As shown in the figure, the protein band in the supernatant gradually became thicker with the increase of induction time, indicating that MpMC5E was expressed soluble in the fermenter and the expression level increased with the increase of induction time.

[0095] After the fermentation, the broken bacterial lysate (total bacteria), supernatant, precipitate, flow-through and pure enzyme solution were subjected to SDS-PAGE electrophoresis according to the same method as above. Figure 1 As shown in the figure (after removing the relevant amino acids), according to the protein electrophoresis diagram, the protein content in the supernatant was greatly improved, which effectively solved the inclusion body problem that occurred during the expression process and successfully achieved the soluble expression of MpMC5E.

[0096] In addition, the inventors also found that the induction temperature has a great influence on the expression of MpMC5E in the host cells. It has a better induction expression effect at a lower temperature. The preferred induction temperature is 15°C to 18°C (16°C in this embodiment). An induction temperature above 20°C is not conducive to its expression in the host cells ( Figure 4 , induction time was 15 h).

[0097] Example 2 Determination of Mannosyl C-5 Epimerase Activity of MpMC5E Recombinant Protease

[0098] 1. Determination of mannuronic acid C-5 epimerase activity

[0099] Mannuronic acid C-5 epimerase can catalyze the conversion of M residues to G, thereby changing the configuration of the polysaccharide chain in alginate. This method uses mannuronic acid (polyM) as a substrate, introduces G residues through epimerase reaction, and then uses alginate lyase coupling reaction that specifically cleaves GM / GG bonds to produce a double bond structure of 4,5-unsaturated uronic acid. This structure has a characteristic absorption peak at a wavelength of 230nm, so the epimerase activity in the sample (i.e., the introduction of G residues into PolyM) is closely related to the obtained ΔA 230 The increase is proportional to the value of A 230 The enzyme activity was quantified by the absorbance change.

[0100] 2. Determination of the Effect of Temperature on MpMC5E Enzyme Activity and Stability

[0101] Determination of Optimal Reaction Temperature: Using polyM as the substrate, the enzyme activity of mannuronate C-5 epimerase MpMC5E was measured at 20, 30, 40, 50, 60, and 70°C in 50 mM phosphate buffer (pH 7.0). Each experiment was performed in triplicate. The highest enzyme activity was defined as 100%, and relative enzyme activities at other temperatures were calculated.

[0102] Temperature stability assay: MpMC5E enzyme solution was incubated at 20, 30, 40, 50, 60, and 70°C for 1 hour. Enzyme activity was measured at the optimal temperature. Each experimental group was set up in triplicate. Relative enzyme activity at each temperature was calculated, with the unincubated enzyme activity as 100%.

[0103] Figure 5 Figure a is the optimal reaction temperature result. The enzyme activity of MpMC5E was tested at temperatures of 20, 30, 40, 50, 60 and 70°C. The enzyme activity of MpMC5E reached the highest at 30°C, and its optimal reaction temperature was 30°C. At temperatures of 30-40°C, the relative enzyme activity of MpMC5E remained above 80%, indicating that MpMC5E has good activity within this temperature range. When the temperature exceeded 40°C, the enzyme activity of MpMC5E began to decline significantly, and at 70°C the relative enzyme activity dropped to below 20%, indicating that the enzyme is more sensitive to high temperatures.

[0104] Figure 5 Figure (b) shows the optimal reaction temperature tolerance results. After incubation at 20, 30, 40, 50, 60, and 70°C for 1 hour, the relative enzyme activity of MpMC5E remained above 80% within the 20°C to 30°C temperature range, indicating good enzyme activity within this temperature range. At incubation temperatures below 30°C, the residual enzyme activity of MpMC5E remained above 80%, demonstrating the enzyme's good stability under low-temperature conditions. With increasing incubation temperature, especially above 40°C, the residual enzyme activity of MpMC5E gradually decreased, reaching approximately 25.6% at 60°C, further confirming that the enzyme is a low-temperature enzyme.

[0105] 3. Determination of the Effect of pH on MpMC5E Enzyme Activity and Stability

[0106] Optimal reaction pH determination: Under the optimal reaction temperature, in different buffer conditions of pH 2.0-10.0, using polyM as substrate, the enzyme activity of MpMC5E at different pH conditions was determined. The highest enzyme activity was defined as 100%, and the relative enzyme activity at other pH conditions was calculated.

[0107] pH stability assay: MpMC5E enzyme solution was diluted to the appropriate multiple using different pH buffers and incubated at 4°C for 12 hours. Enzyme activity was then measured under the optimal conditions, with triplicate replicates performed for each experimental group. The highest enzyme activity was defined as 100%, and relative enzyme activity was calculated under the remaining pH conditions.

[0108] pH significantly influences the efficiency of enzyme catalytic reactions. Generally, enzyme activity varies with pH, but deviations from the optimal pH can easily lead to conformational changes in the enzyme and alter the dissociation state of the active center, resulting in partial or complete loss of enzyme activity. The optimal reaction pH for PmC5A, derived from the marine bacterium Pseudomonas mendocina.sp. DICP-70, is 9.0. Most known mannuronate C-5 epimerases have an optimal pH close to neutral and remain stable within a narrow pH range.

[0109] Figure 6 (a) represents the optimal reaction pH. The enzyme activity of MpMC5E was tested at pH 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0. The enzyme activity of MpMC5E reached its highest at pH 7.0, indicating that its optimal reaction pH is 7.0. In the pH range of 6.0 to 8.0, the relative enzyme activity of MpMC5E can be maintained above 80%, indicating that MpMC5E has good catalytic activity within this pH range. When the pH value is lower than 6.0 and higher than 9.0, the enzyme activity of MpMC5E begins to decrease significantly, indicating that the enzyme is more sensitive to extreme pH values.

[0110] Figure 6 Figure b shows the pH tolerance results. After incubation for 12 hours at different pH conditions, the residual enzyme activity of MpMC5E remained above 80% within the pH range of 6.0 to 8.0, demonstrating good enzyme tolerance within this pH range. Under incubation conditions below pH 7.0, the residual enzyme activity of MpMC5E remained above 80%, demonstrating the enzyme's good stability under neutral to slightly acidic conditions. With increasing or decreasing pH, particularly at pH values below 5.0 and above 9.0, the residual enzyme activity of MpMC5E decreased significantly, further confirming the enzyme's good tolerance within the neutral to slightly alkaline pH range.

[0111] Example 3 Evaluation of the effect of MpMC5E recombinant protease on brown algal oligosaccharides

[0112] The specific steps include:

[0113] After diluting the pure MpMC5E enzyme solution to an appropriate concentration (preferably 0.5 mg / mL to 2 mg / mL, diluted to 1 mg / mL in this example) using PB buffer, 0.5 mL of a 0.5% (w / v) polyMG solution, 20 μL of the diluted enzyme solution, and 480 μL of MOPS buffer (20 mM MOPS, 3.6 mM CaCl2, and 100 mM NaCl, pH 7.0) were mixed. A control group used MOPS buffer instead of the MpMC5E enzyme solution. The mixture was placed in a thermostatic metal bath at 500 rpm and 30°C for 24 hours. After completion of the reaction, the mixture was terminated by heating in a boiling water bath for 10 minutes, followed by centrifugation at 12,000 rpm for 3 minutes. The supernatant was removed and transferred to a 4 mL EP tube and frozen with liquid nitrogen. During the freezing process, the tube mouth should be sealed with parafilm, leaving some air holes. Once the supernatant formed a crystalline state, it was quickly placed in a freeze dryer and lyophilized for 24 hours.

[0114] After lyophilization, the reaction product in the EP tube was dissolved in 0.55 mL of D2O, and 20 μL of 0.3 M TTHA solution (metal ion chelator) was added and mixed thoroughly. The mixture was then centrifuged at 12,000 rpm for 3 minutes. After centrifugation, the supernatant was transferred to an NMR tube and 5 μL of 1% (w / v) TSP solution (internal standard) was added. The sample was finally placed on a 600 M superconducting NMR spectrometer for high-temperature H-NMR spectroscopy at 70°C.

[0115] The NMR hydrogen spectrum was integrated and analyzed to calculate the relative contents of M and G before and after the isomerization reaction. Figure 7 The integration results are shown in Table 2. It can be clearly observed from the H NMR spectrum that M The M peak height decreases and G The height of the G peak increased. From the integrated data in Table 2, it can be seen that the relative contents of G and M in the control substrate brown algae oligosaccharide (polyMG) were 52.7% and 47.3%, respectively. After adding the pure MpMC5E enzyme solution obtained after fermentation and purification in the fermenter for 24 hours, the relative content of G increased from 52.7% to 79.5%, and the M / G ratio decreased from 0.898 to 0.258, indicating that MpMC5E exerted the catalytic activity of mannuronic acid C-5 epimerase, with an isomerization conversion rate of 56.7%, and could isomerize 56.7% of M in brown algae oligosaccharide to G.

[0116] Table 2

[0117]

[0118] The results show that the enzymatic method of the present invention can effectively increase the guluronic acid content in brown algae oligosaccharides, and has important application and development prospects in the targeted preparation of brown algae oligosaccharides.

[0119] The above-described embodiments merely represent several implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for preparing mannuronate C-5 epimerase MpMC5E, characterized in that: The following steps are involved: Cultivating a host cell containing a gene encoding mannuronic acid C-5 epimerase MpMC5E, inducing expression of the MpMC5E encoding gene, and obtaining the mannuronic acid C-5 epimerase MpMC5E; The nucleotide sequence of the MpMC5E encoding gene is shown in SEQ ID NO:

2.

2. The preparation method according to claim 1, wherein The host cell contains a recombinant expression vector into which the MpMC5E encoding gene is inserted.

3. The preparation method according to claim 2, wherein The recombinant expression vector includes an Escherichia coli recombinant expression vector, a yeast recombinant expression vector, and a Bacillus subtilis recombinant expression vector; and / or the host cell includes an Escherichia coli cell, a yeast cell, and a Bacillus subtilis cell.

4. The preparation method according to claim 3, wherein The recombinant expression vector is an Escherichia coli recombinant expression vector, the host cell is an Escherichia coli cell, and the preparation method comprises the following steps: (1) fermenting the Escherichia coli cells to induce expression of the MpMC5E encoding gene after culturing to the logarithmic growth phase; (2) Stop the fermentation, centrifuge the fermentation liquid to collect the bacteria, break the bacteria, and centrifuge to obtain the supernatant.

5. The preparation method according to claim 4, wherein In step (1), IPTG is used to induce the expression of the MpMC5E encoding gene; preferably, the induction conditions are: IPTG 0.1mM to 0.6mM, and / or temperature 15°C to 18°C, and / or pH 6.5 to 7.

5.

6. The preparation method according to claim 4 or 5, characterized in that Stop fermentation after 15 to 20 hours of induction expression; and / or, Step (2) also includes purifying the supernatant.

7. Mannuronate C-5 epimerase MpMC5E obtained by the preparation method according to any one of claims 1 to 6. 8 . Use of the mannuronic acid C-5 epimerase MpMC5E according to claim 7 in catalyzing the conversion of β-D-mannuronic acid to α-L-guluronic acid.

9. A method for catalyzing the conversion of β-D-mannuronic acid to α-L-guluronic acid, characterized in that: The following steps are involved: The mannuronic acid C-5 epimerase MpMC5E as described in claim 7 is added to the substrate to carry out a catalytic reaction.

10. The method according to claim 9, wherein The catalytic reaction temperature is 20°C to 40°C, and the pH value is 6 to 8; and / or, The catalytic reaction time is 20h to 28h; and / or, The substrate includes at least one of alginate and brown algae oligosaccharide.

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