A brown algin lyase, a complex enzyme preparation and application thereof

Abstract

This invention proposes an alginate lyase, a compound enzyme preparation, and their applications, belonging to the field of enzyme engineering technology. It solves the technical problem that a single alginate lyase cannot efficiently prepare alginate oligosaccharides directly from brown algae. The alginate lyase of this invention is isolated from Shewanella. Shewanella sp. HD5 The original alginate lyase gene of *Shewanella* was obtained through heterologous expression. Shewanella sp. HD5 The alginate lyase of this invention was deposited on September 22, 2025, at the China Center for Type Culture Collection, Wuhan, Hubei Province, with accession number CCTCC NO: M20252078. This invention provides a high-yield lyase obtained through heterologous expression. It is combined with cellulase, pectinase, and papain to form a complex enzyme preparation that can be directly applied to the enzymatic hydrolysis of brown algae to obtain uniformly polymerized alginate oligosaccharides. Furthermore, the complex enzyme preparation exhibits higher extraction efficiency of alginate oligosaccharides than a single alginate lyase. The prepared alginate oligosaccharides can be used in food, feed, and pharmaceutical fields.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, and particularly relates to an alginate lyase, a compound enzyme preparation and its application. Background Technology

[0002] Bulbae ( Ascophyllum nodosum ) belongs to the phylum Phaeophyta ( Phaeophyta ), round-shaped class ( Cyclosporeae ), Fucusales ( Fucales ), Fucusaceae ( Fucaceae Alginate (also known as brown-green algae) is slender and brownish-green in color. Because it contains abundant active substances such as alginate, polyphenols, and fucoidan sulfate, it has received widespread attention and systematic research in related fields. Alginate, as one of its core active ingredients, has become a research hotspot. However, its complex structure, large molecular weight, high viscosity, strong polymerization, poor solubility, and low bioavailability greatly limit its application in food, medicine, and agriculture. Therefore, to improve the utilization value of alginate, it is usually necessary to convert it into alginic oligosaccharides with lower molecular weight and simpler structure.

[0003] Currently, there are three main degradation methods for converting alginate into fucoidan: physical degradation, chemical degradation, and biodegradation. Physical methods are limited by the finite release of oligosaccharides and are energy-intensive. While chemical methods can efficiently release oligosaccharides, the chemical reagent residues and wastewater treatment can negatively impact the environment and potentially affect the quality and functionality of the product. Compared to physical and chemical methods, biodegradation offers advantages such as mild reaction conditions and environmental friendliness, but the efficiency is generally low when using a single alginate lyase to enzymatically hydrolyze *Tetraphyta*. Furthermore, pre-treating *Tetraphyta* to extract alginate in industrial production before using it as a raw material to prepare fucoidan not only significantly increases economic costs but also causes alginate loss during extraction, thus reducing the final yield.

[0004] Therefore, how to directly and efficiently extract brown algae oligosaccharides from *Leptochloa crus-galli* has become a key problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] This invention addresses the technical problem that a single alginate lyase cannot efficiently prepare alginate oligosaccharides directly from brown algae. It proposes an alginate lyase, a compound enzyme preparation, and their applications, which have higher extraction efficiency for alginate oligosaccharides. The prepared alginate oligosaccharides can be used in food, feed, medicine, and other fields.

[0006] To achieve the above objectives, the technical solution adopted in this invention is as follows: the alginate lyase is isolated from Shewanella bacteria. Shewanella sp. HD5 The original alginate lyase gene of *Shewanella* was obtained through heterologous expression. Shewanella sp. HD5 It was deposited on September 22, 2025, at the China Center for Type Culture Collection, Wuhan, Hubei Province, with accession number CCTCC NO: M20252078.

[0007] In another aspect, the present invention provides a coding gene for encoding the alginate lyase, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0008] The present invention also provides the application of the alginate lyase in the preparation of alginate oligosaccharides from sodium alginate.

[0009] In one embodiment, the amount of alginate lyase added is 0.5% to 2.5% of the mass of sodium alginate, the enzymatic hydrolysis temperature is 35 to 50°C, the pH is 7.0 to 7.5, and the reaction time is 12 to 24 hours.

[0010] In one embodiment, the extraction rate of the fucoidan oligosaccharide is 60.0%~75.0%, and the degree of polymerization of the fucoidan oligosaccharide is 2~6.

[0011] The present invention also provides a compound enzyme preparation comprising the alginate lyase, wherein the amount of alginate lyase added is 0.5% to 1.0% of the mass of algae powder.

[0012] In one embodiment, the compound enzyme preparation further includes cellulase, pectinase and papain; the amount of cellulase added is 1.0% to 2.0% of the mass of algal powder, the amount of pectinase added is 0.5% to 1.5% of the mass of algal powder, and the amount of papain added is 1.0% to 2.0% of the mass of algal powder.

[0013] The present invention also provides the application of the aforementioned compound enzyme preparation in the direct enzymatic hydrolysis of *Tetraphyta fusiforme* to obtain brown algal oligosaccharides.

[0014] In one embodiment, the enzymatic hydrolysis conditions are: a material-to-liquid ratio of 1:15 to 1:30, a hydrolysis temperature of 35 to 50°C, a pH of 6.5 to 7.5, and a reaction time of 12 to 14 hours. In this technical solution, the material-to-liquid ratio represents the ratio of the mass of algae powder to the volume of pure water as the solvent.

[0015] In one embodiment, the extraction rate of the fucoidan oligosaccharide is 10% to 15%, and the degree of polymerization of the fucoidan oligosaccharide is 2 to 6.

[0016] Compared with the prior art, the advantages and positive effects of the present invention are as follows: the alginate lyase of the present invention is Shewanella bacteria isolated from rotten kelp. Shewanella sp. HD5The high-yield lyase obtained was compounded with cellulase, pectinase and papain in a certain proportion to obtain a complex enzyme preparation, which can be directly applied to the enzymatic hydrolysis of brown algae to obtain brown algae oligosaccharides with uniform degree of polymerization. The extraction rate of brown algae oligosaccharides is 10%~15%, and the degree of polymerization of brown algae oligosaccharides is 2~6. Moreover, the complex enzyme preparation has a higher extraction efficiency of brown algae oligosaccharides than single alginate lyase. The prepared brown algae oligosaccharides can be used in food, feed and medicine and other fields. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of protein electrophoresis of alginate lyase in Example 2 of the present invention; wherein, 1: alginate lyase protein; Figure 2 This is a schematic diagram illustrating the effect of the amount of alginate lyase added on the extraction rate of alginate oligosaccharides in Example 3 of the present invention. Figure 3 This is a schematic diagram illustrating the effect of different reaction times of alginate lyase on the extraction rate of alginate oligosaccharides in Example 3 of the present invention. Figure 4 This is a schematic diagram illustrating the effect of different pH values ​​of alginate lyase on the extraction rate of alginate oligosaccharides in Example 3 of the present invention. Figure 5 This is an HPLC chromatogram showing the relative content distribution of alginic oligosaccharides in sodium alginate under the optimal operating conditions of alginate pyrolytic enzyme in Example 3 of the present invention. Figure 6 This is a schematic diagram illustrating the extraction rate of brown algal oligosaccharides from different geographical populations of *Tetraphyta fuciformis* directly degraded by the compound enzyme preparation in Example 5 of the present invention. Figure 7 This is an HPLC chromatogram showing the relative content distribution of brown algae oligosaccharides in sample one directly degraded by the compound enzyme preparation in Example 5 of this invention. Figure 8 This is an HPLC chromatogram showing the relative content distribution of brown algae oligosaccharides in sample two directly degraded by the compound enzyme preparation in Example 5 of this invention. Figure 9 This is an HPLC chromatogram showing the relative content distribution of brown algae oligosaccharides in sample three directly degraded by the compound enzyme preparation in Example 5 of this invention. Figure 10 This is a schematic diagram comparing the extraction rates of alginate oligosaccharides during enzymatic hydrolysis of the compound enzyme preparation of Example 6 of the present invention with those of the single alginate lyase of Comparative Example 1 and the commercial compound enzyme preparation of Comparative Example 2. Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all 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.

[0019] Because wild-type strains producing alginate lyase contain multiple alginate lyase genes, but the alginate lyases produced by different coding genes have different substrate specificities, it is difficult to produce controllable alginate oligosaccharides through enzymatic hydrolysis using wild-type strains, and the enzyme yield of wild-type strains is low, hindering industrial application. This invention specifically expresses a certain alginate lyase through heterologous expression, obtaining high-yield lyase and alginate oligosaccharides with uniform polymerization degree, meeting the needs of industrial production.

[0020] This invention provides an alginate lyase, a compound enzyme preparation, and their applications. The alginate lyase is Shewanella bacteria isolated from decaying kelp. Shewanella sp. HD5 was prepared and combined with cellulase, pectinase, and papain to obtain a compound enzyme preparation that can be directly used for the degradation of brown algae. This preparation enables highly efficient extraction of algal oligosaccharides, yielding algal oligosaccharides with uniform degree of polymerization. The extraction rate of algal oligosaccharides is 10%–15%, and the degree of polymerization is 2–6. Furthermore, compared with single alginate lyases, this compound enzyme preparation has a higher algal oligosaccharide extraction rate, and the prepared algal oligosaccharides can be used in food, feed, and pharmaceutical fields. (Shewanella) Shewanella sp. HD5 The data was collected in April 2024 in Yantai City, Shandong Province, China.

[0021] To more clearly and in detail introduce the alginate lyase, complex enzyme preparation, and their applications provided in the embodiments of the present invention, the following description is provided in conjunction with specific embodiments. In the following embodiments, molecular biology experimental methods not specifically described are all performed in accordance with the specific methods listed in J. Sambrook's "Molecular Cloning: A Laboratory Manual," 3rd edition, or according to the kit and product instructions; the reagents and biological materials mentioned are commercially available unless otherwise specified.

[0022] Example 1 Construction of a recombinant expression vector for alginate lyase Using two restriction endonucleases, NdeI and XhoI, the original alginate lyase gene was inserted into the pET-30a expression vector to construct a recombinant expression vector fused with a GST tag at the N-terminus and a His tag at the C-terminus. The amino acid sequence of the original alginate lyase is shown in SEQ ID NO: 2, and the amino acid sequence of the original alginate lyase after removing the signal peptide is shown in SEQ ID NO: 3. The recombinant expression vector was transformed into Escherichia coli DH5α competent cells by heat shock. The transformed bacterial culture was plated on LB agar plates containing kanamycin resistance and incubated upside down at 37°C for 12-16 hours. Single colonies grown on the plates were picked for positive clone verification, thus completing the construction of the alginate lyase recombinant expression vector.

[0023] Example 2 Expression of recombinant alginate lyase The recombinant expression vector of alginate lyase was transformed into *E. coli* BL21(DE3) competent cells using the heat shock method. The transformed bacterial culture was plated on LB agar plates containing kanamycin resistance and incubated overnight at 37°C with inverted incubation. Single colonies from the transformation plates were picked and inoculated into liquid medium supplemented with kanamycin resistance and incubated overnight at 37°C with shaking at 220 rpm. The culture was then transferred to fresh liquid medium at a 1:100 volume ratio and incubated again at 37°C with shaking at 220 rpm until cell OD reached the target cell count. 600 The concentration was 0.5-0.8, and isopropyl thio-β-D-galactoside (IPTG) was added to a final concentration of 0.2 mM to induce the expression of the target protein.

[0024] Centrifuge the induced culture medium at 10,000 rpm at room temperature for 2 min and discard the supernatant; resuspend the bacterial pellet in TBS buffer, centrifuge at 4,000 rpm for 10 min and discard the supernatant; resuspend the bacterial pellet in TBS buffer again, add benzyl sulfonyl fluoride (PMSF) and sonicate to obtain protein supernatant.

[0025] Protein purification was performed using a gravity column. The protein supernatant was loaded onto a Ni-NTA affinity chromatography column pre-equilibrated with Ni-NTA Binding-Buffer (20 mM Tris, 0.5 M NaCl, 0 mM imidazole, pH 8.0). The column was washed with 3–5 column volumes (CV) of Binding-Buffer to remove unbound protein. A gradient elution was performed using 20 mL of Ni-NTA Elution-Buffer (20 mM Tris, 0.5 M NaCl, 50 mM / 100 mM / 500 mM imidazole, pH 8.0), and the target protein eluent was collected. The collected protein solution was dialyzed against TBS (20 mM Tris, 0.5 M NaCl, 0 mM imidazole, pH 8.0) at a 1:30 volume ratio. The solution was incubated overnight at 4°C with NaCl (pH 8.0). The next day, the dialysis buffer was changed, and dialysis was continued at 4°C for 8 hours. SDS-PAGE was then performed to verify the results. The dialysis sample solution contained the expressed alginate lyase protein. A schematic diagram of the protein electrophoresis is shown below. Figure 1 As shown in the figure, the molecular weight of alginate lyase protein is between 48 and 63 kDa.

[0026] Example 3: Preparation of fucoidan oligosaccharides by degrading sodium alginate with recombinant alginate lyase. 3.1 Optimal Enzyme Dosage for Enzymatic Hydrolysis Using sodium alginate as a substrate, the amount of alginate lyase added was adjusted to 0.1%, 0.25%, 0.5%, 1%, and 2.5% of the sodium alginate mass, respectively. Enzymatic hydrolysis was carried out at pH 7.5 and 45℃ for 18 hours. The extraction rate of alginate oligosaccharides corresponding to different enzyme dosages was determined. The results of the effect of different enzyme dosages on the extraction rate of alginate oligosaccharides are shown in the attached figure. Figure 2 As shown. (From the appendix) Figure 2 It can be seen that the enzyme concentration is positively correlated with the extraction rate of the enzymatic hydrolysis product, alginate oligosaccharide. That is, as the amount of enzyme added increases, the extraction rate of alginate oligosaccharide increases. When the amount of alginate lyase added reaches 0.5% of the mass of sodium alginate, the extraction rate of alginate oligosaccharide increases slowly. Considering the economic cost, 0.5% is selected as the optimal amount of enzyme added.

[0027] 3.2 Optimal Enzymatic Hydrolysis Time Using sodium alginate as a substrate, the amount of alginate lyase added was adjusted to 0.25% and 0.5% of the sodium alginate mass, respectively. The enzyme was reacted at pH 7.5 and 45℃ for 6 h, 12 h, 18 h, and 24 h, respectively. The effects of different enzyme dosages and reaction times on the extraction rate of alginate oligosaccharides were observed. The results are shown in the attached figure. Figure 3 As shown. (From the appendix) Figure 3 It can be seen that when the amount of alginate lyase added is 0.25% of the mass of sodium alginate, the extraction rate of alginate oligosaccharides shows a trend of first decreasing, then increasing, and then decreasing again with the continuous increase of reaction time, reaching a maximum value at 18 hours. When the amount of alginate lyase added is 0.5% of the mass of sodium alginate, the extraction rate of alginate oligosaccharides shows a trend of first increasing and then decreasing with the continuous increase of reaction time, reaching a maximum value at 18 hours. When the amount of alginate lyase added is 0.25% of the mass of sodium alginate, the extraction rate of alginate oligosaccharides at different reaction times is always lower than the extraction rate of alginate oligosaccharides at the reaction time corresponding to the amount of alginate lyase added is 0.5% of the mass of sodium alginate. Therefore, the optimal enzyme amount is determined to be 0.5%, and the optimal reaction time is 18 hours.

[0028] 3.3 Optimal pH for enzymatic hydrolysis Using sodium alginate as a substrate, the amount of alginate lyase added was adjusted to 0.5% of the sodium alginate mass, and the pH values ​​were 6.0, 6.5, 7.0, 7.5, and 8.0, respectively. The reaction was carried out at 45℃ for 18 h. The results of the effect of different pH values ​​on the extraction rate of alginate oligosaccharides are shown in the attached figure. Figure 4 As shown. (From the appendix) Figure 4 It can be seen that the extraction rate of fucoidan increases with increasing pH, and reaches its highest level when pH=7.5.

[0029] In summary, the optimal conditions for the degradation of sodium alginate by alginate lyase were determined to be: an alginate lyase addition amount of 0.5% of the sodium alginate mass, a reaction time of 18 h, and a pH of 7.5. The HPLC relative content distribution of fucoidan oligosaccharides in sodium alginate degradation under these optimal conditions is shown in the attached figure. Figure 5 As shown, the extraction rate of brown algae oligosaccharides can reach 70.00%, and the DP is 2~6.

[0030] Example 4: Formulation of compound enzyme preparations The optimal addition amounts of cellulase, pectinase, and papain were determined through single-factor experiments. The optimal ratio of raw materials for the compound enzyme preparation was verified to be: 0.6% alginate lyase, 1.82% cellulase, 1.2% pectinase, and 1.5% papain. The optimal enzymatic hydrolysis conditions were: a material-to-liquid ratio of 1:25, a pH of 7.22, a temperature of 45℃, and a reaction time of 18 h.

[0031] Example 5: Preparation of brown algae oligosaccharides by degrading brown algae with a compound enzyme preparation Using the compound enzyme preparation described in Example 4, three samples (sample 1, sample 2, and sample 3) from different geographical populations of *Leptochloa crus-galli* were degraded, and the extraction rate of brown algae oligosaccharides was determined. The results are shown in the appendix. Figure 6 As shown in the attached figure, the HPLC relative content distribution of brown algal oligosaccharides in different geographical populations of *Tetracentron sinense* is as follows. Figure 7-9 As shown, the material-to-liquid ratio was 1:25, the pH was 7.22, the temperature was 45℃, and the reaction time was 18 hours. (See attached...) Figure 6 It can be seen that the extraction rates of alginate oligosaccharides from Sample 1, Sample 2, and Sample 3 are: Sample 1 > Sample 3 > Sample 2. (From Appendix...) Figure 7-9 It can be seen that the *Alternaria* samples from Sample 1, Sample 2, and Sample 3 can all be effectively hydrolyzed by the compound enzyme preparation of this invention to obtain a series of brown algae oligosaccharides with a DP of 2-6.

[0032] Example 6 Sample 2 was placed in an oven and dried at 60°C. Then, it was subjected to ultrafine pulverization to obtain *Alternaria latifolia* powder. An appropriate amount of the above *Alternaria latifolia* powder was added to the compound enzyme preparation described in Example 4. After reacting for 18 hours at a material-to-liquid ratio of 1:25, a pH of 7.22, and a temperature of 45°C, the extraction rate of brown algae oligosaccharides was determined.

[0033] Comparative Example 1 Sample 2 was placed in an oven and dried at 60°C. Then, the powder of *Alternaria latifolia* was processed by ultra-fine grinding. An appropriate amount of the above *Alternaria latifolia* powder was taken, and alginate lyase was added. After reacting for 18 hours at a material-to-liquid ratio of 1:25, pH 7.22, and temperature of 45°C, the extraction rate of alginate oligosaccharides was determined.

[0034] Comparative Example 2 Sample 2 was placed in an oven and dried at 60°C. Then, the powder of *Alternaria latifolia* was processed by ultra-fine grinding. An appropriate amount of the above *Alternaria latifolia* powder was taken and a commercial compound enzyme preparation (algal core 01 cell wall breaking enzyme purchased from a certain biotechnology company in Qingdao) was added. After reacting for 18 hours at a material-to-liquid ratio of 1:25, pH 7.22, and temperature of 45°C, the extraction rate of brown algae oligosaccharides was determined.

[0035] The results of extracting brown algae oligosaccharides from Example 6, Comparative Example 1, and Comparative Example 2 are compared as shown in the appendix. Figure 10 As shown, by appendix Figure 10 As can be seen, compared with the single alginate lyase in Comparative Example 1, the alginate oligosaccharide extraction rate of the compound enzyme preparation in Example 6 of the present invention is significantly higher than that in Comparative Example 1. This indicates that the use of cellulase, pectinase, papain, and alginate lyase as a compound enzyme preparation in the present invention has a positive impact on the alginate oligosaccharide extraction rate. Compared with the commercial compound enzyme preparation in Comparative Example 2, the alginate oligosaccharide extraction rate of the compound enzyme preparation in Example 6 of the present invention is significantly higher than that of the commercial compound enzyme preparation, proving that the compound enzyme preparation has high commercial application value.

[0036] The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, evolutions, or improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope of the present invention.

Claims

1. An alginate lyase, characterized in that, The alginate lyase was isolated from Shewanella. Shewanella sp. HD5 The original alginate lyase gene of *Shewanella* was obtained through heterologous expression. Shewanella sp. HD5 It was deposited on September 22, 2025, at the China Center for Type Culture Collection, Wuhan, Hubei Province, with accession number CCTCC NO: M20252078.

2. A gene encoding a gene, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO: 1 and is used to encode the alginate lyase of claim 1.

3. The application of the alginate lyase as described in claim 1 in the preparation of alginate oligosaccharides from sodium alginate.

4. The application of the alginate lyase according to claim 3 in the preparation of fucoidan oligosaccharides from sodium alginate, characterized in that, The amount of alginate lyase added is 0.5% to 2.5% of the mass of sodium alginate, the enzymatic hydrolysis temperature is 35 to 50°C, the pH is 7.0 to 7.5, and the reaction time is 12 to 24 hours.

5. The application of the alginate lyase according to claim 3 in the preparation of fucoidan oligosaccharides from sodium alginate, characterized in that, The extraction rate of the brown algae oligosaccharide is 60.0%~75.0%, and the degree of polymerization of the brown algae oligosaccharide is 2~6.

6. A compound enzyme preparation, characterized in that, The alginate lyase according to claim 1 is included, wherein the amount of alginate lyase added is 0.5% to 1.0% of the mass of the algae powder.

7. The compound enzyme preparation according to claim 6, characterized in that, The compound enzyme preparation further includes cellulase, pectinase and papain; the amount of cellulase added is 1.0% to 2.0% of the mass of algae powder, the amount of pectinase added is 0.5% to 1.5% of the mass of algae powder, and the amount of papain added is 1.0% to 2.0% of the mass of algae powder.

8. The application of the compound enzyme preparation as described in claim 7 in the direct enzymatic hydrolysis of *Tetraphyta fusiforme* to obtain brown algae oligosaccharides.

9. The application of the compound enzyme preparation according to claim 8 in the direct enzymatic hydrolysis of *Tetraphyta fusiforme* to obtain brown algal oligosaccharides, characterized in that, The enzymatic hydrolysis conditions are as follows: material-to-liquid ratio of 1:15 to 1:30, enzymatic hydrolysis temperature of 35 to 50°C, pH of 6.5 to 7.5, and reaction time of 12 to 14 hours.

10. The application of the compound enzyme preparation according to claim 9 in the direct enzymatic hydrolysis of *Tetraphyta fusiforme* to obtain brown algal oligosaccharides, characterized in that, The extraction rate of the brown algae oligosaccharide is 10%~15%, and the degree of polymerization of the brown algae oligosaccharide is 2~6.

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