Alginate lyase and application thereof

Abstract

The invention provides a compound enzyme system with alginate lyase activity. The compound enzyme system is selected from any two or three of alginate lyase VSAly7A, alginate lyase VSAly7B and alginate lyase VSAly7D, and the alginate lyase VSAly7A, the alginate lyase VSAly7B and the alginate lyase VSAly7D are selected from any two or three of alginate lyase VSAly7A, alginate lyase VSAly7B and alginate lyase VSAly7D. Compared with SEQ ID No.1, the amino acid sequence of the VSAly7A has at least 90% of sequence identity; compared with the amino acid sequence of SEQ ID No.3, the amino acid sequence of the VSAly7B has at least 90% of sequence identity; compared with the amino acid sequence of SEQ ID No.5, the amino acid sequence of the VSAly7D has at least 90% of sequence identity.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an alginate lyase and an application thereof. Background Art

[0002] Algin is the main component of the cell wall of brown algae, accounting for about 40% of the dry weight of brown algae. It provides algae with flexibility and stability to cope with ocean currents. Algin is a polyanionic carbohydrate formed by the polymerization of α-L-guluronic acid (G) and β-D-mannuronic acid (M) through 1,4-glycosidic bonds. According to the different arrangements of M and G, alginate is divided into three types: polyMG, polyG and polyM. The strength and flexibility of different types of alginate mainly depend on the content of G and M in the monomer. The G-rich area can react with Ca 2+ Specific binding forms an "egg-box" structure, which promotes the cross-linking of two antiparallel polysaccharide chains, forming a three-dimensional gel network, thereby enhancing the rigidity of alginate. The ratio of M to G varies not only between different brown algae species, but also in different parts of the same species. Furthermore, the ratio of M to G in alginate is closely related to the growth stage, growth environment, light intensity, growing season, and harvest time of the brown algae.

[0003] Alginate oligosaccharides (AOS) are oligomers with a degree of polymerization (DP) between 2 and 25, produced by the degradation of high-molecular-weight alginate. Compared to alginate, AOS have a lower molecular weight, higher water solubility, and lower solution viscosity, making them widely used in medicine, cosmetics, health supplements, and agriculture. AOS can be obtained through various methods, including physical, chemical, and enzymatic methods. Physical degradation primarily involves gamma irradiation, ultraviolet irradiation, ultrasonic treatment, and thermal degradation; chemical degradation involves the destruction of the chain structure of alginate using hydrochloric acid, hydrogen peroxide, and potassium persulfate; and enzymatic methods involve the production of unsaturated algal oligosaccharides through a β-elimination mechanism using alginate lyase. Compared to physical and chemical methods, AOS produced by enzymatic methods exhibit superior biological activity. Furthermore, enzymatic methods offer numerous advantages, including high specificity, excellent safety, and environmental friendliness.

[0004] Algin lyases identified so far are primarily derived from a variety of organisms, including seaweed, mollusks, marine echinoderms, bacteria, fungi, and viruses. Marine bacteria are the organisms with the most characterized alginate lyases. In the CAZy database, alginate lyases are classified into 15 polysaccharide lyase (PL) families, including PL5, PL6, PL7, PL8, PL14, PL15, PL17, PL18, PL31, PL34, PL36, PL38, PL39, PL41, and PL44. Studies have shown that alginate lyases from bacteria are primarily distributed in the PL5, PL6, PL7, PL15, and PL17 families, while alginate lyases from marine animals and viruses are primarily distributed in the PL14 family. Based on their substrate specificity, alginate lyases are classified as PolyG-specific alginate lyases (EC 4.2.2.11), PolyM-specific alginate lyases (EC 4.2.2.3), and bifunctional alginate lyases (EC 4.2.2.). Based on their catalytic modes, alginate lyases can be divided into endo- and exo-type alginate lyases. Endo-type alginate lyases can randomly cleave the alginate backbone, producing alginic oligosaccharides of varying degrees of polymerization. Exo-type alginate lyases, primarily composed of alginic oligosaccharide lyases, can only gradually cleave sugar chains from the ends of alginate, producing either fucoidan or fucoidan. Most of the alginate lyases characterized to date are endo-type, while reports of exo-type alginate lyases are relatively rare.

[0005] Because alginate lyases have shown great potential in the preparation of AOS, and reports have demonstrated their potential application in laboratory-based AOS production, screening for efficient alginate-degrading bacteria and alginate lyases is of great significance. The inventors have identified a highly efficient alginate-degrading bacterium, a new species of Vibrio. Through genomic analysis, a potential alginate lyase gene was identified. In-depth analysis of the extracellular alginate lyase revealed its basic properties and degradation pattern. Furthermore, a combination of extracellular enzymes can improve the degradation rate of kelp, providing new ideas for the rapid degradation of brown algae. Summary of the Invention

[0006] In one aspect, the present invention provides a composite enzyme system having alginate lyase activity, wherein the composite enzyme system is selected from any two or three of alginate lyase VSAly7A, alginate lyase VSAly7B and alginate lyase VSAly7D.

[0007] Furthermore, the amino acid sequence of VSAly7A has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity compared to SEQ ID No.1.

[0008] Furthermore, the amino acid sequence of VSAly7B has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity compared to SEQ ID No. 3.

[0009] Furthermore, the amino acid sequence of VSAly7D has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity compared to SEQ ID No.5.

[0010] In one embodiment, the VSAly7A, VSAly7B, and VSAly7D are derived from Vibrio.

[0011] In a preferred embodiment, the amino acid sequence of the VSAly7A is shown as SEQ ID No.1; the amino acid sequence of the VSAly7B is shown as SEQ ID No.3; and the amino acid sequence of the VSAly7D is shown as SEQ ID No.5.

[0012] In one embodiment, the complex enzyme system includes alginate lyase VSAly7A and alginate lyase VSAly7B.

[0013] In one embodiment, the complex enzyme system includes alginate lyase VSAly7A and alginate lyase VSAly7D.

[0014] In one embodiment, the complex enzyme system includes alginate lyase VSAly7B and alginate lyase VSAly7D.

[0015] In one embodiment, the complex enzyme system includes alginate lyase VSAly7A, alginate lyase VSAly7B and alginate lyase VSAly7D.

[0016] In one embodiment, in the composite enzyme system, alginate lyase VSAly7A, alginate lyase VSAly7B and alginate lyase VSAly7D are compounded in an equimolar ratio or an equal enzyme activity ratio.

[0017] In one embodiment, in the composite enzyme system, the molar ratio of alginate lyase VSAly7A, alginate lyase VSAly7B and alginate lyase VSAly7D is 1:1:1.

[0018] In one embodiment, in the composite enzyme system, the ratio of the enzyme activities of alginate lyase VSAly7A, alginate lyase VSAly7B and alginate lyase VSAly7D is 1:1:1.

[0019] On the other hand, the present invention also provides the use of the above-mentioned complex enzyme system in degrading alginate, or in preparing brown algae oligosaccharides.

[0020] On the other hand, the present invention also provides use of the above-mentioned complex enzyme system in preparing a preparation for degrading alginate.

[0021] On the other hand, the present invention also provides a preparation for degrading alginate, which comprises the above-mentioned composite enzyme system.

[0022] On the other hand, the present invention also provides a method for degrading alginate or preparing algal oligosaccharides, which comprises the step of degrading alginate using the above-mentioned composite enzyme system.

[0023] In one embodiment, the fucoidan oligosaccharide is selected from one or any combination of fucoidan monosaccharide, fucoidan biose, and fucoidan triose.

[0024] In one embodiment, the alginate is derived from kelp.

[0025] In one embodiment, the composite enzyme system degrades alginate under the following conditions: temperature 30°C-40°C, pH 7.0-8.0, and salt concentration 0.1M-0.3M. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 .Primary sequence alignment of PL7 family alginate lyases.

[0027] Figure 2 .Analysis of the optimal temperature, optimal pH and optimal NaCl concentration of recombinant alginate lyase.

[0028] Figure 3 .Determination of the relative enzyme activity of recombinant alginate lyase against different substrates.

[0029] Figure 4 .Analysis of the end products of alginate degradation.

[0030] Figure 5 .Analysis of the minimum recognizable substrate and catalytic mode of VSAly7A.

[0031] Figure 6 .Analysis of the minimum recognizable substrate and catalytic mode of VSAly7B.

[0032] Figure 7 .Analysis of the minimum recognizable substrate and catalytic mode of VSAly7D.

[0033] Figure 8 .Analysis of the minimum recognizable substrate and catalytic mode of VSAly7E.

[0034] Figure 9 .VSAly7A, VSAly7B and VSAly7D are combined to degrade alginate and kelp. Implementation Method

[0035] The present invention will be further described below with reference to the following embodiments. The following description is merely a preferred embodiment of the present invention and does not limit the present invention in any other form. Any person skilled in the art may utilize the above disclosed technical content to make equivalent embodiments with equivalent variations. Any simple modification or equivalent variation of the following embodiments made in accordance with the technical essence of the present invention without departing from the content of the present invention shall fall within the scope of protection of the present invention.

[0036] Example 1. Materials and methods

[0037] 1.1 Experimental Materials

[0038] PrimeStar Mixture was purchased from TaKaRa; Taq DNA polymerase and gel recovery kit were purchased from Novezan Biotechnology Co., Ltd.; general DNA product purification kit and plasmid mini-preparation kit were purchased from Tiangen Biochemical Technology Co., Ltd.; restriction endonucleases and T4 DNA ligase were purchased from Thermo Fisher Scientific; SOSO enzyme was purchased from Qingke Biotechnology Co., Ltd.; Ni Sepharose™ 6Fast Flow was purchased from Cytiva; brown algae oligosaccharides were purchased from Qingdao Bozhi Huili Biotechnology Co., Ltd.; kelp was from Weihai, Shandong; and other biochemical reagents were purchased from Shanghai Shenggong Bioengineering Co., Ltd., Sigma-Aldrich, and Beijing Dingguo Changsheng Biotechnology Co., Ltd.

[0039] 1.2 Bioinformatics analysis

[0040] Clustal Omega (https: / / www.ebi.ac.uk / jdispatcher / msa / clustalo) was used for multiple sequence alignment; 16S rRNA and whole genome phylogenetic trees were drawn using IQ-tree (https: / / iqtree.github.io / ); ANI and dDDH were calculated using EZbiocloud (https: / / www.ezbiocloud.net / ); phylogenetic tree beautification was performed using iTol (https: / / itol.embl.de / ); the presence of signal peptides in proteins was predicted using SignalP-5.0 (https: / / services.healthtech.dtu.dk / services / SignalP-5.0 / ); the domain composition of alginate lyase was analyzed using SMART (https: / / smart.embl.de / ); and the ChiPlot online website (https: / / w The domain structure of alginate lyase was mapped using the CRISPR / Cas9 (www.chiplot.online / conserved_motif_plot.html) library. The alginate lyase family information was obtained through CAZy (https: / / www.cazy.org / Polysaccharide-Lyases.html). Protein structure modeling was performed using AlphaFold2 (https: / / alphafoldserver.com / ). Primary sequence alignment of alginate lyase was performed using ESPript3.0 (https: / / espript.ibcp.fr / ESPript / cgi-bin / ESPript.cgi).

[0041] 1.3 Screening and identification of alginate-degrading bacteria

[0042] Put the rotten kelp leaves into the enrichment medium and enrich them at 4℃ for 10 days. 7 , 10 6 , 10 5 , 10 4 , 10 3 CFU / mL ratio, 50 μL of the diluted bacterial solution was spread on 2216E medium and cultured at 28°C. Single colonies were picked every day and placed on a 96-well plate containing 2216E liquid medium, cultured at 28°C for 1 day, and 2 μL of bacterial solution was respectively placed on an M9 plate with alginate as the sole carbon source and an M9 plate containing 20% ​​casamino acids. After two days of culture at 28°C, 10% C 21 H 33C2N was used to develop the degradation zones for 10 minutes. Strains with distinct clear zones were selected and their clear zones were measured. Strains obtained from the initial screening were streaked onto a 2216E solid plate to confirm pure culture and rescreened using the same method as the initial screening. The clear zone sizes from the initial and rescreening measurements were compared to identify strains with true alginate degradation ability.

[0043] 1.4 Heterologous expression of alginate lyase

[0044] (1) Transformation of expression plasmid: 1 μL recombinant plasmid and 50 μL E. coli BL21 (DE3) competent cells were mixed by gentle pipetting on ice. After ice bath for 30 min, heat shock was performed at 42°C for 90 s, ice bath was performed for 2 min, 1 mL LB liquid culture medium was added and the cells were resuspended at 37°C and 200 rpm for 60 min. After centrifugation at 3,500 rpm for 5 min, the supernatant was discarded and the cells were resuspended. The cells were spread on LB solid plates containing Kan resistance and cultured at 37°C overnight.

[0045] (2) Pick a single clone for activation and expansion: Pick a single clone and inoculate it into 5 mL of liquid LB medium containing kan (50 ng / μL). Cultivate at 37°C with shaking at 200 rpm for 8 h until the OD600 reaches approximately 1.0. Then, inoculate 1% of the clone into 800 mL of LB medium (Kan concentration is 50 ng / μL) for expansion. After culturing for 2-3 h, when the OD600 reaches 0.8, add 0.1 mM IPTG and induce expression at 16°C for 20 h.

[0046] (3) Bacteria collection and crushing

[0047] Centrifuge the culture at 8,000 rpm for 10 minutes in a high-capacity centrifuge to collect the cells. Resuspend the cells in 10 mL of Tris-HCl (50 mM Tris, 150 mM NaCl, pH 8.0) and sonicate on ice (power: 35 W, sonication for 2 seconds, off for 2 seconds, for 25 minutes). Centrifuge at 11,000 rpm for 35 minutes at 4°C. Collect the supernatant and precipitate. Store the supernatant in a refrigerator at 4°C until use.

[0048] (4) Purification and detection of target protein

[0049] The nickel column was rinsed with 10 column volumes (V) of ddH2O to remove 20% ethanol. The nickel column was equilibrated with Balance Buffer (50mM Tris, 150mM NaCl, pH 8.0) at 10V. The supernatant obtained by ultrasonic disruption and centrifugation was passed through a 0.22μm filter and loaded onto the nickel column. Weakly bound contaminants were eluted with Wash Buffer (50mM Tris, 150mM NaCl, 20mM Imidazole, pH 8.0) at 10V. The target protein was eluted with Elution Buffer (50mM Tris, 150mM NaCl, 300mM Imidazole, pH 8.0) at 5V. The supernatant, precipitate, flow-through, and eluted protein obtained in each of the above steps were subjected to SDS-PAGE electrophoresis (12% separating gel, 5% stacking gel) to verify the protein purification efficiency. After electrophoresis, the samples were stained with Coomassie Brilliant Blue R-250 for 2 h and then destained with a destaining solution.

[0050] (5) PD-10 replacement protein buffer

[0051] After electrophoresis of the eluted protein, select the eluate containing the highly purified target protein and perform ultrafiltration and concentration using a 15mL PALL 3K ultrafiltration centrifuge tube at 3,500 rpm at 4°C. Store the concentrate in a refrigerator at 4°C until use. Use a PD-10 desalting column to remove high concentrations of imidazole from the target protein, following the PD-10 instructions.

[0052] (6) Purity detection and preservation of target protein

[0053] Use NanoDrop to measure the purity of the target protein. After measurement, aliquot the target protein into sterile 1.5 mL EP tubes, indicate the concentration, snap-freeze with liquid nitrogen, and store at -80°C.

[0054] 1.5 Analysis of Enzymatic Properties of PL7 Family Alginate Lyases

[0055] Alginate lyase activity was determined using ultraviolet absorption spectroscopy. The principle behind this is that the unsaturated uronic acid produced by alginate lyase degradation exhibits a characteristic absorption peak at 235 nm. The specific steps are as follows: 20 μL of recombinant alginate lyase (0.1 mg / mL) was mixed with 180 μL of 0.3% (w / v) alginate substrate and incubated under optimal conditions for 10 minutes. After the reaction, the mixture was boiled in a boiling water bath for 10 minutes, centrifuged at 12,000 rpm for 5 minutes to remove protein precipitates, and 150 μL of the supernatant was added to a 96-well microtiter plate. The absorbance of the product at 235 nm was measured.

[0056] The optimal reaction conditions were determined by UV absorption spectroscopy. Specifically, 20 μL of recombinant alginate lyase (0.1 mg / mL) was mixed with 180 μL of 0.3% alginate substrate (Tris-HCl buffer) and reacted for 10 minutes under different conditions. The reaction was inactivated in a boiling water bath for 10 minutes, followed by centrifugation at 12,000 rpm for 5 minutes to remove protein precipitates. 150 μL of the supernatant was then added to a 96-well microtiter plate, and the absorbance of the product was measured at 235 nm. The maximum enzyme activity was defined as 100%, and the relative activity of the recombinant alginate lyase at different temperatures was calculated. Each experiment was performed in triplicate and repeated three times.

[0057] 1.6 Analysis of enzymatic hydrolysis products of PL7 family alginate lyase

[0058] The final product of the enzymatic hydrolysis of recombinant alginate lyase was analyzed by high performance liquid chromatography (HPLC). The specific steps are as follows: 100 μL of recombinant alginate lyase (1 mg / mL) was mixed with 100 μL of 0.3% alginate substrate and reacted at the optimal temperature for 24 hours. After the reaction, the mixture was boiled in a boiling water bath for 10 minutes to terminate the reaction, and the residual protein precipitate was removed by centrifugation at 12,000 rpm for 10 minutes. 200 mM NH4HCO3 was used as the mobile phase, and the Superdex peptide 10 / 300GL gel filtration column was equilibrated at a flow rate of 0.3 mL / min. Then 15 μL of the supernatant after the reaction was added to the gel filtration column at a flow rate of 0.3 mL / min, and the change in absorbance at 235 nm was monitored. The molecular weight of the generated oligosaccharides was further detected by electrospray ionization mass spectrometry.

[0059] 1.7 Analysis of degradation patterns of PL7 family alginate lyases

[0060] To determine the minimum substrate for recombinant alginate lyase, 40 μL of recombinant alginate lyase (1 mg / mL) was mixed with 10 μL of 1 mg / mL mannuronic acid oligosaccharides of varying degrees of polymerization (DP 2-4) and 10 μL of 1 mg / mL guluronic acid oligosaccharides. The mixture was reacted under optimal conditions for 24 hours and then terminated by heating in a boiling water bath. The reaction products were analyzed by HPLC.

[0061] 100 μL of recombinant alginate lyase (0.01 mg / mL) was mixed with 100 μL of guluronic acid oligosaccharides (DP 4-6) and reacted under optimal conditions. 30 μL of the reaction mixture was sampled at different time intervals (5 minutes, 2 hours, and 24 hours). After each sampling, the reaction mixture was immediately inactivated in a boiling water bath for 10 minutes. The reaction products at different time points were analyzed by HPLC.

[0062] 1.8 Main enzyme systems synergistically degrade alginate and kelp

[0063] (1) Protein profiling to identify the main extracellular degradation enzyme system

[0064] The extracellular fermentation broth of alginate-degrading bacteria was passed through a 3KDa ultrafiltration membrane and ultrafiltered to 10-20 mL. Then, 5 g of trichloroacetic acid (TCA), 0.05 g of dithiothreitol (DTT), and 50 mL of acetone solution were added to the concentrate, and the mixture was allowed to stand overnight at -20 ° C for precipitation; the protein precipitate was collected by centrifugation at 8,000 rpm for 20 min. The precipitate was washed with 80% acetone for 20-30 min, and then centrifuged at 8,000 rpm for 20 min to collect the precipitate, and this step was repeated twice. The precipitate was further washed with 100% acetone for 20 min to thoroughly remove the residual TCA solution in the protein sample. After washing, the precipitate was collected by centrifugation at 8,000 rpm for 20 min and dried in a 65 ° C oven to obtain protein powder. The resulting protein powder was dissolved in ultrapure water and the protein concentration was determined using a NanoDrop. 30 μg of protein solution was added to 50 μL of denaturation buffer (0.5 M Tris-HCl, 2.75 mM EDTA, 6 M guanidine hydrochloride, pH 8.0) to denature the protein. The denatured protein solution was then added with 30 μL of 1 M DTT and incubated at 37°C for 2 h to reduce disulfide bonds. The reduced protein solution was then added with 50 μL of 1 M iodoacetamide (IA) and incubated in the dark for 1 h to alkylate the cysteines. The treated sample was transferred to a cleaned Microcon YM-10 3K ultrafiltration tube, and 360 μL of 25 mM NH₄HCO₃ was added. The sample was centrifuged for 10-15 min. This step was repeated four times. The ultrafiltration tube was then inverted, centrifuged at 3,000 rpm for 3 min, and the sample was transferred to a 200 μL EP tube. The washed protein was added with 0.5 μg / μL trypsin at a ratio of 1:25 (w / w) and digested overnight at 37°C. After digestion, the protein was inactivated at 105°C for 10 minutes. The sample was desalted using a C18 Ziptip column: first, the C18 Ziptip column was repeatedly rinsed with 50% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA). After the rinse, the sample was blown to allow the protein sample to adsorb on the column. Then, 0.1% TFA was used to remove impurities in the sample. Finally, the sample was eluted with 50% ACN and 0.1% TFA. After freeze-drying the sample, 10 μL of ultrapure water was added and the protein was detected by MS.

[0065] (2) The main extracellular enzymes synergistically degrade alginate

[0066] The main extracellular enzymes (VSAly7A, VSAly7B and VSAly7D) were mixed in equal molar ratios for alginate degradation experiments. The specific steps were as follows: 20 μL of the mixed enzymes (total molar amount of 8.91×10 -10 molar, VSAly7A, VSAly7B and VSAly7D were 2.97×10 -10 After mixing 180 μL of 0.3% alginate substrate (0.1 mol / L) with 180 μL of 0.3% alginate, the reaction was incubated at 30°C for 10 minutes. After completion of the reaction, the cells were inactivated in a boiling water bath for 10 minutes and centrifuged at 12,000 rpm for 5 minutes to remove the protein precipitate. 150 μL of the supernatant was added to a 96-well microtiter plate, and the absorbance of the product was measured at 235 nm.

[0067] (3) The main extracellular enzymes synergistically degrade kelp

[0068] The main extracellular enzymes (VSAly7A, VSAly7B, and VSAly7D) were mixed at equal enzyme activity ratios for alginate degradation experiments. One gram of kelp sample was weighed into a 100-mL sterile Erlenmeyer flask. A composite alginate lyase system with a total enzyme activity of 600 U (VSAly7A, VSAly7B, and VSAly7D were mixed at equal enzyme activity ratios, with 200 U of each added) was then added to the flask. The flask was then diluted to 30 mL with Tris-HCl buffer (50 mM Tris, 150 mM NaCl, pH 8.0) and the kelp was degraded at 40°C and 200 rpm. Every 24 hours, the mixture was centrifuged at 11,000 rpm for 10 minutes. The supernatant after enzymatic hydrolysis and the kelp sediment after degradation were collected. The supernatant (seaweed liquid fertilizer) was stored at -80°C for later use. The sediment was dried in a 65°C oven and weighed. Samples were taken every 12 hours, twice in a row, with three replicates each time. If cellulase was required, 0.3% cellulase was added to the above system and the kelp was degraded and sampled under the same conditions.

[0069] Example 2. Results and Discussion

[0070] 2.1 Screening and identification of alginate-degrading microorganisms

[0071] By enriching rotten kelp leaves, 10 strains of bacteria with alginate degradation capabilities were screened. Ultimately, a strain of alginate-degrading bacteria with the largest alginate degradation circle was obtained, a new species of Vibrio sp. Bioinformatics analysis revealed that the Vibrio genome encodes a total of 12 alginate lyases belonging to five different alginate lyase families. Among them, the alginate lyases of the PL7 family include VSAly7A, VSAly7B, VSAly7C, VSAly7D, and VSAly7E.

[0072] like Figure 1 As shown, although VSAly7A, VSAly7B, VSAly7C, VSAly7D, and VSAly7E all belong to the PL7_5 family of alginate lyases and share conserved PL7 family sequence modules, such as Q*H, R*E*R*M, and YFKAG*Y*Q, their primary sequences differ. AlphaFold2 structural prediction analysis revealed that, compared to other enzymes, VSAly7A possesses two sequences above the catalytic cleft. While both VSAly7B and VSAly7C contain a CBM domain, the linker between the CBM and CD domains differs, with the linker being longer in VSAly7B compared to VSAly7C. Furthermore, VSAly7C and VSAly7E possess a long loop at the substrate-binding non-reducing end, which may be involved in substrate binding or release. These unique structures may confer distinct biological properties upon the different alginate lyases.

[0073] The enzymatic properties of VSAly7A, VSAly7B, VSAly7D, and VSAly7E were further studied below.

[0074] Among them, the amino acid sequence and nucleotide sequence of VSAly7A are shown as SEQ ID No.1 and SEQ ID No.2 respectively; the amino acid sequence and nucleotide sequence of VSAly7B are shown as SEQ ID No.3 and SEQ ID No.4 respectively; the amino acid sequence and nucleotide sequence of VSAly7D are shown as SEQ ID No.5 and SEQ ID No.6 respectively; the amino acid sequence and nucleotide sequence of VSAly7E are shown as SEQ ID No.7 and SEQ ID No.8 respectively.

[0075]

[0076]

[0077]

[0078] 2.2 Analysis of basic enzymatic properties of extracellular PL7 family alginate lyases

[0079] like Figure 2 As shown, the optimal reaction temperature for VSAly7A, VSAly7B, and VSAly7D is 40°C, while that for VSAly7E is 30°C. VSAly7A has an optimal reaction pH of 7.0, making it a neutral alginate lyase; VSAly7B and VSAly7E have an optimal pH of 8.0, making them alkaline alginate lyases; and VSAly7D has an optimal pH of 5.0, making it an acidic alginate lyase. The optimal NaCl concentration for VSAly7A is 0.1 M, for VSAly7E 0.2 M, and for VSAly7B and VSAly7D 0.3 M. Different alginate lyases have different optimal reaction conditions, suggesting that these enzymes exhibit environmental synergy in substrate degradation.

[0080] The specific enzymatic activities of four alginate lyases were determined using alginate, polyM and polyG as substrates. Figure 3 As shown in the figure, except for VSAly7B and VSAly7E, all other alginate lyases are polyG-preferring alginate lyases. Compared with the other three alginate lyases, VSAly7E has the lowest specific enzyme activity towards polyM, polyG, and alginate, which are 50.6±0.2U / mg, 71.1±2.7U / mg, and 46.7±7.5U / mg, respectively.

[0081] 2.3 Analysis of extracellular PL7 family alginate lyase hydrolysis products

[0082] The hydrolysis products of five alginate lyases were analyzed and determined by HPLC and MS. Figure 4 As shown in the figure, the main degradation products of alginate by VSAly7A are fucoidan and fucosilicate, while the main degradation products of alginate by VSAly7B, VSAly7D, and VSAly7E are fucoidan and fucosilicate. The diversity of their degradation products suggests that they may have different catalytic modes.

[0083] 2.4 Analysis of the catalytic mode of extracellular PL7 family alginate lyase

[0084] (1) Analysis of the minimal substrate recognition and catalytic mechanism of VSAly7A

[0085] To determine the minimum substrate that VSAly7A can recognize, the degradation products of VSAly7A under G-type and M-type disaccharide and trisaccharide substrates were detected. Figure 5As shown in the figure, when G2 and M2 were used as substrates, no product was detected after 24 hours of reaction, indicating that VSAly7A could not degrade G2 or M2. When G3 or M3 were used as substrates, enzymatic hydrolysis produced unsaturated disaccharides ΔG and ΔM, indicating that the minimum substrate that VSAly7A can degrade is a trisaccharide.

[0086] (2) Analysis of the minimal substrate recognition and catalytic mechanism of VSAly7B

[0087] To determine the minimum substrate size that VSAly7B can recognize, the degradation products of VSAly7B in the presence of G-type and M-type disaccharide, trisaccharide and tetrasaccharide substrates were detected. Figure 6 As shown in the figure, when G2, M2 and G3, M3 were used as substrates, no product was detected after 24 hours of reaction, indicating that VSAly7B cannot degrade G2, G3 or M2, M3. When G4 and M4 were used as substrates, enzymatic hydrolysis produced ΔGG and ΔMM, indicating that the minimum substrate that VSAly7B can degrade is a tetrasaccharide.

[0088] (3) Analysis of the minimal substrate recognition and catalytic mechanism of VSAly7D

[0089] To determine the minimum substrate that VSAly7D can recognize, the degradation products of VSAly7D under G-type and M-type disaccharide and trisaccharide substrates were detected. Figure 7 As shown in the figure, when G2, M2, G3, and M3 were used as substrates, no product was detected after 24 hours of reaction, indicating that VSAly7D cannot degrade G2, G3, or M2, M3. When G4 was used as a substrate, enzymatic hydrolysis produced ΔGG, indicating that the minimum substrate that VSAly7D can degrade is a tetrasaccharide.

[0090] (4) Analysis of the minimal substrate recognition and catalytic mechanism of VSAly7E

[0091] To determine the minimum substrate that VSAly7E can recognize, the degradation products of VSAly7E under G-type and M-type disaccharide and trisaccharide substrates were detected. Figure 8 As shown in the figure, when G2 and M2 were used as substrates, no product was detected after 24 hours of reaction, indicating that VSAly7E could not degrade G2 or M2. When G3 or M3 were used as substrates, enzymatic hydrolysis produced unsaturated disaccharides (ΔG and ΔM), indicating that the minimum substrate that VSAly7E can degrade is a trisaccharide.

[0092] 2.5 Cooperative degradation of alginate and kelp by major extracellular enzymes

[0093] To identify the key alginate lyases in the fermentation broth, the relative abundance of extracellular alginate lyases was further analyzed by mass spectrometry. Figure 9As shown in Figure a, VSAly7B was the most abundant enzyme in the extracellular supernatant, accounting for approximately 36% of the total extracellular alginate-degrading enzymes. This was followed by VSAly7A and VSAly7D, which accounted for 24% and 22% of the total extracellular alginate lyases, respectively. The remaining alginate lyase components combined accounted for 18% of the total extracellular alginate lyases. Therefore, the mass spectrometry results of the extracellular secreted proteins suggest that VSAly7A, VSAly7B, and VSAly7D may be key enzymes in alginate degradation. Combined with analysis of the degradation mechanism of alginate lyases, these three enzymes may have a synergistic effect in their catalytic mode.

[0094] VSAly7A, VSAly7B and VSAly7D were compounded in equal molar ratios. Figure 9 As shown in Figure b, when compounded in an equimolar ratio, the theoretical enzyme activity was 2360±3.2 U / mg, while the actual enzyme activity was 3540±150.4 U / mg. Therefore, the enzyme activity of the compound enzyme system was increased by 45%, indicating that VSAly7A, VSAly7B and VSAly7D did have a synergistic effect in the degradation of alginate.

[0095] The degradation efficiency of VSAly7A, VSAly7B and VSAly7D on the natural substrate kelp was determined after they were mixed with equal enzyme activity ratios. Figure 9 c and Figure 9 As shown in Figure d, after adding 3,000 U of cellulase, the degradation rate of kelp by the three alginate lyase enzymes combined was 47% after 12 hours of degradation; after 24 hours of degradation, the degradation rate of kelp by the three alginate lyase enzymes was 50%. This means that after 12 hours of degradation, the enzyme combination can degrade most of the soluble substances in kelp. Moreover, compared with the theoretical degradation rate (based on the simple addition of the theoretical degradation rates of the three enzymes), the actual degradation efficiency of kelp after the three enzyme combination increased by 40% after 12 hours and by 20% after 24 hours, indicating that the three alginate lyases also have a synergistic effect on the degradation of the natural substrate kelp.

Claims

1. A composite enzyme system having alginate lyase activity, wherein the composite enzyme system is selected from any two or three of alginate lyase VSAly7A, alginate lyase VSAly7B, and alginate lyase VSAly7D; The amino acid sequence of the VSAly7A has at least 90% sequence identity compared with SEQ ID No.1; the amino acid sequence of the VSAly7B has at least 90% sequence identity compared with SEQ ID No.3; and the amino acid sequence of the VSAly7D has at least 90% sequence identity compared with SEQ ID No.

5.

2. The complex enzyme system according to claim 1, characterized in that The VSAly7A, VSAly7B and VSAly7D are derived from Vibrio.

3. The complex enzyme system according to claim 1, characterized in that The complex enzyme system comprises alginate lyase VSAly7A, alginate lyase VSAly7B and alginate lyase VSAly7D.

4. The complex enzyme system according to claim 3, characterized in that In the complex enzyme system, alginate lyase VSAly7A, alginate lyase VSAly7B and alginate lyase VSAly7D are compounded in an equal molar ratio or an equal enzyme activity ratio.

5. A preparation for degrading alginate, comprising the composite enzyme system according to any one of claims 1 to 4.

6. Use of the complex enzyme system according to any one of claims 1 to 4 in degrading alginate, or in preparing brown algal oligosaccharides.

7. Use of the complex enzyme system according to any one of claims 1 to 4 in the preparation of a preparation for degrading alginate.

8. A method for degrading alginate or preparing algal oligosaccharides, comprising the step of degrading alginate using the complex enzyme system according to any one of claims 1 to 4.

9. The method according to claim 8, characterized in that The brown algae oligosaccharide is selected from one or any combination of brown algae monosaccharide, brown algae biose and brown algae triose.

10. The method according to claim 8 or 9, characterized in that The alginate is derived from kelp.

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