Alginate lyase alg17g and related biomaterials, methods of making, and uses

Abstract

The present application relates to the field of biotechnology, in particular to alginic acid lyase Alg17G and related biological materials, preparation method and application. The present application clones alginic acid lyase gene Alg17G from Shewanella haliotis BP-1, heterologously expresses in E. coli by constructing a recombinant expression vector, and analyzes the enzymatic properties and products of the alginic acid lyase Alg17G. The alginic acid lyase Alg17G belongs to the PL17 family, has obvious preference for polyM substrate, has good pH stability and thermal stability, and can be used for preparing alginic acid source monosaccharide or enzymatic product. The enzymatic product has strong in vitro free radical scavenging capacity, and has potential application value in the green preparation of functional brown algae oligosaccharide; Ca 2+ , Ba 2+ Or Mg 2+ Salt can improve the enzyme activity of alginic acid lyase Alg17G.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to alginate lyase Alg17G and related biomaterials, preparation methods, and applications. Background Technology

[0002] Alginate, also known as brown alginic acid, is a natural linear polysaccharide derived from brown algae and some bacteria. Its main chain consists of β-D-mannuronic acid (M) linked to its C5 epimer α-L-guluronic acid (G) via 1,4-glycosidic bonds, and exists in the form of polyM, polyG, or polyMG blocks. Alginate accounts for up to 40% of the dry weight of brown algae, making it one of the most abundant polysaccharides in the ocean. Its efficient development and utilization are of great significance for the high-value utilization of marine biological resources.

[0003] Alginate oligosaccharides (AOS) are oligosaccharide products formed by enzymatic hydrolysis or chemical degradation of alginate. They possess various biological activities, including immunomodulation, antioxidant, antibacterial, anti-inflammatory, and prebiotic effects, and have attracted widespread attention in recent years. Currently, methods for preparing AOS from alginate degradation mainly include physical, chemical, and enzymatic methods. While physical methods (such as ultrasonic and microwave treatment) and chemical methods (such as acid hydrolysis and oxidative degradation) are simple to operate, they suffer from problems such as uneven product polymerization degree distribution, numerous byproducts, and environmental pollution. In contrast, enzymatic methods offer advantages such as mild reaction conditions, high substrate specificity, good product uniformity, and environmental friendliness, and are considered a promising green preparation technology. Therefore, the discovery and characterization of novel and highly efficient alginate lyases are crucial for promoting the industrial application of AOS.

[0004] Alginate lyase (EC 4.2.2.-) cleaves the glycosidic bonds in the alginate chain via a β-elimination mechanism, forming a C4=C5 unsaturated bond at its non-reducing end. Based on their mode of action, alginate lyases can be classified into endonucleases that randomly cleave the substrate chain and exonucleases that stepwise release degradation products from the substrate chain ends. Based on substrate preference, they can be classified into polymannuronic acid (polyM)-preferring, polyguluronic acid (polyG)-preferring, and bifunctional types. In the Carbohydrate Active Enzyme Database (CAZy), alginate lyases are mainly distributed in several families, including PL5, PL6, PL7, PL14, PL15, PL17, and PL18. Currently reported alginate lyases mainly originate from marine bacteria, fungi, algae, and invertebrates, with marine bacteria being the primary source, especially *Pseudoalteromonas*, *Shewanella*, and *Vibrio*.

[0005] The applicant previously isolated a strain of *Shewanella haliotis* BP-1 from decaying Sargassum fusiforme. Its fermentation broth exhibited alginate degradation activity, suggesting that this strain may contain alginate lyase. However, the limited yield of natural enzymes makes it difficult to meet the needs of in-depth research and application development. Therefore, predicting the alginate lyase encoding gene through genomic sequence analysis, and further cloning, heterologously expressing, and purifying it to obtain high-purity recombinant alginate lyase, is an important technical approach to address the insufficient yield of natural enzymes and expand the high-value utilization of alginate enzyme resources. It also provides a foundation for subsequent enzyme engineering and industrial applications. Summary of the Invention

[0006] The purpose of this invention is to address the problem of limited production of naturally sourced alginate lyase, which is insufficient to meet the needs of in-depth research and application development. This invention provides an alginate lyase Alg17G, its encoding gene, a recombinant expression vector, engineered bacteria, a preparation method, and its applications. By cloning and heterologously expressing the Alg17G gene fragment after removing the signal peptide coding sequence, high-purity recombinant alginate lyase can be obtained, providing a new enzyme resource for the high-value utilization of alginate and laying the foundation for subsequent enzyme engineering and industrial applications.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] This invention provides an alginate lyase Alg17G, the amino acid sequence of the full-length alginate lyase Alg17G protein is shown in SEQ ID NO:1; the amino acid sequence of the mature Alg17G protein after removing the N-terminal signal peptide is shown in SEQ ID NO:2.

[0009] The present invention also includes biomaterials related to the aforementioned alginate lyase Alg17G, characterized in that the biomaterials comprise any one of the following (1)-(5):

[0010] (1) A DNA molecule encoding SEQ ID NO:1, wherein the nucleic acid sequence of the DNA molecule is shown in SEQ ID NO:3;

[0011] (2) A DNA molecule encoding SEQ ID NO:2, wherein the nucleic acid sequence of the DNA molecule is shown in SEQ ID NO:4;

[0012] (3) An expression cassette containing the DNA molecule described in (2);

[0013] (4) A recombinant expression vector containing the DNA molecule described in (2), or a recombinant expression vector containing the expression cassette described in (3);

[0014] (5) A recombinant microorganism containing the DNA molecule described in (2), or a recombinant microorganism containing the expression cassette described in (3), or a recombinant expression vector microorganism containing the expression cassette described in (4).

[0015] Furthermore, the biological material is a recombinant expression vector of Alg17G, and the recombinant expression vector is pET30a-Alg17G.

[0016] This invention also includes a method for preparing the alginate lyase Alg17G, comprising the following steps: using the genomic DNA of Shewanella haliotis BP-1 as a template, amplifying the Alg17G gene fragment after removing the signal peptide coding sequence; ligating the gene fragment into an expression vector to obtain the recombinant expression vector pET30a-Alg17G; transforming the recombinant expression vector into competent Escherichia coli cells, screening for positive transformants to obtain engineered bacteria; culturing the engineered bacteria and adding IPTG to induce expression; collecting the bacterial cells; and obtaining the alginate lyase Alg17G after disruption, separation, and purification.

[0017] The present invention also includes a method for improving the activity of the alginate lyase Alg17G, the method comprising: adding Ca to a reaction system containing the alginate lyase Alg17G. 2+ Salt, Mg 2+ Salt or Ba 2+ Salt.

[0018] Furthermore, the Ca 2+ Salt, Mg 2+ Salt or Ba 2+ The final concentration of the salt is 1 mmol / L; the Ca 2+ The salt is CaCl2, and the Mg... 2+ The salt is MgCl2, and the Ba 2+ The salt is BaCl2.

[0019] The present invention also includes the application of the alginate lyase Alg17G in the preparation of alginate hydrolysate with free radical scavenging activity.

[0020] This invention also includes the application of the alginate lyase Alg17G in the degradation of polymannuronic acid, sodium alginate and / or alginate to prepare alginate-derived monosaccharides.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention obtains the Alg17G encoding gene of alginate lyase from marine Shewanella haliotis BP-1, and achieves heterologous expression and purification of Alg17G in Escherichia coli by constructing a recombinant expression vector containing a sequence that removes the signal peptide encoding sequence, thus solving the problem of limited natural enzyme production.

[0023] The alginate lyase Alg17G obtained in this invention belongs to the PL17 family of alginate lyases. It has a clear preference for polyM substrates, good pH stability and thermal stability, and can be used for the enzymatic degradation of alginate or polymannuronic acid.

[0024] This invention discovers Ca 2+ Salt, Mg 2+ Salt or Ba 2+ Salt can increase the enzyme activity of alginate lyase Alg17G, which helps to improve its utilization efficiency in alginate degradation reactions.

[0025] The Alg17G enzymatic hydrolysate prepared by this invention has in vitro free radical scavenging ability and can be used to prepare alginate enzymatic hydrolysate with antioxidant activity. At the same time, Alg17G can be used to degrade polymannuronic acid, sodium alginate or alginate to prepare alginate-derived monosaccharides, which has good application value. Attached Figure Description

[0026] Figure 1 The image shows the SDS-PAGE analysis results of purified recombinant enzyme Alg17G. In the figure, M represents the protein molecular weight standard; 1 represents the total protein of uninduced cells; 2 represents the total protein of cells after IPTG induction; 3 represents the supernatant protein after sonication; and 4 represents the protein after Nitrogen peroxide (NiO2) reaction. 2+ 5. Recombinant Alg17G protein purified by affinity chromatography; 6. Recombinant Alg17G protein purified by molecular sieve chromatography.

[0027] Figure 2 The figure shows the effect of pH and temperature on the activity of recombinant alginate lyase Alg17G; in the figure, a represents the optimal pH; b represents pH stability; c represents the optimal reaction temperature; and d represents temperature stability.

[0028] Figure 3 The figure shows the effect of metal ions and chemical reagents on the activity of recombinant Alg17G; in the figure, a represents metal ions; b represents chemical reagents.

[0029] Figure 4 The graph shows the relative enzyme activities of recombinant Alg17G on different substrates.

[0030] Figure 5The UHPLC-Q-Exactive analysis results of the products obtained by degrading different substrates with recombinant Alg17G are shown in the figure. In the figure, a is the polyM enzymatic hydrolysis product; b is the polyG enzymatic hydrolysis product; and c is the sodium alginate enzymatic hydrolysis product.

[0031] Figure 6 The figure shows the antioxidant activity results of the recombinant Alg17G enzymatic hydrolysis product; in the figure, a represents ABTS. + Free radical scavenging rate; b is the DPPH free radical scavenging rate. Detailed Implementation

[0032] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0033] Unless otherwise stated, each feature disclosed in this specification is merely one example of a series of equivalent or similar features.

[0034] Example 1

[0035] This embodiment provides the alginate lyase Alg17G and its encoding gene. The full-length amino acid sequence of the alginate lyase Alg17G is shown in SEQ ID NO:1, and the nucleotide sequence encoding the full-length Alg17G is shown in SEQ ID NO:3. The Alg17G protein is predicted to contain a signal peptide at its N-terminus; the amino acid sequence of the mature Alg17G protein after removing the signal peptide is shown in SEQ ID NO:2, and the nucleotide sequence encoding the mature Alg17G protein is shown in SEQ ID NO:4.

[0036] The Alg17G gene in this embodiment originates from the marine Shewanella haliotis BP-1 isolated and preserved in the applicant's laboratory. This strain was isolated from a sample of decaying Sargassum seaweed from Weizhou Island, Beihai, Guangxi Zhuang Autonomous Region, and is preserved in the applicant's laboratory.

[0037] Example 2

[0038] This embodiment is used to construct the recombinant expression plasmid pET30a-Alg17G expressing the mature alginate lyase Alg17G and its engineered bacteria. The specific scheme is as follows:

[0039] 1. Genomic DNA Preparation: Genomic DNA was extracted from Shewanella haliotis BP-1 strain according to the BioSpin Bacteria Genomic DNA Extraction Kit instructions. DNA integrity was assessed using a 1% (w / v) agarose gel, and DNA concentration and purity were determined using a NanoDrop 2000c.

[0040] 2. Primer Design: Based on the whole genome annotation results of *Shewanella haliotis* BP-1 strain, the predicted alginate lyase coding gene sequence was selected, and the signal peptide coding sequence was removed. Combined with the multiple cloning site of the expression vector pET30a, BamHI and XhoI restriction sites were introduced into the primers to design PCR primers. The primer sequences are as follows:

[0041] Alg17G-F:CG GGATCC CGCCCGTCACTGGTGC (underlined site is BamHI)

[0042] Alg17G-R: CCG CTCGAG GTCTTTATTGACTTTA (underlined site is XhoI).

[0043] 3. Gene cloning and expression vector construction: Using Shewanella haliotis BP-1 genomic DNA as a template, the Alg17G gene fragment after removing the signal peptide coding sequence was amplified using specific primers containing BamHI and XhoI restriction sites. PCR reaction conditions were: 98 ℃ pre-denaturation for 3 min; 95 ℃ denaturation for 15 s, 55 ℃ annealing for 10 s, 72 ℃ extension for 20 s, for a total of 31 cycles; final extension at 72 ℃ for 10 min. The PCR product was detected and purified by 1% agarose gel electrophoresis, and then digested with the expression vector pET30a using BamHI and XhoI, respectively. The target fragment and vector fragment were recovered and ligated using T4 DNA ligase to obtain the recombinant expression plasmid pET30a-Alg17G. The recombinant plasmid was transformed into E. coli DH5α competent cells, positive clones were screened and sequenced for verification; the recombinant plasmid with the correct sequence was transformed into E. coli BL21(DE3) competent cells for subsequent protein expression.

[0044] Example 3

[0045] This example demonstrates the protein expression and purification of the recombinant plasmid pET30a-Alg17G:

[0046] 1. Induction of recombinant protein expression: Successfully transformed positive clones were inoculated into LB liquid medium containing kanamycin (final concentration 50 μg / mL) and cultured overnight at 37 ℃ and 200 r / min. Then, 1% of the clones were inoculated into fresh LB medium (containing kanamycin at a final concentration of 50 μg / mL) and cultured until OD (end-stage reaction). 600 When the concentration reaches 0.5-0.6, add IPTG to a final concentration of 0.5 mmol / L and induce expression overnight at 16 ℃.

[0047] 2. Purification of recombinant proteins: Induced bacterial cells were collected, washed with 50 mmol / L Tris–HCl buffer (pH 7.5), and resuspended in the same buffer containing 20 mmol / L imidazole and 0.15 mmol / L protease inhibitor. Cells were disrupted by sonication, and the supernatant was collected after centrifugation at 12000 r / min for 30 min. The supernatant was purified sequentially by Ni–Fast affinity chromatography and Superdex™ 75 10 / 300GL molecular sieve chromatography, and concentrated using a 30 kDa molecular weight cutoff ultrafiltration tube. Protein purity was analyzed by SDS-PAGE, and protein concentration was determined using the Bradford method. All protein purification operations were performed at 4℃.

[0048] The results are as follows Figure 1 As shown. SDS-PAGE analysis revealed a distinct and specific protein band at approximately 80.0 kDa, consistent with the theoretical molecular weight of the recombinant fusion protein formed from the mature Alg17G protein and its vector-derived tag. (Ni...) 2+ After purification by affinity chromatography and molecular sieve chromatography, recombinant Alg17G protein with high purity was obtained. The purified recombinant protein was used for subsequent enzymatic property determination.

[0049] Example 4

[0050] This embodiment measures the enzyme activity of the purified protein and analyzes the recombinant enzyme, as detailed below:

[0051] (1) Enzyme activity was determined using the DNS method: The reaction system consisted of 290 μL of 0.1% (w / v) PolyM substrate solution (50 mmol / L glycine-sodium hydroxide buffer, pH 9.0), 10 μL of enzyme solution (0.2 mg / mL), and the reaction was carried out in a water bath at the optimal reaction temperature for 20 min. Then, 300 μL of DNS was added to terminate the reaction, and the mixture was heated in a boiling water bath for 5 min for color development. After cooling to room temperature, the absorbance was measured at 540 nm. Enzyme activity unit (U) definition: Under the above conditions, the amount of enzyme required to generate 1 μmol of reducing sugar (calculated using the glucose standard curve) per minute is defined as 1 enzyme activity unit.

[0052] (2) Determination of optimal reaction pH and pH stability: Different pH buffers were prepared (pH 3.5–12.0): Acetic acid-sodium acetate buffer (50 mmol / L) was used for pH 4.0–6.0, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (50 mmol / L) was used for pH 6.0–8.5, and glycine-sodium hydroxide buffer (50 mmol / L) was used for pH 8.5–12.0. Using 0.1% (w / v) polyM as substrate, the reaction was carried out at the optimal temperature for 20 min, and enzyme activity was measured. The enzyme activity under the optimal pH conditions was taken as 100%, and relative enzyme activity was calculated. pH stability determination: Recombinant enzyme Alg17G was placed in different pH buffers and incubated at 4 ℃ for 24 h. After incubation at the optimal reaction conditions for 20 min, residual enzyme activity was measured. The untreated enzyme activity was taken as 100%, and relative enzyme activity was calculated.

[0053] The enzyme activity of recombinant alginate lyase Alg17G was measured under different pH conditions as follows: Figure 2 As shown in Figure a: the enzyme exhibits low activity in acidic (pH 4.0-6.0) and neutral (pH 6.0-8.5) buffer systems, but its activity significantly increases under alkaline (pH 8.5-9.5) conditions. Its optimal reaction pH is 9.0, and the relative enzyme activity remains above 90% within the pH range of 8.5–9.5, indicating that recombinant Alg17G is a slightly alkaline alginate lyase. To further evaluate its pH stability, the enzyme was incubated in different pH buffers at 4°C for 24 h, and the residual enzyme activity was measured. The results are shown below. Figure 2 As shown in b, recombinant Alg17G maintains high stability within a pH range of 3.5–11.5, with residual enzyme activity remaining above 95%. Notably, treatment in an alkaline buffer system (glycine-sodium hydroxide buffer, pH 8.5–11.5) resulted in a slight increase in recombinant enzyme activity, possibly related to this condition's ability to maintain the enzyme's active conformation or enhance substrate binding. These results indicate that recombinant Alg17G exhibits excellent pH stability and a significant alkalinity preference, providing potential advantages for its industrial applications in alkaline environments.

[0054] (3) Determination of optimal reaction temperature and temperature stability: Using 0.1% (w / v) polyM as substrate, enzyme activity was measured within the range of 20–60 °C under optimal pH conditions for 20 min. The relative enzyme activity was calculated with the enzyme activity under optimal temperature conditions as 100%. Temperature stability determination: The recombinant enzyme was incubated in the optimal pH buffer at 25–60 °C for 30 min, and then reacted under optimal reaction conditions for 20 min to determine the residual enzyme activity. The relative enzyme activity was calculated with the untreated enzyme activity as 100%.

[0055] The results obtained by measuring the enzyme activity of recombinant alginate lyase Alg17G under different temperature conditions are as follows: Figure 2 As shown in Figure c: The optimal reaction temperature for recombinant Alg17G is 45℃. Within the temperature range of 30-50℃, its relative enzyme activity remains at a high level; however, when the temperature exceeds 50℃, the enzyme activity begins to decrease, retaining only about 10% of the relative enzyme activity at 60℃. This indicates that recombinant Alg17G exhibits high catalytic activity under mesophilic conditions, while higher temperatures inhibit its enzyme activity. The results of thermal stability analysis are as follows... Figure 2 As shown in Figure d, the residual enzyme activity was measured after incubating the enzyme at different temperatures for 30 min. The results showed that recombinant Alg17G exhibited good thermostability in the range of 30-50℃, with residual enzyme activity remaining above 80%. When the incubation temperature was increased to 55℃, the residual enzyme activity decreased to approximately 55%, and further decreased to approximately 30% after treatment at 60℃. This indicates that recombinant Alg17G is a typical mesophilic enzyme, exhibiting good catalytic activity and thermostability below 50℃, but is prone to thermal inactivation above 50℃, possibly related to decreased protein structural stability and conformational changes.

[0056] (4) Effects of metal ions and chemical reagents on recombinant enzyme activity: In a reaction system containing 0.1% (w / v) polyM, different metal ions (NaCl, KCl, MgCl2, CaCl2, CoCl2, BaCl2, MnCl2, CuCl2, NiCl2, ZnCl2, FeCl2, FeCl3) were added to achieve final concentrations of 1 mmol / L and 5 mmol / L, respectively. The reaction system without added metal ions served as the control. The chemical reagent treatment groups were treated with ethanol, isopropanol, Tween 20, DMSO, SDS, Triton X-100, β-mercaptoethanol (β-ME), and EDTA, respectively, to achieve final concentrations of 0.5% and 1% (v / v), respectively. The system without added chemical reagents served as the control. An equal volume of enzyme solution was added to each reaction system, and the reaction was carried out at the optimal reaction temperature for 20 min. Enzyme activity was then measured. The relative enzyme activity was calculated with the control group's enzyme activity as 100%.

[0057] The effects of different metal ions at final concentrations of 1 mM and 5 mM on the activity of recombinant Alg17G enzyme were investigated, and the results are as follows: Figure 3 As shown in a. Under 1 mM conditions, Ca 2+ Ba 2+ and Mg 2+ It showed a significant activating effect on recombinant Alg17G, with relative enzyme activities increasing to 135%, 131%, and 130%, respectively; Na + and K +It also has a certain promoting effect, with enzyme activities reaching 106% and 102%, respectively. In comparison, Mn 2+ Co 2+ Cu 2+ Fe 3+ Ni 2+ Zn 2+ and Fe 2+ All showed varying degrees of inhibition, with Fe²⁺ almost completely inhibiting enzyme activity. When the metal ion concentration increased to 5 mM, the recombinant Alg17G exhibited significantly different responses to different metal ions. 2+ and Mg 2+ The activation effect was somewhat weakened, but the enzyme activity remained high; Na + and K + The promoting effect was further enhanced, increasing to 121% and 109% respectively, of which Na + It exhibits a certain concentration-dependent activation effect; while Ba 2+ The promoting effect was significantly reduced, and enzyme activity decreased to 51%. Furthermore, Mn... 2+ Co 2+ Cu 2+ Fe 3+ Ni 2+ Zn 2+ and Fe 2+ The inhibitory effect was further enhanced, among which Ni 2+ Zn 2+ Fe 2+ and Fe 3+ It almost completely inhibits enzyme activity, while Mn 2+ Co 2+ and Cu 2+ The relative enzyme activities decreased to 16%, 10%, and 4%, respectively. These results indicate that Ca... 2+ Ba 2+ and Mg 2+ Divalent metal ions may help maintain the active conformation of recombinant Alg17G or promote substrate binding, while some heavy metal ions may lead to decreased enzyme activity by disrupting enzyme structure or interfering with the action of catalytic residues. The effects of different chemical reagents on the activity of recombinant Alg17G enzyme at final concentrations of 0.5% and 1.0% are shown below. Figure 3As shown in b, at a concentration of 0.5%, ethanol and Tween 20 had a slight promoting effect on enzyme activity, while DMSO had virtually no significant effect. Isopropanol, SDS, Triton X-100, EDTA, and β-mercaptoethanol all exhibited varying degrees of inhibition. When the concentration increased to 1.0%, the effects of each chemical reagent on enzyme activity showed a generally consistent trend, with SDS, Triton X-100, EDTA, and β-mercaptoethanol showing further enhanced inhibitory effects, indicating that recombinant Alg17G is relatively sensitive to surfactants and reducing agents.

[0058] Example 5

[0059] Substrate preference analysis was performed on the recombinase Alg17G, as detailed below:

[0060] Under optimal reaction conditions, using sodium alginate, polyG, and polyM as substrates, the reaction was carried out for 20 min, and enzyme activity was measured. The highest enzyme activity was taken as 100%, and the relative enzyme activity was calculated.

[0061] Enzyme activity using polyM as substrate was defined as 100%, and the results are as follows: Figure 4 As shown, the relative enzyme activities of recombinant enzyme Alg17G to polyG and sodium alginate were 10.2% and 3.5%, respectively. This indicates that recombinant Alg17G exhibits a significant substrate preference for polyM, with its catalytic activity for polyM being approximately 10 times that of polyG, while its activity for sodium alginate is even lower. These results suggest that recombinant Alg17G is a typical polyM-preferred alginate lyase.

[0062] Example 6

[0063] The degradation products of recombinase Alg17G are analyzed as follows:

[0064] Under optimal reaction pH conditions, 0.3 mg / mL of recombinant enzyme was mixed with equal volumes of 0.5% (w / v) sodium alginate, polyM, and polyG, and reacted in a 40 ℃ water bath for 72 h, followed by heating in a boiling water bath for 10 min to terminate the reaction. The reaction solution was centrifuged at 12000 r / min, and the supernatant was filtered through a 0.22 μm filter membrane. The reaction system with inactivated enzyme was used as a control. The products were analyzed using an ultra-high resolution mass spectrometer (UltiMate 3000 UHPLC-Q Exactive, Thermo Fisher Scientific). HPLC conditions: Column: ACQUITY UPLC HSS T3 C18 (2.1 mm × 100 mm, 1.8 μm); Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: acetonitrile; Flow rate: 0.4 mL / min; Column temperature: 35 ℃; Injection volume: 1 μL. Mass spectrometry conditions: Electrospray ionization (HESI-II); Spray voltage: 3.50 kV; Sheath gas: Nitrogen, flow rate: 30 L / min; Auxiliary gas: Nitrogen, flow rate: 10 L / min, temperature: 350 ℃; Ion transmission tube temperature: 320 ℃; Mass spectrometry data acquisition mode: Full scan of primary precursor ions plus data-dependent scan of secondary daughter ions (Full MS / dd MS2); Scan mode: Negative ion scan; Primary precursor ion full scan resolution: 70000; Maximum injection time: 100 ms; Mass scan range: 150 m / z; Secondary mass spectrometry resolution: 17500; Trigger threshold: 1.0 e5; Maximum injection time: 50 ms; Normalized collision energy: 30, 60, 90.

[0065] Mass spectrometer results as follows Figure 5 As shown in the figure, the degradation product spectra of different substrates exhibit significant differences, consistent with the substrate-specific analysis results. When polyM was used as the substrate, saturated and unsaturated oligosaccharides with degrees of polymerization (DP) of 1-6 were detected. The saturated monosaccharide (DP1) showed the highest signal intensity, followed by unsaturated monosaccharides (Δ signal) and oligosaccharides (DP2-DP3). The signals of DP4-DP6 decreased sequentially, indicating that the products were predominantly monosaccharides. Figure 5 a). When polyG was used as a substrate, oligosaccharides of DP1-DP4 were detected, with stronger signals from unsaturated disaccharides (Δ oligosaccharides) and trisaccharides (Δ and trisaccharides), while weaker signals were observed from monosaccharides (DP1) and unsaturated tetrasaccharides (Δ and unsaturated). Compared to polyM, the proportion of oligosaccharides with intermediate degree of polymerization was higher, indicating that recombinant Alg17G had a lower degree of degradation of polyG and significant accumulation of intermediate products. Figure 5b). When sodium alginate was used as a substrate, although a series of ionic signals of oligosaccharides with different degrees of polymerization could be detected, the overall signal intensity was significantly lower than that of the polyM and polyG groups, indicating that the recombinant Alg17G had the lowest degradation efficiency for sodium alginate. Figure 5 c). Based on the overall product distribution characteristics, recombinant Alg17G mainly produces monosaccharides, accompanied by a small amount of unsaturated oligosaccharide intermediates, indicating that it has typical characteristics of exo-alginic acid lyases, while also exhibiting a significant substrate preference for polyM. Considering the β-elimination reaction mechanism of PL-type alginate lyases, it is speculated that the unsaturated monosaccharides containing C4=C5 unsaturated bonds in the initial product can undergo further non-enzymatic transformation, leading to the dominance of saturated monosaccharide signals in UHPLC-Q-Exactive detection.

[0066] Example 7

[0067] The antioxidant activity of the degradation products of recombinant alginate lyase Alg17G was determined as follows:

[0068] 1. Product sample preparation: 30 μL of Alg17G enzyme solution (7.72 mg / mL) was added to 480 μL of 2% (W / V) polyM substrate solution and reacted in a 40 ℃ water bath for 72 h. The reaction was then terminated by boiling in a water bath for 10 min. After cooling, the product was used for antioxidant activity determination.

[0069] 2. DPPH free radical scavenging capacity assay: Prepare a 0.16 mg / mL DPPH solution (anhydrous ethanol). The concentration of the enzymatic hydrolysis product was calculated based on the initial polyM concentration in the reaction system. Add 20 μL of different concentrations of enzymatic hydrolysis product to 220 μL of DPPH solution, incubate in the dark for 30 min, and then measure the absorbance at 517 nm. Use the reaction buffer as a blank control and vitamin C (VC) as a positive control. DPPH scavenging rate (%) = (A0 - A...) / (A0 - A0 ... s ) / A0×100%. Where A0 is the absorbance value of the blank control, A s This represents the absorbance of the sample.

[0070] 3. Determination of ABTS free radical scavenging ability: Equal volumes of ABTS (7 mM) solution and K2S2O8 (2.45 mM) solution were mixed and incubated in the dark for 16 h. The mixture was then diluted with PBS (pH 7.0) until the absorbance at A734 nm was 0.7 ± 0.020. This diluted solution was used as the ABTS concentration. + Working solution. Take 10 μL of enzyme digestion products of different concentrations and add them to 220 μL of ABTS. + After incubation at 37 °C for 15 min in the working solution, the absorbance was measured at 734 nm. The reaction buffer was used as a blank control, and VC as a positive control. ABTS +The formula for calculating the clearance rate (%) is the same as above.

[0071] The results obtained are as follows Figure 6 As shown in the figure: the enzymatic hydrolysis products affect ABTS. + Both free radicals and DPPH free radicals exhibited significant scavenging ability, showing a good concentration-dependent effect. Calculations showed that they were effective against ABTS. + EC50 of free radical scavenging rate 50 The concentration was 0.033 mg / mL, and the free radical scavenging rate reached 90% when the product concentration was 0.069 mg / mL; the EC50 of the DPPH free radical scavenging rate was... 50 At a concentration of 0.36 mg / mL, the free radical scavenging rate was 89.70% when the product concentration was 1.08 mg / mL. These results indicate that the Alg17G enzymatic hydrolysate possesses strong in vitro free radical scavenging ability. This activity may be related to the high proportion of low-polymerization oligosaccharides in its product, and the C4=C5 double bond in the unsaturated uronic acid structure may also participate in the free radical scavenging process.

[0072] In summary, this application cloned the Alg17G encoding gene of the PL17 family alginate lyase from *Shewanella haliotis* BP-1, and achieved heterologous expression of recombinant Alg17G by expressing the coding sequence after removing the signal peptide. The enzyme exhibits an optimal reaction temperature of 45 °C, an optimal pH of 9.0, good stability within the pH range of 3.5-11.5, and good thermostability below 50 °C. Under 1 mmol / L Ca... 2+ Ba 2+ and Mg 2+ Alg17G exhibits significant activation activity. Alg17G shows a clear substrate preference for polyM, and its enzymatic hydrolysates are predominantly monosaccharides, exhibiting exolytic degradation characteristics. Alg17G hydrolysates possess strong in vitro free radical scavenging capabilities. In conclusion, Alg17G can serve as a candidate enzyme resource for preparing alginate-derived monosaccharides and alginate hydrolysates with antioxidant activity.

[0073] The above description is a detailed description of the preferred embodiments of the present invention. However, the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modifications made under the technical spirit of the present invention should fall within the patent scope covered by the present invention.

Claims

1. An alginate lyase Alg17G, characterized in that, The amino acid sequence of the full-length alginate lyase Alg17G protein is shown in SEQ ID NO:1; the amino acid sequence of the mature Alg17G protein after removing the N-terminal signal peptide is shown in SEQ ID NO:

2.

2. Biomaterials related to the alginate lyase Alg17G of claim 1, characterized in that, The biomaterial includes any one of the following (1)-(5): (1) A DNA molecule encoding SEQ ID NO:1, wherein the nucleic acid sequence of the DNA molecule is shown in SEQ ID NO:3; (2) A DNA molecule encoding SEQ ID NO:2, wherein the nucleic acid sequence of the DNA molecule is shown in SEQ ID NO:4; (3) An expression cassette containing the DNA molecule described in (2); (4) A recombinant expression vector containing the DNA molecule described in (2), or a recombinant expression vector containing the expression cassette described in (3); (5) A recombinant microorganism containing the DNA molecule described in (2), or a recombinant microorganism containing the expression cassette described in (3), or a recombinant expression vector microorganism containing the recombinant expression vector described in (4).

3. The biomaterial according to claim 2, characterized in that, The biological material is a recombinant expression vector, and the recombinant expression vector is pET30a-Alg17G.

4. A method for preparing the alginate lyase Alg17G according to claim 1, characterized in that, The procedure includes the following steps: using the genomic DNA of Shewanella haliotis BP-1 as a template, amplifying the Alg17G gene fragment after removing the signal peptide coding sequence, ligating the gene fragment into an expression vector to obtain the recombinant expression vector pET30a-Alg17G; transforming the recombinant expression vector into competent Escherichia coli cells, screening for positive transformants to obtain engineered bacteria; culturing the engineered bacteria and adding IPTG to induce expression, collecting the bacterial cells, and obtaining the alginate lyase Alg17G after disruption, separation, and purification.

5. A method for improving the activity of the alginate lyase Alg17G according to claim 1, characterized in that, The method is as follows: Ca is added to the reaction system containing the alginate lyase Alg17G. 2+ Salt, Mg 2+ Salt or Ba 2+ Salt.

6. The method according to claim 5, characterized in that, The Ca 2+ Salt, Mg 2+ Salt or Ba 2+ The final concentration of the salt is 1 mmol / L; the Ca 2+ The salt is CaCl2, and the Mg... 2+ The salt is MgCl2, and the Ba 2+ The salt is BaCl2.

7. The use of the alginate lyase Alg17G of claim 1 in the preparation of alginate hydrolysate with free radical scavenging activity.

8. The use of the alginate lyase Alg17G of claim 1 in the preparation of alginate-derived monosaccharides by degrading polymannuronic acid, sodium alginate and / or alginate.

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