Xylanase OUC-Xyn1, its coding gene, recombinant engineering bacteria and application

Abstract

The application discloses a xylanase OUC-Xyn1, a coding gene thereof, a recombination engineering bacterium and application, and belongs to the technical field of functional enzymes. The amino acid sequence of the xylanase OUC-Xyn1 is shown as SEQ ID NO. 1, and the coding gene sequence is shown as SEQ ID NO. 2. The enzyme belongs to GH 11 family, the optimal reaction temperature is 70 DEG C, and the optimal pH is 5.0; under the conditions of 40-60 DEG C for 4 hours or under the conditions of pH 4.0-8.0 for 48 hours, the enzyme activity can still be kept high. Without any mutation, the specific enzyme activity of the enzyme can reach 2432.25+ / -28.08 U / mg. The OUC-Xyn1 is synergistically used with pectinase and cellulase on the giant kelp powder, and the alginate yield is increased from 28.88+ / -0.40% to 41.42+ / -0.61%. The application has important industrial application value in the field of enzyme green extraction of alginate.

Description

Technical Field

[0001] This invention relates to xylanase OUC-Xyn1, its encoding gene, recombinant engineered bacteria and their applications, belonging to the field of functional enzyme technology. Background Technology

[0002] Xylan is an abundant hemicellulose component found in nature, widely distributed in the cell walls of terrestrial plants and some seaweed. Xylanase specifically hydrolyzes the β-1,4 glycosidic bonds of the xylan backbone to produce xylooligosaccharides and xylose, which have important industrial applications in feed, food, papermaking, textiles, and bioenergy. Xylanases from different sources exhibit significant differences in catalytic properties, thermal stability, and pH stability, making the discovery of novel xylanases with superior enzymatic properties a crucial research area.

[0003] In recent years, some patented studies have effectively improved xylanase activity through site-directed mutagenesis and other techniques combined with recombinant engineered bacteria (such as *E. coli* and *Trichoderma reesei* expression systems). For example, patent CN 122081282 A studied a xylanase with an activity of 245.17 U / mL obtained from *E. coli* through site-directed mutagenesis; patent CN 121874160 A studied a xylanase with a specific activity of 1457 U / mg obtained from *Trichoderma reesei* through single-point mutagenesis. However, site-directed mutagenesis relies on known wild-type enzymes as templates for modification, and mutations are often accompanied by uncertainties such as changes in enzymatic properties or decreased expression levels. In contrast, directly mining novel xylanase genes from the NCBI database using bioinformatics to achieve efficient heterologous expression of xylanase without any mutations is an effective strategy that avoids these uncertainties.

[0004] Giant kelp is an important raw material for extracting alginate. Traditional alginate extraction methods mostly employ acid-base methods, which suffer from problems such as harsh conditions, easy degradation of alginate molecular chains, decreased viscosity, and high wastewater treatment pressure. Enzyme-assisted extraction can specifically degrade algal cell wall components under mild conditions, effectively protecting the alginate structure and improving yield, making it a green alternative process. Current enzyme-assisted extraction mainly uses cellulase or alginate lyase, but insufficient attention has been paid to the enzymatic hydrolysis of hemicellulose components (such as xylan) in the algal cell wall.

[0005] In addition to cellulose, seaweed cell walls also contain abundant hemicellulose (including xylan). Xylanase can synergistically disrupt cell wall structure by degrading the hemicellulose skeleton, promoting the release of alginate. Although some studies have mentioned xylanase in compound enzyme formulations, there is a lack of systematic research on the gene mining, recombinant engineered bacteria construction, and application processes of xylanase specifically for the extraction of alginate from giant kelp, and a lack of targeted technical solutions.

[0006] Therefore, developing xylanases with the ability to efficiently degrade hemicellulose in giant kelp cell walls, along with their engineered strains and extraction processes, is of great significance for improving the extraction efficiency and quality of alginate and promoting the green upgrading of the alginate industry. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a novel xylanase OUC-Xyn1 and its encoding gene, a recombinant vector containing the encoding gene, recombinant engineered bacteria, and the application of this xylanase in combination with cellulase and pectinase in the extraction of alginate from giant kelp. This invention aims to solve the problems of insufficient exploitation of natural xylanase resources and the lack of application solutions for its use in the extraction of alginate from giant kelp.

[0008] The technical solution adopted in this invention is as follows: In a first aspect, the present invention provides a xylanase OUC-Xyn1, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] Furthermore, the xylanase OUC-Xyn1 belongs to the 11th family of glycoside hydrolases (GH 11).

[0010] In a second aspect, the present invention provides a gene encoding the xylanase OUC-Xyn1 described in the first aspect, the nucleotide sequence of which is shown in SEQ ID NO. 2.

[0011] Furthermore, the nucleotide sequence is an optimized sequence based on the codon preference of the host cell, and the host is Escherichia coli BL 21(DE 3).

[0012] Thirdly, the present invention provides a recombinant expression vector carrying the gene encoding xylanase OUC-Xyn1 as described in the second aspect.

[0013] Furthermore, the recombinant expression vector is pET 28a(+)-OUC-Xyn1.

[0014] Fourthly, the present invention provides a recombinant engineered bacterium whose genome contains the gene encoding xylanase OUC-Xyn1 as described in the second aspect.

[0015] Furthermore, the recombinant engineered bacteria are E. coli BL21(DE3) / OUC-Xyn1.

[0016] Fifthly, the present invention provides the application of the xylanase OUC-Xyn1 described in the first aspect, the recombinant expression vector described in the third aspect, or the recombinant engineered bacteria described in the fourth aspect in the extraction of alginate from giant kelp, wherein the extraction is a synergistic extraction of xylanase OUC-Xyn1 with pectinase and cellulase.

[0017] Further, the extraction conditions are as follows: giant kelp powder passes through a 40-60 mesh sieve, OUC-Xyn1 is added at a rate of 600-1200 U / g, cellulase is added at a rate of 600-1200 U / g, pectinase is added at a rate of 600-1200 U / g, reaction temperature is 30-50℃, pH is 4.0-8.0, solid-liquid ratio is 1:15-1:55, and reaction time is 1-5 h.

[0018] Furthermore, the giant kelp powder is sieved through a 40-mesh sieve, the amount of OUC-Xyn1 added is 1200 U / g, the amount of cellulase added is 1000 U / g, the amount of pectinase added is 1000 U / g, the reaction temperature is 40℃, the reaction pH is 7.0, the solid-liquid ratio is 1:35, and the reaction time is 3 h.

[0019] In a sixth aspect, the present invention provides an enzyme preparation comprising the xylanase OUC-Xyn1 described in the first aspect.

[0020] Furthermore, the enzyme preparation also contains pharmaceutically or industrially acceptable carriers, auxiliaries, or excipients.

[0021] In a seventh aspect, the present invention also provides a method for extracting alginate, comprising co-treating giant kelp powder with the xylanase OUC-Xyn1 described in the first aspect, along with pectinase and cellulase.

[0022] Further, the extraction conditions are as follows: sieving of giant kelp powder, addition of OUC-Xyn1 at 600~1200 U / g, addition of cellulase at 600~1200 U / g, addition of pectinase at 600~1200 U / g, reaction temperature at 30~50℃, pH at 4.0~8.0, solid-liquid ratio at 1:15~1:55, and reaction time at 1~5 h.

[0023] Furthermore, after enzymatic hydrolysis, the algae were inactivated in a boiling water bath for 15-20 min, centrifuged (8000 rpm, 15 min), and the supernatant was collected. The remaining algal residue was replaced with 3% (w / v) Na2CO3 at 60°C for 3 h, and centrifuged again (8000 rpm, 15 min), and the supernatant was collected. The two supernatants were combined, diluted with deionized water (solid-to-liquid ratio 1:120), and the pH was adjusted to neutral. 10% CaCl2 was added to form a gel. After rinsing with deionized water, the gel was treated with 1.0 M HCl for 2-4 h for the first decalcification, and then treated with 0.5 M HCl for 2-4 h for the second decalcification. The gel was precipitated with 2 volumes of anhydrous ethanol. Finally, the precipitated sample was dried at 50±2°C to constant weight to obtain the finished alginate product.

[0024] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: (1) A novel xylanase of the GH 11 family is provided: This invention expresses and studies xylanase OUC-Xyn1 obtained based on the glycoside hydrolase family 11 protein with GenBank accession number WP_303264977.1, enriching the enzyme resource library of xylanase.

[0025] (2) Excellent enzymatic properties and high enzyme activity: The optimal temperature of OUC-Xyn1 is 70℃, the optimal pH is 5.0, and it still has high enzyme activity after treatment at 40~60℃ for 4 h or at pH 4.0~8.0 for 48 h. The specific enzyme activity can reach 2432.25 ± 28.08 U / mg without any mutation.

[0026] (3) Significant effect in the extraction of alginate from giant kelp: Currently, there are few public reports on xylanase for enzymatic extraction of alginate from giant kelp and its application. This invention is the first to apply OUC-Xyn1 to the green extraction process of alginate from giant kelp, synergistically treating giant kelp powder with pectinase and cellulase, and the alginate yield is increased by 43.40% compared with that without the addition of compound enzymes.

[0027] (4) High industrial application value: The OUC-Xyn1 of the present invention has important industrial application and economic value in the enzymatic green extraction of alginate. Attached Figure Description

[0028] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0029] Figure 1Schematic diagram of SDS-PAGE electrophoresis of purified xylanase OUC-Xyn1.

[0030] Figure 2 Schematic diagram of the optimal temperature for xylanase OUC-Xyn1.

[0031] Figure 3 Schematic diagram of the thermal stability of xylanase OUC-Xyn1.

[0032] Figure 4 Schematic diagram of the optimal pH for xylanase OUC-Xyn1.

[0033] Figure 5 Schematic diagram of pH stability of xylanase OUC-Xyn1.

[0034] Figure 6 Schematic diagram showing the effect of xylanase OUC-Xyn1, pectinase, and cellulase addition on the extraction of alginate from giant kelp.

[0035] Figure 7 Schematic diagram of optimized conditions for the synergistic extraction of alginate from giant kelp using xylanase OUC-Xyn1 in conjunction with pectinase and cellulase. A1, A2, and A3 represent cellulase additions of 600 U / g, 800 U / g, and 1000 U / g, respectively; B1, B2, and B3 represent xylanase OUC-Xyn1 additions of 800 U / g, 1000 U / g, and 1200 U / g, respectively; and C1, C2, and C3 represent pectinase additions of 600 U / g, 800 U / g, and 1000 U / g, respectively. Detailed Implementation

[0036] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and premise of the invention.

[0037] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0038] Example 1: Design and synthesis of the gene encoding xylanase OUC-Xyn1 Based on the amino acid sequence information of glycosidase family 11 protein with GenBank accession number WP_303264977.1, the mature peptide sequence was obtained after removing its N-terminal signal peptide.

[0039] Based on the aforementioned mature peptide sequence and the codon preference of the host *E. coli*, the coding gene was codon optimized for efficient expression in *E. coli*. The optimized xylanase OUC-Xyn1 coding gene contains 588 bases, as shown in SEQ ID NO. 2, and encodes 196 amino acids, as shown in SEQ ID NO. 1.

[0040] The gene encoding the xylanase OUC-Xyn1 shown in SEQ ID NO. 2 was synthesized in its entirety by Qingke Biotechnology Co., Ltd.

[0041] Example 2 Construction of recombinant expression vector The encoding gene of xylanase OUC-Xyn1 obtained in Example 1 (nucleotide sequence as shown in SEQ ID NO. 2, amino acid sequence as shown in SEQ ID NO. 1) was inserted between the BamHI restriction site and the XhoHI restriction site of the pET 28a(+) vector to construct a recombinant expression plasmid named pET28a-OUC-Xyn1.

[0042] The specific steps are as follows: Primers were designed and synthesized according to the target sequence. The target gene fragment was amplified by PCR, identified by electrophoresis, and then purified. Simultaneously, the pET-28a(+) target vector was linearized by double digestion with restriction endonucleases BamHI and XhoI (1 μL of restriction enzyme 1 and 2 μL of restriction enzyme 2 were added to the system). After electrophoresis identification, the fragment was purified and ready for use. The purified target fragment was mixed with the linearized vector and ligated using the Trelief SoSoo Cloning Kit homologous recombination arm at 50℃ for 30 min. The ligation product was then transformed into *E. coli* TOP10 competent cells for activation and evenly spread on LB agar plates containing 50 μg / mL kanamycin, and incubated overnight at 37℃. The next day, single clones were randomly picked from the plates for colony PCR using a 2X T5 Super PCR Mix reaction system. Positive clones were screened by gel electrophoresis. Positive clones were selected and inoculated into LB broth containing 50 μg / mL kanamycin. The cultures were incubated overnight at 37°C and 220 rpm using a shaker. Recombinant plasmids were then extracted by centrifugation using an alkaline lysis method. Finally, the extracted plasmids were sequenced using Sanger sequencing, and the sequencing results confirmed that the inserted sequence was completely identical to SEQ ID NO. 2. The correctly constructed recombinant expression plasmid was named pET28a-OUC-Xyn1 and stored at -20°C for later use.

[0043] Example 3 Construction of recombinant engineered bacteria The recombinant expression plasmid pET28a-OUC-Xyn1 constructed in Example 2 was transformed into the expression host. E. coli BL21(DE3) competent cells were plated on LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37°C. The following day, single colonies were picked and inoculated into LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C with shaking at 220 rpm for 12 h. Plasmids were extracted and double-enzyme digested (BamHI and XhoHI) for verification. The digestion products showed the release of the target band on 1% agarose gel electrophoresis. Sequencing verification showed that the inserted fragment was completely identical to SEQ ID NO. 2. The correctly verified positive clone was named... E. coli Add BL21(DE3) / pET28a-OUC-Xyn1 to a final concentration of 15% glycerol and store at -80°C for later use.

[0044] Example 4 Preparation of xylanase OUC-Xyn1 The recombinant Escherichia coli constructed in Example 3 E. coli After activating BL21(DE3) / pET28a-OUC-Xyn1 in 5 mL LB liquid medium (containing 100 μg / mL kanamycin) for 12 h, it was inoculated into 100 mL LB liquid medium (containing 100 μg / mL kanamycin) at a 1% inoculum size and cultured at 37℃ and 200 rpm for 5 h. When the OD600 value of the bacterial culture was 0.6~0.8, 1‰ IPTG (100 mM, final concentration 0.1 mM) was added and the culture was induced at 20℃ for 16-20 h to express xylanase OUC-Xyn1.

[0045] After fermentation, the fermentation broth was centrifuged at 8000 rpm for 10 min, and the cells were collected. The cells were resuspended in 50 mM pH 7.0 Tris-HCl buffer and then sonicated in an ice-water bath at 300 W for 3 s on and 3 s off for a total of 30 min. After that, the cells were centrifuged again at 8000 rpm for 15 min, and the supernatant was collected as the crude enzyme solution.

[0046] The His-tagged fusion protein in the crude enzyme solution was purified using a Ni-NTA affinity chromatography column. The specific steps are as follows: The Ni-NTA column was equilibrated with imidazole-free Buffer A (0 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl). After loading the sample, a gradient elution was performed sequentially with elution buffers containing 20 mM imidazole, 50 mM imidazole, and 100 mM imidazole (each elution buffer contained 500 mM NaCl and 50 mM Tris-HCl) to remove weakly binding contaminating proteins. Then, the target protein was eluted with elution buffer containing 200 mM imidazole (200 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl), and the eluted fractions from this stage were collected. The column was then rinsed with Buffer B (500 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl), reequilibrated with Buffer A, rinsed with deionized water, sealed with 20% ethanol, and stored at 4°C.

[0047] The fraction collected by elution with 200 mM imidazole was concentrated by ultrafiltration using a 10 kDa ultrafiltration tube, and the imidazole in the eluent was replaced with deionized water to obtain a purified recombinant xylanase OUC-Xyn1 solution.

[0048] Protein concentration was determined using the Bradford method, and protein purity and molecular weight were determined by SDS-PAGE. Results are as follows: Figure 1As shown: After affinity purification, electrophoretically pure recombinant protein was obtained with a molecular weight of approximately 25 kDa, consistent with the theoretical molecular weight of xylanase OUC-Xyn1, indicating that the target pure enzyme was successfully obtained. The pure enzyme was stored at 4°C for later use.

[0049] Example 5: Determination of specific enzyme activity of xylanase OUC-Xyn1 The standard method for determining the activity of xylanase OUC-Xyn1 is as follows: A total system of 200 μL contains: 100 μL of 0.5% (w / v) beech xylan, 10 μL of OUC-Xyn1, and 90 μL of 50 mM CBS buffer (pH 5.0). The reaction solution is incubated at 70°C for 5 min.

[0050] DNS method for determining the specific activity of xylanase OUC-Xyn1: After the reaction, 300 μL of DNS reagent (3,5-dinitrosalicylic acid) was added to the reaction solution and placed in a boiling water bath for 10 min for color development. The absorbance at 540 nm was measured using a microplate reader. An equal volume of inactivated enzyme (boiled for 15 min) was used as a control reaction instead of the active enzyme. A standard curve was plotted using standard xylose solution (0.05–0.20 mg / mL), and the reducing sugar yield (mg / mL) was calculated. Each experimental group was repeated three times, and the results are expressed as mean ± standard deviation.

[0051] Enzyme activity unit (U) is defined as the amount of enzyme required to generate 1 μmol of reducing sugar (calculated as xylose) from xylan per minute at 70°C and pH 5.0 (using beech xylan as substrate).

[0052] The Bradford method for protein concentration determination: The Bradford protein assay kit, sourced from Beyotime Biotechnology Co., Ltd., is based on the property that Coomassie Brilliant Blue G-250 dye enhances absorbance at 595 nm after binding to proteins. The standard curve uses bovine serum albumin (BSA) as a control, with a concentration gradient of 0.075–1.500 mg / mL. Protein concentration is calculated by substituting the absorbance of the measured samples into the standard equation. Each experimental group was tested in triplicate, and results are expressed as mean ± standard deviation.

[0053] The enzyme activity of OUC-Xyn1 was determined to be 2432.25 ± 28.08 U / mg.

[0054] Example 6 Determination of optimal reaction conditions The effects of temperature and pH on the relative enzyme activity of OUC-Xyn1 were determined using the method described in Example 5.

[0055] Determination of optimal temperature: At temperatures of 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, and 80℃, all components except the enzyme (substrate solution, CBS buffer) were pre-incubated for 5 min at each test temperature. Then, the OUC-Xyn1 purified enzyme prepared in Example 4 was added to initiate the reaction, and enzyme activity was measured according to the method in Example 5. The relative enzyme activity at each temperature was calculated with the highest enzyme activity defined as 100%. Results are as follows: Figure 2 As shown, the optimal temperature for OUC-Xyn1 is 70℃.

[0056] Temperature stability determination: Recombinant xylanase OUC-Xyn1 was incubated at 40℃, 50℃, 60℃, and 70℃ for 0–240 min (samples were taken at different time points). The substrate solution and CBS buffer (without enzyme) were pre-incubated at the enzyme's optimal reaction temperature of 70℃ for 5 min. Then, enzyme solutions treated at different temperatures and times were added, and the reaction was initiated according to the method in Example 5, and residual enzyme activity was measured. The relative enzyme activity under each treatment condition was calculated with the enzyme activity without incubation (0 min) as 100%. Results are as follows: Figure 3 As shown, OUC-Xyn1 can still maintain high enzyme activity after incubation at 40℃~60℃ for 240 min.

[0057] Determination of optimal pH: Different pH buffer solutions were prepared: 50 mM citrate buffer (pH 4.0–6.0), 50 mM phosphate buffer (pH 6.0–7.0), 50 mM Tris-HCl buffer (pH 7.0–8.0), and 50 mM glycine-NaOH buffer (pH 8.0–10.0). The effect of different pH values ​​on the relative enzyme activity of OUC-Xyn1 was determined at 70°C. Buffer solutions with pH values ​​from 4.0 to 10.0 were selected as the different pH buffer solutions for the reaction. Enzyme activity was determined according to the method in Example 5, and the relative enzyme activity at each pH was calculated with the highest enzyme activity as 100%. The results are as follows: Figure 4 As shown, the optimal pH for OUC-Xyn1 is 5.0.

[0058] pH stability determination: Recombinant xylanase OUC-Xyn1 was placed in buffer solutions with pH values ​​of 4.0, 5.0, 6.0, 7.0, and 8.0, and incubated at 4°C for 0–48 h (samples were taken at different time points). The substrate solution and corresponding pH buffer solutions (without enzyme) were pre-incubated at the enzyme's optimal reaction temperature of 70°C for 5 min. Then, enzyme solutions treated with different pH values ​​were added, and the reaction was initiated according to the method in Example 5, and residual enzyme activity was measured. The relative enzyme activity under each treatment condition was calculated with the enzyme activity before incubation (0 h) as 100%. The results are as follows: Figure 5 As shown, OUC-Xyn1 maintained high enzyme activity after incubation at pH 4.0–8.0 for 48 h, indicating that the enzyme has excellent pH stability.

[0059] Example 7: Extraction process of brown alginic acid from giant kelp Preparation of giant kelp powder: Giant kelp Lessonia Nigrescens (LN) The sample was obtained from Qingdao Mingyue Seaweed Group. The giant kelp was washed with distilled water to remove salt, soaked for 12 h, and then dried in a 65℃ oven to constant weight. It was then pulverized using a traditional Chinese medicine pulverizer and passed through a 40-60 mesh sieve (0.250 mm < pore size < 0.425 mm). The powder that passed through the sieve was collected and sealed for later use.

[0060] Alginate extraction: Add the above algal powder to 35 mL / g deionized water, 600-1200 U / g cellulase, 600-1200 U / g pectinase, and 600-1200 U / g recombinant xylanase, adjust the pH to 4.0-8.0, and perform enzymatic hydrolysis at 30-50°C for 1-5 h. After enzymatic hydrolysis, inactivate the enzymes in a boiling water bath for 15-20 min, and centrifuge (8000 rpm, 15 min) to obtain the supernatant and the remaining algal residue. Collect the supernatant for later use; add 3% (w / v) Na2CO3 to the remaining algal residue and replace it at 60°C for 3 h, then centrifuge again (8000 rpm, 15 min) and collect the supernatant. The two supernatants were combined and diluted with deionized water (solid-to-liquid ratio 1:120). The pH was adjusted to neutral, and 10 (w / v)% CaCl2 was added to form a gel. After rinsing with deionized water, the gel was treated with 1 M HCl for 2-4 h for the first decalcification, and then treated with 0.5 M HCl for 2-4 h for the second decalcification. The gel was then precipitated with 2 volumes of anhydrous ethanol. Finally, the precipitated sample was dried at 50 ± 2°C to constant weight to obtain the alginate product. The alginate yield was then weighed and determined.

[0061] ; In the formula, W A To determine the mass (g) of the extracted alginate, W S The weight of the giant kelp powder raw material is (g).

[0062] Example 8: Synergistic extraction of brown algin from giant kelp using xylanase OUC-Xyn1 in combination with pectinase and cellulose. Determination of single enzyme addition: Weigh 1.0 g of the giant kelp powder treated in Example 7, add 35 mL of deionized water, add 600~1200 U / g OUC-Xyn1, 600~1200 U / g cellulase, and 600~1200 U / g pectinase. The reaction temperature is 40℃, the pH is 7.0, and the reaction time is 4 h. Extract alginate according to the alginate extraction process in Example 7.

[0063] The results are as follows Figure 6 As shown, the alginate yield in the control group without added compound enzymes was 28.88 ± 0.40%. In the single enzyme addition assay, the alginate yield reached 35.60 ± 0.71% when xylanase OUC-Xyn1 was added at 1000 U / g; 34.00 ± 0.26% when pectinase was added at 800 U / g; and 32.28% ± 0.38% when cellulase was added at 800 U / g. Compared with the groups without added enzymes, the alginate yield showed a significant increase, and the alginate yield tended to stabilize after reaching its maximum value.

[0064] Determination of the amount of compound enzyme added: Weigh 1.0 g of the giant kelp powder treated in Example 7, add deionized water at a solid-liquid ratio of 1:35, and add xylanase OUC-Xyn1 (800 U / g, 1000 U / g, 1200 U / g, corresponding to B1, B2, B3), pectinase (600 U / g, 800 U / g, 1000 U / g, corresponding to C1, C2, C3) and cellulase (600 U / g, 800 U / g, 1000 U / g, corresponding to A1, A2, A3), respectively. The reaction temperature is 40℃, pH 7.0, and the reaction time is 3 h. Extract alginate according to the process in Example 7.

[0065] The results are as follows Figure 7 As shown, the synergistic effect of the three enzymes—xylanase, pectinase, and cellulase—at concentrations of 1200 U / g, 1000 U / g, and 1000 U / g, respectively, increased the alginate yield to 41.42 ± 0.61%, significantly higher than that of the single-enzyme system. This result confirms that enzyme synergy can efficiently degrade the complex structure of giant kelp cell walls, significantly releasing alginate; furthermore, the optimized ratio of the three enzymes further improved substrate conversion efficiency and extraction rate, providing scalable process parameters for industrial-scale green extraction.

[0066] In summary, the xylanase OUC-Xyn1 of the present invention has high specific enzyme activity and good stability; at the same time, the OUC-Xyn1 of the present invention can synergistically work with pectinase and cellulase to improve the extraction efficiency of alginate from giant kelp, and has high industrial and economic value.

[0067] The above embodiments are preferred embodiments of the present invention, but the implementation of the present invention is not limited to the above embodiments. Any modifications, alterations, substitutions, equivalent replacements or improvements made without departing from the spirit and principle of the present invention should be covered within the protection scope of the present invention.

Claims

1. A xylanase OUC-Xyn1, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

2. The gene encoding the xylanase OUC-Xyn1 of claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that, Carrying the gene as described in claim 2.

4. A recombinant engineered bacterium, characterized in that, The genome contains the gene described in claim 2.

5. The application of the xylanase OUC-Xyn1 according to claim 1 in the extraction of alginate from giant kelp, characterized in that, The extraction was performed by synergistic extraction of xylanase OUC-Xyn1 with pectinase and cellulase. The extraction conditions were as follows: the amount of xylanase OUC-Xyn1 added in the three-enzyme synergistic system was 1200 U / g, the amount of cellulase added was 1000 U / g, the amount of pectinase added was 1000 U / g, the reaction temperature was 40℃, the pH was 7.0, the solid-liquid ratio was 1:35, and the reaction time was 3 h.

6. The application according to claim 5, characterized in that, The giant kelp powder was passed through a 40-mesh sieve.

7. A method for extracting alginate, characterized in that, The step includes synergistically treating giant kelp powder with the xylanase OUC-Xyn1 described in claim 1, along with pectinase and cellulase; The addition amounts of xylanase OUC-Xyn1 were 1200 U / g, cellulase 1000 U / g, and pectinase 1000 U / g. The reaction temperature was 40℃, the pH was 7.0, the solid-liquid ratio was 1:35, and the reaction time was 3 h.

8. An enzyme preparation, characterized in that, It contains the xylanase OUC-Xyn1 as described in claim 1.

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