PL6 family alginate lyase Aly94 as well as coding gene and application thereof

Abstract

The invention relates to PL6 family alginate lyase Aly94 as well as a coding gene and application thereof, and belongs to the technical field of enzyme engineering. According to the algin lyase Aly94 disclosed by the invention, algin degradation is in an incision enzyme mode; when sodium alginate is degraded by the alginate lyase Aly94, the final products are mainly unsaturated trisaccharide and unsaturated tetrasaccharide, and a small amount of unsaturated disaccharide, pentasaccharide, hexasaccharide and heptasaccharide are generated; when algin lyase Aly94 is used for degrading polyguluronic acid (Poly G), a final product is mainly unsaturated trisaccharide, and a small amount of unsaturated disaccharide and unsaturated tetrasaccharide are generated; when algin lyase Aly94 is used for degrading polymannuronic acid (Poly M), the final products are mainly unsaturated trisaccharide and unsaturated tetrasaccharide, and then unsaturated disaccharide, and a small amount of unsaturated pentasaccharide, hexasaccharide and heptasaccharide are generated. The alginate lyase Aly94 disclosed by the invention can be applied to preparation of alginate oligosaccharide and research on structures and functions of alginate, and has a wide application prospect in the fields of medicines, food industry and the like.

Description

Technical Field

[0001] The invention relates to a PL6 family alginate lyase Aly94 and a coding gene and application thereof, belonging to the technical field of enzyme engineering. Background Art

[0002] Alginate, as the main structural component of the outer layer of the brown algae cell wall, is a linear polyanionic acidic polysaccharide formed by β-D-mannuronic acid (β-D-Mannuronate, M) and its C5 diastereomer α-L-guluronic acid (α-L-Guluronate, G) connected by β-1,4 glycosidic bonds. These two uronic acids form three characteristic domains through homopolymerization or random copolymerization: poly β-D-mannuronic acid segment (poly M), poly α-L-guluronic acid segment (poly G) and alternating blocks of mannuronic acid and guluronic acid (poly MG / GM).

[0003] The structural characteristics of alginate are regulated by many factors. The species, growth stage, geographical distribution and harvesting season of brown algae can significantly affect its M / G ratio. This ratio not only determines the mechanical properties of the algae (giving it toughness and structural rigidity), but also directly affects the physical and chemical properties of alginate, such as viscosity, gel properties and ion selectivity. It is worth noting that alginate extracted from different tissue parts of brown algae also has significant differences in uronic acid composition, which provides a structural basis for its functional diversity. Based on its unique rheological properties, alginate has been widely used as a stabilizer and thickener in the food industry, as a drug carrier in the medical field, and as a moisturizing ingredient in cosmetics. Recent studies have also found that alginate as a dietary fiber can regulate metabolic disorders, and its hydrogel form also shows important value in wound dressings and active substance encapsulation.

[0004] After alginate is converted into alginate oligosaccharides (AOS) by physical crushing, chemical hydrolysis and enzymatic degradation, its application field has been further expanded. Compared with alginate, AOS has a lower molecular weight, better water solubility and bioavailability, and exhibits significant antioxidant, immunomodulatory, antibacterial and anti-inflammatory biological activities. Studies have found that the functional properties of AOS are closely related to its uronic acid composition, degree of polymerization (DP) and specific domain distribution. However, there is still a gap in the systematic analysis of the structure-activity relationship of AOS, and its application potential needs to be developed through in-depth structural biology research and functional verification.

[0005] In the study of alginate degradation enzyme system, no traditional hydrolase has been found so far, and it mainly relies on the lyase system. According to the difference in substrate specificity, alginate lyase can be divided into three categories: poly M-specific lyase, poly G-specific lyase and bifunctional lyase. Phylogenetic analysis shows that this type of enzyme is mainly distributed in 24 polysaccharide lyase families such as PL-5, PL-6, and PL-7. Different family members show significant differences in three-dimensional structure and catalytic mechanism. Its mechanism of action follows the β-elimination reaction, forming a C4-C5 unsaturated double bond at the non-reducing end of the product. The generated characteristic product 4-deoxy-L-erythro-4-enol pyranosiduronic acid (DEH) can significantly improve the physicochemical properties of AOS.

[0006] This study innovatively used metagenomic technology to successfully screen and obtain a new alginate lyase gene from sea mud samples. aly94 (ORF20394). After obtaining the Aly94 protein through heterologous expression, protein purification, enzymatic property characterization and anti-Pseudomonas aeruginosa biofilm activity were systematically carried out. In-depth research on this enzyme not only provides a new perspective for the study of the molecular mechanism of alginate lyase, but also lays a theoretical foundation for clinical anti-biofilm treatment and high-value utilization of alginate. Summary of the invention

[0007] In view of the deficiencies in the prior art, the present invention provides a PL6 family alginate lyase Aly94 and its encoding gene and application, wherein the enzyme has highly efficient enzymatic activity for degrading alginate.

[0008] The technical solution of the present invention is as follows: A kind of alginate lyase Aly94, the amino acid sequence of which is shown as SEQ ID NO.2.

[0009] The alginate lyase Aly94 of the present invention is an endo-enzyme. When the alginate lyase Aly94 degrades sodium alginate, the final product is mainly unsaturated trisaccharides and unsaturated tetrasaccharides, and a small amount of unsaturated disaccharides, pentasaccharides, hexasaccharides and heptasaccharides are produced; when the alginate lyase Aly94 degrades polyguluronic acid (poly G), the final product is mainly unsaturated trisaccharides, and a small amount of unsaturated disaccharides and unsaturated tetrasaccharides are produced; when the alginate lyase Aly94 degrades polymannuronic acid (poly M), the final product is mainly unsaturated trisaccharides and unsaturated tetrasaccharides, followed by unsaturated disaccharides, and a small amount of unsaturated pentasaccharides, hexasaccharides and heptasaccharides are also produced. A coding gene can encode the above-mentioned alginate lyase Aly94.

[0010] Preferably according to the present invention, the nucleotide sequence of the encoding gene is shown as SEQ ID NO.1.

[0011] A recombinant expression vector is provided, in which the coding gene of the alginate lyase Aly94 is inserted.

[0012] Preferably according to the present invention, the expression vector is an Escherichia coli expression vector, a yeast expression vector, a Bacillus subtilis expression vector, a lactic acid bacteria expression vector, a Streptomyces expression vector, a bacteriophage vector, a filamentous fungus expression vector, a plant expression vector, an insect expression vector or a mammalian cell expression vector.

[0013] A recombinant cell, in which the coding gene of the alginate lyase Aly94 or the recombinant expression vector is inserted into the host cell.

[0014] Preferably according to the present invention, the host cell is an Escherichia coli host cell, a yeast host cell, a Bacillus subtilis host cell, a lactic acid bacteria host cell, an actinomycete host cell, a filamentous fungus host cell, an insect cell or a mammalian cell.

[0015] Further preferably, the Escherichia coli host cell is Escherichia coli BL21, Escherichia coli JM 109 or Escherichia coli DH5α; the yeast host cell is Saccharomyces cerevisiae , Pichia pastoris or Kluyveromyces Iactis ; The Bacillus subtilis host cell is Bacillus subtilis R25 or Bacillus subtilis 9920; the lactic acid bacteria host cell is Lactic acid bacteria COCC101; the actinomycete host cell is Streptomyces spp; the filamentous fungal host cell is Trichoderma viride , Trichoderma reesei , Aspergillus niger or Aspergillus nidulans ; The insect cells are Bombyxmori or Antharaea eucalypti; the mammalian cells are Chinese hamster ovary cells CHO, baby hamster kidney cells BHK or Chinese hamster lung cells CHL.

[0016] The use of the recombinant cell in the recombinant expression of the alginate lyase Aly94.

[0017] Application of the alginate lyase Aly94 in the preparation of alginate oligosaccharides.

[0018] The application of the alginate lyase Aly94 in combating Pseudomonas aeruginosa biofilm.

[0019] Beneficial effects: 1. The alginate lyase Aly94 in the present invention is isolated from the sea mud metagenomic library, is easy to heterologously express and purify, has stable physicochemical properties, high degradation activity, and has the potential for industrial application.

[0020] 2. The alginate lyase Aly94 in the present invention is a new type of alginate lyase and has a high enzyme activity on alginate.

[0021] 3. The alginate lyase Aly94 in the present invention degrades alginate in an endo-enzyme mode; when the alginate lyase Aly94 degrades sodium alginate, the final product is mainly unsaturated trisaccharides and unsaturated tetrasaccharides, and produces a small amount of unsaturated disaccharides, pentasaccharides, hexasaccharides and heptasaccharides; when the alginate lyase Aly94 degrades polyguluronic acid (Poly G), the final product is mainly unsaturated trisaccharides, and produces a small amount of unsaturated disaccharides and unsaturated tetrasaccharides; when the alginate lyase Aly94 degrades polymannuronic acid (Poly M), the final product is mainly unsaturated trisaccharides and unsaturated tetrasaccharides, followed by unsaturated disaccharides, and also produces a small amount of unsaturated pentasaccharides, hexasaccharides and heptasaccharides. The alginate lyase Aly94 in the present invention can be applied to the preparation of alginate oligosaccharides and the study of the structure and function of alginate, and has broad application prospects in the fields of medicine and food industry.

[0022] 4. The alginate lyase Aly94 in the present invention can effectively remove the biofilm formed by Pseudomonas aeruginosa. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the functional module analysis diagram of alginate lyase Aly94.

[0024] Figure 2 This is the evolutionary tree analysis diagram of alginate lyase Aly94.

[0025] Figure 3 The polyacrylamide gel electrophoresis profile of the expression and purification samples of alginate lyase Aly94; wherein: lane 1, protein molecular weight standard, sample volume 5 µL, band sizes from top to bottom are 180 kDa, 130 kDa, 100 kDa, 70 kDa, 55 kDa, 40 kDa, 35 kDa, 25 kDa, 15 kDa; lane 2, control group strain cell culture solution, sample volume 5 µL; lane 3, recombinant strain cell culture solution, sample volume 5 µL; lane 4, recombinant strain cell culture supernatant, sample volume 4 µL; lane 5, Aly94 enzyme solution purified by nickel column, sample volume 5 µL.

[0026] Figure 4 This is the effect curve of temperature on the enzyme activity of alginate lyase Aly94.

[0027] Figure 5 This is the effect curve of pH on the enzyme activity of alginate lyase Aly94.

[0028] Figure 6 This is the curve showing the effect of temperature on the stability of alginate lyase Aly94.

[0029] Figure 7 This is a bar graph showing the effects of metal ions and other compounds on the enzyme activity of alginate lyase Aly94.

[0030] Figure 8 This is the effect curve of NaCl concentration on the enzyme activity of alginate lyase Aly94.

[0031] Fig. 9 The figures are high performance liquid chromatograms of degradation products of different substrates degraded by alginate lyase Aly94; in the figures: A is an HPLC chromatogram with sodium alginate as substrate, B is an HPLC chromatogram with polyguluronic acid (Poly G) as substrate, and C is an HPLC chromatogram with polymannuronic acid (Poly M) as substrate; among them, DP2 is an unsaturated disaccharide; DP3 is an unsaturated trisaccharide; DP4 is an unsaturated tetrasaccharide; DP5 is an unsaturated pentasaccharide; DP6 is an unsaturated hexasaccharide; DP7 is an unsaturated heptasaccharide.

[0032] Fig.10 The HPLC chromatograms of the degradation products of sodium alginate after degradation by alginate lyase Aly94 at different times; in the figure: Di, unsaturated disaccharide; Tetra, unsaturated tetrasaccharide; Hexa, unsaturated hexasaccharide; Octa, unsaturated octasaccharide; Deca, unsaturated decasaccharide.

[0033] Fig.11 This is a bar graph showing the clearance effect of alginate lyase Aly94 on the established Pseudomonas aeruginosa biofilm. DETAILED DESCRIPTION

[0034] The following examples are provided to fully disclose some common techniques for implementing the present invention, but are not intended to limit the scope of application of the present invention. Unless otherwise specified, molecular weight in the present invention refers to weight average molecular weight, and temperature refers to degrees Celsius. The reagents and materials involved in the examples are all common commercially available products unless otherwise specified.

[0035] Source of experimental materials: Escherichia coli DH5α strain was provided by Vazyme Biotech, Inc. (Nanjing, China). Sodium alginate, polymannuronic acid (poly M), and polyguluronic acid (poly G) were obtained from Sigma-Aldrich. All chemical reagents mentioned in this article were provided by China National Pharmaceutical Group Co., Ltd. (Beijing, China).

[0036] Sources of biological materials: The sea mud samples were collected from the Hutouya intertidal zone in Yantai, Shandong (N37°18'31", E119°09'27"), and the sample collection time was July 8, 2021.

[0037] Example 1 Sequence Analysis of the Gene and Protein of Alginate Lyase Aly94 The present invention uses metagenomic technology to successfully screen and obtain a new alginate lyase gene from sea mud samples aly94 (ORF20394), using Bio-Edit version 7.0.9 to calculate the alginate lyase gene aly94 The GC content of Aly94 protein was 4.347.77% and 3.324.76% respectively. The theoretical molecular weight (Mw) and isoelectric point (pI) of Aly94 protein were determined using ExPASy online software. To determine whether Aly94 protein contains a signal peptide, prediction was performed using SignalP 5.0 server. Phylogenetic analysis and amino acid alignment were performed using MEGA version 7.0. To gain a deeper understanding of the enzyme function and sequence similarity, the amino acid sequence was searched using NCBI (National Center for Biotechnology Information, http: / / www.ncbi.nlm.nih.gov / ) and Basic Local Alignment Search Tool (BLAST, http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) for functional annotation and sequence similarity analysis.

[0038] NCBI analysis results show alginate lyase genes in metagenomic libraries aly94 The gene coding region is 1563 bp long, and its nucleotide sequence is shown in SEQ ID NO.1. Alginate lyase gene aly94 The encoded alginate lyase Aly94 consists of 520 amino acids, and its amino acid sequence is shown in SEQ ID NO.2. The theoretical molecular weight of the protein is about 58.68 kDa. The results of online analysis using BLAST software showed that alginate lyase Aly94 was similar to the existing active Vibrio splendidus The whole genome sequence of polysaccharide lyase 6 family protein The gene encodes a polysaccharide lyase 6 family protein consisting of 538 amino acids (NCBI sequence number: WP_116871951.1) with 99% homology.

[0039] The structural information of alginate lyase Aly94 was analyzed using the Simple Modular Architecture Research Tool (SMART, http: / / smart.embl-heidelberg.de / ). The results showed that the 1st to 17th amino acids at the N-terminus were signal peptide sequences, and the 61st to 466th amino acid sequence belonged to the PL6 family, such as Figure 1 As shown. By analyzing the neighbor-joining tree of Aly94 protein and other types of alginate lyases that have been identified, Aly94 protein is in the same evolutionary branch as the PL6 family, as shown in Figure 2 shown.

[0040] Example 2 Heterologous expression and purification of Aly94 protein The ORF20394 gene fragment was amplified by PCR, and the primers were as follows: Forward primer Aly94-F: 5'-ATCTGTATACATATGTGTGGCTCAACGGGTAGCAGTGTTAACAC-3' (SEQ ID NO. 3); Reverse primer Aly94-R: 5'-GTGGTGGTGCTCGAGTTTTTTAATTTGGTACTGTGCGCCGGTG-3' (SEQ ID NO. 4); PrimeSTAR HS DNA polymerase was purchased from Solebio Biotechnology, and the PCR reaction system was operated according to the product instructions provided by the company.

[0041] The reaction conditions of PCR amplification were as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 40 s, annealing at 60°C for 30 s, extension at 72°C for 2 min, 35 cycles; extension at 72°C for 10 min, and stabilization at 4°C for 15 min.

[0042] After the ORF20394 gene fragment was amplified by PCR, the recombinase Exnase II, pET-30a expression vector and PCR amplification product were used to prepare the recombination reaction system. The recombination reaction was completed by reacting at 37°C for 30 min, and the in vitro cyclization of the two linear DNAs was achieved to obtain the pET-30a-Aly94 recombinant vector. The recombinant vector was directly transformed into the Escherichia coli DH5α strain and then spread on a solid plate of LB medium containing 50 µg / mL kanamycin and cultured at 37°C for 12-14 h. A single clone was picked and transferred to liquid LB medium containing 50 µg / mL kanamycin. After culturing at 37°C and 200 rpm for 12-14 h, the forward primer Aly94-F and the reverse primer Aly94-R were used to verify the bacterial liquid PCR. The result showed that the amplified product of the correct size was obtained, which preliminarily proved that the constructed recombinant plasmid was correct. 20 µL of the recombinant plasmid was taken out and sent to Sangon Biotech Co., Ltd. for sequencing to verify whether the positive clone was correct. The recombinant enzyme Exnase II was purchased from Solebio Biotech, and the pET-30a expression vector was purchased from Novagen, USA. The reaction system of enzyme and substrate, reaction temperature and reaction time were all in accordance with the instruction manual.

[0043] The colonies were spread on solid LB medium plates on a clean bench and cultured overnight at 37°C. After the culture was completed, a single colony was picked and inoculated into fresh LB liquid medium supplemented with 50 µg / mL kanamycin and cultured at 37°C and 200 rpm until the cell density reached 0.6-0.8 (OD 600 ). When the desired cell density is reached, the mixture is cooled to 16°C and IPTG (isopropylthiogalactoside) is added to the medium at a final concentration of 0.05 mM. The culture is shaken at 200 rpm for 16-18 h to induce the synthesis of the target protein Aly94. The induced bacterial solution is centrifuged at 8000 rpm and 4°C for 8 min, the supernatant is discarded, and the bacteria are resuspended in pre-cooled Buffer A. The centrifugation step is repeated once and resuspended again. Buffer A consists of 50 mM Tris-HCl and 150 mM NaCl, pH 8.0. Next, the bacterial solution resuspended in pre-cooled Buffer A is subjected to ultrasonic treatment (4 s, 8 s interval, 50% power, 30 min) to break it. The resulting mixture is centrifuged at 15,000 × g and 4°C for 30 min.

[0044] The supernatant was collected and introduced into a column filled with Ni-sepharose™ 6 Fast Flow (manufactured by GE Healthcare). Before injection, the affinity chromatography column was equilibrated with 10 V0 of Buffer A solution, and then 5 V0 of Buffer A solution containing 5 mM imidazole was added to the affinity chromatography column to remove impurities and prevent nonspecific adsorption. The target protein Aly94 was eluted in order from low to high concentration using Buffer A solution containing imidazole linear concentrations of 0 to 500 mM, and the eluate at 50 mM was collected. SDS-PAGE was performed using 12% (w / v) gel to ensure the purity and molecular weight of the purified Aly94 protein. The test results are shown in Figure 2. Figure 3 As shown in the figure, the purity of the purified Aly94 protein reached 95%. To ensure the removal of impurities, dialysis was performed overnight at 4°C in 800 mL of Buffer A. After overnight dialysis, the dialysis solution was replaced every 3 h and repeated twice. After dialysis, the purified protein in the dialysis bag (cutoff size 3000 Da) was collected and the concentration of the purified Aly94 protein was quantitatively determined using the BCA protein detection kit produced by Sangon Biotech, Shanghai, China, and the corresponding concentration of alginate lyase Aly94 enzyme solution was prepared for subsequent experiments.

[0045] Example 3 Substrate specificity analysis of alginate lyase Aly94 (activity test) In order to explore the substrate specificity of alginate lyase Aly94, we used a variety of polysaccharides as substrates, including hyaluronic acid (HA, 3 mg / mL), chondroitin sulfate A (CSA, 10 mg / mL), sodium alginate (5 mg / mL), poly M (3 mg / mL) and poly G (3 mg / mL). In order to completely degrade the polysaccharide substrate, a suitable reaction system was prepared, including: sodium phosphate buffer (NaH2PO4-Na2HPO4buffer, pH8.0), substrate, ddH2O, alginate lyase Aly94 enzyme solution (0.05 mg / mL), and the negative control used the same amount of boiled inactivated enzyme solution, which was placed in a 30 ℃ constant temperature water bath for overnight reaction. After the overnight reaction, it was boiled in boiling water for 10 min, followed by an ice water bath for 5 min. Centrifuge at 12000 rpm, 4°C, 10 min in a desktop high-speed centrifuge. After centrifugation, take the supernatant and add an equal volume of 3,5-dinitrosalicylic acid (DNS) solution. Boil for 10 min, and the solution will appear yellow-brown in corresponding shades. Take 200 μL of the solution in a 96-well plate, and measure the absorbance with an enzyme reader at a wavelength of 540 nm. The absorbance is analyzed. The results show that alginate lyase Aly94 can degrade sodium alginate, poly M, and poly G.

[0046] Example 4 Analysis of the enzymatic properties of alginate lyase Aly94 1. Effect of temperature on enzyme activity First, 3 mg / mL sodium alginate solution, 50 mM NaH2PO4-Na2HPO4 buffer solution at pH 8.0, alginate lyase Aly94 enzyme solution (0.05 mg / mL) and deionized water were mixed in a ratio of 10:10:3:7 (volume ratio), and reacted at 0℃, 10℃, 20℃, 30℃, 40℃, 50℃, 60℃, and 70℃ for 30 min. After the reaction, the same volume of DNS solution was added and boiled in boiling water for 10 min to inactivate the reaction, and then placed in an ice water bath for 5 min. Finally, centrifuge in a desktop high-speed centrifuge at 4℃ and 12000 rpm for 10 min. After centrifugation, 200 μL of the solution was added to a 96-well plate and the absorbance was measured at 540nm. The absorbance was measured using an enzyme reader, and the absorbance was analyzed to detect the enzyme activity. The optimal enzyme activity was defined as 100% relative enzyme activity (relative activity). The test results are shown in Figure 4As shown, the relative enzyme activity of alginate lyase Aly94 towards sodium alginate is above 90% at around 40°C. When the reaction temperature is above 40°C, the enzyme activity of alginate lyase Aly94 decreases rapidly; among them, the alginate lyase Aly94 reaches the maximum enzyme activity towards sodium alginate at 40°C, indicating that the optimal reaction temperature of alginate lyase Aly94 towards sodium alginate is 40°C.

[0047] The method for determining the activity of alginate lyase Aly94 is to measure the absorbance change per minute at 540 nm. The enzyme activity unit U is the amount of enzyme required to convert a certain amount of substrate into product within a certain period of time under certain conditions. This experiment uses the DNS method to measure the amount of reducing sugar generated by the enzyme-catalyzed reaction to indirectly reflect the activity of the enzyme, and then calculate the enzyme activity unit.

[0048] 2. Effect of pH on enzyme activity First, 3 mg / mL sodium alginate solution, reaction buffer, alginate lyase Aly94 enzyme solution (0.05 mg / mL) and deionized water were mixed in a ratio of 10:10:3:7 (volume ratio). The reaction buffer included 50 mM NaAc-Hac buffer (pH5.0-6.0), 50 mM NaH2PO4-Na2HPO4 buffer (pH6.0-8.0), and 50 mM Tris-HCl buffer (pH7.0-10.0). The reaction was carried out at the optimal temperature for 30 min. After the reaction, the DNS method was used to measure the absorbance at a wavelength of 540 nm using an enzyme reader. The absorbance was analyzed to determine the enzyme activity. The optimal enzyme activity was defined as 100% relative enzyme activity (relative activity). The test results are shown in Figure 2. Figure 5 As shown, the enzymatic activity of alginate lyase Aly94 towards sodium alginate was the highest in NaH2PO4-Na2HPO4 buffer (pH 7.0).

[0049] 3. Effect of temperature on enzyme stability 3 mg / mL sodium alginate solution, 150 mM NaH2PO4-Na2HPO4 buffer solution with a pH of 7.0, and alginate lyase Aly94 enzyme solution (0.05 mg / mL) after heat treatment at different temperatures (0℃-70℃) for 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h were mixed with deionized water in a ratio of 10:10:3:7 (volume ratio), and reacted at the optimal temperature for 30 min. The DNS method was used to measure the absorbance with a microplate reader at a wavelength of 540 nm. The absorbance was analyzed and the enzyme activity was determined. The enzyme activity of alginate lyase Aly94 before heat treatment was defined as 100% relative enzyme activity. The test results are shown in Figure 6 As shown, after being treated at 0-20℃ for 24 h, alginate lyase Aly94 still maintained more than 80% relative enzyme activity for sodium alginate; after being treated at 30℃ for 12 h, alginate lyase Aly94 still maintained more than 50% relative enzyme activity for sodium alginate; this indicates that temperature can affect the stability of alginate lyase Aly94, and the stability of alginate lyase Aly94 is better at 0-30℃.

[0050] 4. Effects of metal ions and other compounds on enzyme activity 3 mg / mL sodium alginate solution, 150 mM NaH2PO4-Na2HPO4 buffer solution with pH 7.0, alginate lyase Aly94 enzyme solution (0.05 mg / mL) and deionized water were mixed in a ratio of 10:10:3:7 (volume ratio), and then different metal ions or compounds were added to the reaction system with a final concentration of 5 mM. The reaction was carried out at the optimal temperature for 30 min. After the reaction was completed, the DNS method was used to measure the absorbance with a microplate reader at a wavelength of 540 nm. The absorbance was analyzed and the enzyme activity was detected. The enzyme activity without the addition of metal ions was defined as 100% relative enzyme activity. The test results are shown in the following table. Figure 7 As shown, no metal ions and chemicals can act as enzyme promoters.

[0051] 5. Effect of NaCl concentration on enzyme activity 3 mg / mL sodium alginate solution, 150 mM pH7.0 NaH2PO4-Na2HPO4 buffer, alginate lyase Aly94 enzyme solution (0.05 mg / mL) and deionized water were mixed in a ratio of 10:10:3:7 (volume ratio), and then different final concentrations of NaCl were added to the reaction system. The final concentrations of NaCl added were 0 mM, 1 mM, 2 mM, 5 mM, 10 mM, 20 mM, 50 mM, 100 mM, 250 mM, 500 mM and 1000 mM, respectively. The reaction was carried out at the optimal temperature for 30 min. After the reaction, the DNS method was used to measure the absorbance at a wavelength of 540 nm using an enzyme reader. The absorbance was analyzed and the enzyme activity was determined. The enzyme activity with a final NaCl concentration of 0 mM was defined as 100% relative enzyme activity. The test results are as follows: Figure 8 As shown in the figure, when the final concentration of NaCl is 20 mM, the enzyme activity of alginate lyase Aly94 is the highest. After 20 mM, the enzyme activity gradually decreases with the increase of NaCl concentration, indicating that low concentration NaCl can be used as an enzyme promoter, but too high NaCl concentration will inhibit the enzyme activity of alginate lyase Aly94.

[0052] Example 5 Determination of the activity of alginate lyase Aly94 3 mg / mL sodium alginate solution, 150 mM NaH2PO4-Na2HPO4 buffer solution with pH 7.0, alginate lyase Aly94 enzyme solution (0.05 mg / mL) and deionized water were mixed in a ratio of 10:10:3:7 (volume ratio), and the reaction was carried out at the optimal temperature for 30 s, 1 min, 2 min, and 5 min, respectively. An equal amount of boiled alginate lyase Aly94 enzyme solution was added to the negative control. After the reaction time was up, the DNS method was used to measure the absorbance at a wavelength of 540 nm using a microplate reader, and the absorbance was analyzed to determine the enzyme activity.

[0053] The enzyme activities of polymannuronic acid (poly M) and polyguluronic acid (poly G) were determined according to the above method.

[0054] Definition of enzyme activity unit (U): the amount of enzyme required to generate 1 μmol of product containing unsaturated double bonds per minute.

[0055] At the same time, the protein content of alginate lyase Aly94 enzyme solution was determined using a protein quantification kit purchased from Kangwei Century Company. The results showed that the enzyme activities of alginate lyase Aly94 on sodium alginate, polymannuronic acid (poly M), and polyguluronic acid (poly G) were 2.620 U / mg, 3.043 U / mg, and 3.421 U / mg, respectively.

[0056] Example 6 HPLC analysis of degradation products of different substrates by alginate lyase Aly94 Alginate lyase Aly94 used sodium alginate (3 mg / mL), poly M (3 mg / mL) and poly G (3 mg / mL) as substrates, respectively. The substrate, 150 mM pH7.0 NaH2PO4-Na2HPO4 buffer, alginate lyase Aly94 enzyme solution (0.05 mg / mL) and deionized water were mixed in a ratio of 10:10:3:7 (volume ratio), and reacted for 48 h under the optimal reaction conditions, and 1 μL of undiluted enzyme solution (0.05 μg / μL) was added in the middle. After the reaction was completed, it was inactivated in a constant temperature water bath at 100℃ for 20 min, iced for 5 min, ddH2O was added to 200 μL, and the reaction solution was filtered using a syringe and a 0.22 μm microporous filter membrane to obtain the final product. The reaction products were analyzed by high performance liquid chromatography (HPLC). The HPLC analysis conditions were as follows: gel column: Superdexpeptide 10 / 300 GL (GE); mobile phase: 0.2 M ammonium bicarbonate; flow rate: 0.4 mL / min; detection conditions: UV 232 nm.

[0057] Test results such as Fig. 9 As shown in FIG. 1 , when alginate lyase Aly94 degrades alginate polysaccharide, the final product is a series of unsaturated oligosaccharides, including but not limited to unsaturated disaccharides to unsaturated heptasaccharides. Fig. 9 As shown in A, when alginate lyase Aly94 degrades sodium alginate, the final products are mainly unsaturated trisaccharides and unsaturated tetrasaccharides, and a small amount of unsaturated disaccharides, pentasaccharides, hexasaccharides and heptasaccharides are produced; Fig. 9 As shown in Figure B, when alginate lyase Aly94 degrades polyguluronic acid (poly G), the final product is mainly unsaturated trisaccharides, and a small amount of unsaturated disaccharides and unsaturated tetrasaccharides are produced; Fig. 9 As shown in Figure C, when alginate lyase Aly94 degrades polymannuronic acid (poly M), the final products are mainly unsaturated trisaccharides and unsaturated tetrasaccharides, followed by unsaturated disaccharides, and a small amount of unsaturated pentasaccharides, hexasaccharides and heptasaccharides.

[0058] Example 7 Degradation pattern of alginate lyase Aly94 5 mg / mL sodium alginate solution, 50 mM NaH2PO4-Na2HPO4 buffer solution with a pH of 7.0, alginate lyase Aly94 enzyme solution (0.05 mg / mL) and deionized water were mixed in a ratio of 10:10:3:7 (volume ratio), and reacted at 40°C for 0 min, 5 min, 30 min, 2 h, 8 h, and 24 h. 30 μL of the reaction solution was taken at each time point, and an equal amount of boiled alginate lyase Aly94 enzyme solution was added to the negative control. The reaction solution taken out at each time point was inactivated in a constant temperature water bath at 100°C for 20 min. After the reaction, ddH2O was added to 200 μL, and the reaction solution was filtered using a syringe and a 0.22 μm microporous filter membrane. The degradation products after different enzymatic hydrolysis times were selected for HPLC analysis. The HPLC analysis conditions were as follows: gel column: Superdex peptide 10 / 300 GL (GE); mobile phase: 0.2 M ammonium bicarbonate; flow rate: 0.4 mL / min; detection conditions: UV 232 nm. Test results such as Fig.10 As shown, when alginate lyase Aly94 degrades sodium alginate, the final products are unsaturated disaccharides, tetrasaccharides, hexasaccharides, octasaccharides and decasaccharides, indicating that alginate lyase Aly94 efficiently degrades alginate in a typical endo-cutting manner.

[0059] Example 8 Clearance of established Pseudomonas aeruginosa biofilm by alginate lyase Aly94 Pseudomonas aeruginosa PAO1 (Beijing Biobio Biotech) was picked up into LB liquid culture medium, and after overnight culture at 37°C and 200 rpm, 100 μL of bacterial solution was transferred to 100 mL of fresh LB liquid culture medium for moderate dilution. Take a clean sterile 96-well plate, add 200 μL of diluted bacterial solution to each well, and culture it in a 37°C incubator to form a biofilm. After the biofilm is formed, the bacterial solution is aspirated, and the well plate is washed with PBS buffer (pH8.0), and methanol solution is added to fix the biofilm. Then, 200 μL of alginate lyase Aly94 enzyme solution with concentrations of 0 mg / mL, 0.5 mg / mL, 1 mg / mL, and 2 mg / mL were added to the wells, respectively, and reacted at 30°C overnight. After the enzymatic reaction, the Pseudomonas aeruginosa biofilm was stained with crystal violet. After the staining, the OD value was measured at 600 nm using an enzyme reader to analyze the residual degree of the Pseudomonas aeruginosa biofilm. Fig.11 As shown, alginate lyase Aly94 has a clearing effect on Pseudomonas aeruginosa biofilm, and as the concentration of alginate lyase Aly94 increases, the degree of biofilm residue decreases and the clearing effect increases.

Claims

1. An alginate lyase Aly94, characterized in that: The amino acid sequence of the alginate lyase Aly94 is shown in SEQ ID NO.

2.

2. A coding gene, characterized in that The encoding gene can encode the alginate lyase Aly94 described in claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence of the coding gene is shown as SEQ ID NO.

1.

4. A recombinant expression vector, characterized in that: Insertion into expression vector The coding gene according to claim 2 or claim 3.

5. The recombinant expression vector according to claim 4, characterized in that The expression vector is an Escherichia coli expression vector, a yeast expression vector, a Bacillus subtilis expression vector, a lactic acid bacteria expression vector, a Streptomyces expression vector, a bacteriophage vector, a filamentous fungus expression vector, a plant expression vector, an insect expression vector or a mammalian cell expression vector.

6. A recombinant cell, characterized in that The encoding gene according to claim 2 or claim 3 or the recombinant expression vector according to claim 4 is inserted into the host cell.

7. The recombinant cell according to claim 6, characterized in that The host cell is an Escherichia coli host cell, a yeast host cell, a Bacillus subtilis host cell, a lactic acid bacteria host cell, an actinomycete host cell, a filamentous fungus host cell, an insect cell or a mammalian cell.

8. Use of the recombinant cell according to claim 6 or claim 7 in recombinantly expressing the alginate lyase Aly94 according to claim 1.

9. Use of the alginate lyase Aly94 according to claim 1 in the preparation of alginate oligosaccharides.

10. Use of the alginate lyase Aly94 according to claim 1 in resisting Pseudomonas aeruginosa biofilm.

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