A brown alga pectin lyase mutant e180q, a coding gene, a recombinant expression vector and a genetically engineered bacterium

Abstract

The application discloses a brown alga gelolysis enzyme mutant E180Q, a coding gene, a recombination expression carrier and a gene engineering bacteria, and belongs to the technical field of gene engineering. The application takes brown alga gelolysis enzyme PpAly7A as a starting template, and obtains a mutant with improved thermal stability through site-directed mutation and combined mutation. The thermal stability is improved by destroying the salt bond of Glu180-Arg178 (E180Q), the half-life of the mutant at 50 DEG C is improved to 7.246 days compared with wild enzyme PpAly7A, and the site can be used as a new target for subsequent rational design.

Description

[0001] This case is a divisional application of application number 202610090327.5 (application date: January 23, 2026, invention patent). (Name: A mutant of alginate lyase, encoding gene, recombinant expression vector and genetically engineered bacterium). Technical Field

[0002] This invention relates to the field of genetic engineering technology, specifically to an alginate lyase mutant E180Q, its encoding gene, recombinant expression vector, and genetically engineered bacteria. Background Technology

[0003] Alginate is a natural polysaccharide extracted from brown algae and is an important marine organic carbon source. It exists in the cell walls of brown algae in the form of calcium, sodium, and magnesium salts, accounting for approximately 30% to 60% of the dry weight of brown algae. It is mainly found in the cell walls of hundreds of species of brown algae, including kelp, kombu, giant kelp, staghorn kelp, fucus vesiculosus, and Sargassum. It is primarily composed of two monomers: β-D-mannuronic acid (M unit) and α-L-guluronic acid (G unit). These two monomers are uniformly or heterogeneously linked by 1,4-glycosidic bonds, forming three different blocks: homopolymer M block (poly M), homopolymer G block (poly G), and mixed blocks (alternating M and G units). Alginate possesses a variety of unique physical and chemical properties, such as thickening, gelling, film-forming, biodegradability, and biocompatibility, thus finding wide applications in various fields such as food, pharmaceuticals, cosmetics, papermaking, textiles, and biomaterials. However, its large molecular weight, poor water solubility, and low bioavailability have limited its application. Alginate can be degraded into alginate oligosaccharides (AOS) with double bonds at the non-reducing ends through physical, chemical, and enzymatic methods. AOS is an oligomer composed of 2-25 monomers, overcoming the shortcomings of alginate application. Moreover, it can be used as a therapeutic and growth promoter for plants, and has antioxidant, antitumor, and blood sugar and lipid-regulating effects.

[0004] Alginate lyases are a class of polysaccharide lysins that break glycosidic bonds between alginate molecules through an elimination mechanism, generating alginate oligosaccharides with unsaturated double bonds at their non-reducing ends. The alginate lyase method can prepare oligosaccharides with specific structures and biological activities, featuring high catalytic efficiency, mild reaction conditions, good substrate specificity, and energy-saving and environmentally friendly characteristics, making it widely applicable in industry, agriculture, food, and medicine. However, naturally derived alginate lyases typically suffer from poor thermal stability, which to some extent limits their potential for industrial application. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide an alginate lyase mutant E180Q, its encoding gene, a recombinant expression vector, and a genetically engineered bacterium.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides an alginate lyase mutant, wherein the alginate lyase mutant has a mutation site of E180Q compared with alginate lyase PpAly7A, and the amino acid sequence of alginate lyase PpAly7A is shown in SEQ ID NO:2.

[0007] A second aspect of the invention provides a gene encoding the alginate lyase mutant.

[0008] A third aspect of the invention provides a recombinant expression vector carrying the gene.

[0009] Furthermore, pET series plasmids were used as expression vectors.

[0010] In a fourth aspect, the present invention provides a genetically engineered bacterium containing the gene or the recombinant expression vector.

[0011] Furthermore, BL21 is used as the expression host.

[0012] In a fifth aspect, the invention provides the use of the alginate lyase mutant, the gene, the recombinant expression vector, or the genetically engineered bacteria in the degradation of Pseudomonas aeruginosa biofilms.

[0013] The beneficial effects of this invention are: This invention constructed a series of mutants by disrupting the salt bonds of the wild-type enzyme PpAly7A, and explored the mechanism by which salt bonds affect the thermostability of alginate lyase. This invention enriches the diversity of alginate lyases, promotes the understanding of their structure and function, and provides a theoretical basis for modifying the enzyme's thermostability. Disruption of the Glu180-Arg178 salt bond (E180Q) improved thermostability; the half-life of this mutant at 50°C was increased to 7.246 days compared to the wild-type enzyme PpAly7A, and this site can serve as a new target for subsequent rational design. This invention provides a theoretical basis and a clear target for the molecular modification and industrial application of alginate lyase, and has significant economic and social benefits. Attached Figure Description

[0014] Figure 1 This is an SDS-PAGE electrophoresis image of PpAly7A, where lane 1 is PpAly7A.

[0015] Figure 2The image shows an SDS-PAGE electrophoresis image of salt bond mutants. The samples tested were salt bond mutants E25Q, E37Q, E81Q, E209Q, D201N, and E180Q.

[0016] Figure 3 The effect of temperature on the alginate lyase PpAly7A was tested. Figure 3 In Figure A, PpAly7A was tested for catalytic activity at 0-70 °C and for thermal stability at 0, 10, 20, 30, 40, 50, 60, and 70 °C for 1 hour. Figure 3 B represents the thermal stability test of PpAly7A.

[0017] Figure 4 The effect of salt bonds on the alginate lyase PpAly7A was tested, among which... Figure 4 A represents the salt bond mutation site. Figure 4 B represents the temperature stability of PpAly7A and its salt bond mutant at 50℃, and WT represents the alginate lyase PpAly7A.

[0018] Figure 5 This study tested the substrate preference of alginate lyase and mutants, where 7A is the alginate lyase PpAly7A.

[0019] Figure 6 The degradation of biofilms by alginate lyase and mutants is represented by 7A, which is alginate lyase PpAly7A. Detailed Implementation

[0020] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0021] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0022] Unless otherwise specified, all experimental materials used in the embodiments of this invention are conventional experimental materials in the art and can be purchased through commercial channels. Enzymes and reagents: Kanamycin and isopropyl-β-D-thiogalactoside (IPTG) were purchased from Beijing Solarbio Science & Technology Co., Ltd. Protein molecular weight standard markers, high-fidelity Taq enzyme, plasmid extraction kit, and restriction endonuclease DpnI were purchased from Nanjing Novizan Biotechnology Co., Ltd. Electrophoresis gel preparation kits were purchased from Shaanxi Zhonghui Hecai Biomedical Technology Co., Ltd. Imidazole was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and protein concentration assay kits were purchased from Shanghai Sangon Biotech Co., Ltd. Alginate was purchased from Qingdao Juyuan Algae Industry Group Co., Ltd., and polyM and polyG blocks (purity > 97%) were purchased from Qingdao Juyuan Algae Industry Group Co., Ltd. Strains, plasmids, and culture media: Luria-Bertani (LB) medium consisted of 10 g / L Tryptone, 5 g / L Yeast exact (purchased from Oxoid, USA), and 10 g / L NaCl (purchased from Sinopharm Chemical Reagent Co., Ltd.). When preparing solid medium, 13-16 g of agar powder (purchased from Beijing Solarbio Science & Technology Co., Ltd.) was added to each 1 L of LB medium. *E. coli* BL21(DE3) competent cells and *E. coli* DH5α competent cells were ordered from Nanjing Novizan Biotechnology Co., Ltd. The synthesis of the optimized sequence and the construction of the recombinant plasmid pET-24a(+)-Aly5A were outsourced to Tianyi Huayu Biotechnology Co., Ltd.

[0023] Example 1: Construction of recombinant expression vector 1. Construction of the recombinant expression vector for alginate lyase PpAly7A A strain of *Pseudomonas aeruginosa* encoding the alginate lyase gene PpAly7A was screened from samples collected from the Weifang Tamarix and Cistanche deserticola demonstration base. Pseudomonas plecoglossicida The PpAly7A gene sequence (code: AXM95248.1) of alginate lyase was downloaded from the NCBI nucleotide database, codon optimized, and synthesized. The PpAly7A gene consists of an open gene reading frame of 681 bp, encoding a 227-amino acid protein, namely the alginate lyase PpAly7A.

[0024] The optimized nucleotide sequence of the alginate lyase gene PpAly7A (SEQ ID NO: 1): ATGACCGTTAACATTAACAACCTGACCATTACCACCCCGGTGCCGACCAGCCCGACCAACCCAGTGGCCCTGGAACTGACCGGTGCCGAAGCCATTGCCCAGCTGCCGGAAGTTGTGAAAGTGCTGAGCGACGGCAGCATTCGCTTCTCAGCCCCTACCAAAGGCGCAAGCAGCAAAAGCACCCATCGTACCCGTTGCGAATGGAAAGAACCGGTTTACTGGAGCCTGGCAAGCGCAGATGAACACATAAATCTGCAGGAAATGACCCTGACCAAAGTGAATAGTGCACAAAAAGTGGTTATCAGCCAGCTGCATGTGAAGGATGATGACAGTCCGCCGGTTAAAGTTTTTTGGAGCAAAGGTAATATCACCTTAGGATTCCGCTCTACCTTCAATCAGGCGAGTCCCACCAATACCACCCTGTTAAAAGGTGTTCCATTAGGAGCAAAATTTAAAGTTACCATTCGCGCGTTAGCAAGTGGCGCCCTGACCGTTACCGCAGAATGTAATGGCCACGCAGGTTCAAGCGGCCGCCTGGAAATGGATAGCAGTTGGCGGAGCAGTCTGCTGAATTTTCATGGTGGTGTTTATAATCAAATCGATTATAGTGACTCGACCCCGGCAGAAGATGGCAGTGTTTGTATTATTAGCAAACTGACGCTGACCCATAGTGACAGCAAT Amino acid sequence of alginate lyase PpAly7A (SEQ ID NO: 2): MTVNINNLTITTPVPTSPTNPVALELTGAEAIAQLPEVVKVLSDGSIRFSAPTKGASSKSTHRTRCEWKEPVYWSLASADEHINLQEMTLTKVNSAQKVVISQLHVKDDDSPPVKVFWSKGNITLGFRSTFNQASPTNTTLLKGVPLGAKFKVTIRALASGALTVTAECNGHAGSSGRLEMDSSWRSSLLNFHGGVYNQIDYSDSTPAEDGSVCIISKLTLTHSDSN The gene was cloned into the pET-24a expression vector and transformed into *E. coli* DH5α competent cells. The transformation process was as follows: *E. coli* DH5α competent cells were removed from the -80℃ freezer and placed on ice for 20 minutes. 10 μL of PCR product was added, and the cells were placed on ice for 30 minutes. A heat shock at 42℃ for 90 seconds was performed, followed by another 3 minutes on ice. Then, 900 μL of LB medium was added, and the cells were incubated at 37℃ and 180 rpm for 1 hour. After 1 hour of incubation, the bacterial culture was centrifuged at 3000 rpm for 5 minutes, and 900 μL of supernatant was discarded. The remaining supernatant was resuspended with the bacterial culture and evenly spread on LB agar plates. After incubation at 37℃ overnight, positive clones were picked for DNA sequencing. The correctly sequenced recombinant expression plasmid was named pET24a(+)-PpAly7A, and the plasmid was extracted using a plasmid extraction kit (Novozymes). The recombinant expression plasmid was transformed into the expression strain *E. coli* BL21, and the transformants were spread on LB agar plates and incubated overnight at 37℃. A recombinant expression vector for alginate lyase PpAly7A was obtained.

[0025] 2. Construction of mutants The present invention constructs six salt bond mutants, including E25Q, E37Q, E81Q, E180Q, E209Q, and D201N.

[0026] The Visual Molecular Dynamics (VMD) website predicted that PpAly7A has several potential salt bonds: Arg63-Asp201, GLU37-ARG48, GLU67-HIS193, GLU180-ARG178, GLU209-LYS54, GLU67-ARG65, GLU81-ARG156, and GLU25-LYS69. In order to disrupt the salt bonds, the glutamic acid Glu (E) and aspartic acid Asp (D) residues in the salt bonds were replaced with glutamine Gln (Q) and asparagine Asn (N) residues, respectively, to construct 6 salt bond mutants: E25Q, E37Q, E81Q, E180Q, E209Q, and D201N.

[0027] 3. Construction of mutant recombinant expression vector First, the mutant E25Q expression vector was constructed (using mutant E25Q as an example, others follow the same principle). Using the constructed recombinant plasmid pET24a(+)-PpAly7A as a template, PCR amplification was performed using primers E25Q-F and E25Q-R. The PCR reaction was carried out under the following conditions: 95 ℃ pre-denaturation for 3 min; 95 ℃ denaturation for 15 s; 60 ℃ annealing for 15 s; 72 ℃ extension for 60 s, 72 ℃ extension for 5 min, for 30 cycles. The PCR reaction volume was 20 μL (Table 1). The template DNA of the PCR product was digested with 0.5 μL of DPNⅠ. The digested PCR product was transformed into *E. coli* DH5α competent cells, and the transformation process was the same as in step 1 above. Positive clones were selected for DNA sequencing. Using the expression vector pET24a(+)-E25Q corresponding to the mutant E25Q as a template, the expression vector pET24a(+)-E37Q corresponding to the salt-bonded mutant E37Q was constructed. The experimental procedure was the same as that for expression vector pET24a(+)-E25Q, except that the amplification primers were changed to E37Q-F and E37Q-R, finally obtaining the expression vector pET24a(+)-E37Q corresponding to the salt-bonded mutant E37Q. Expression vectors pET24a(+)-E81Q (mutant E81Q), pET24a(+)-E180Q (mutant E87Q), pET24a(+)-E209Q (mutant E209Q), and pET24a(+)-D201N (mutant D201N) were obtained using the same method. Recombinant expression plasmids with correct sequencing results were extracted using a plasmid extraction kit (Novozymes). The plasmid was transformed into competent E. coli BL21 cells, and the transformants were plated on LB agar plates and incubated overnight at 37°C.

[0028] Salt bond mutant primer sequences: E25Q-F:CCCAGTGGCCCTGCAGCTGACCGGTGCCG (SEQ ID NO:3) E25Q-R:CGGCACCGGTCAGCTGCAGGGCCACTGGG(SEQ ID NO:4) E37Q-F:ATTGCCCAGCTGCCGCAGGTTGTGAAAGTGCTGA(SEQ ID NO:5) E37Q-R:TCAGCACTTTCACAACCTGCGGCAGCTGGGCAAT(SEQ ID NO:6) E81Q-F:CTGGCAAGCGCAGATCAGCACATAAATCTGCAGG(SEQ ID NO:7) E81Q-R:CCTGCAGATTTATGTGCTGATCTGCGCTTGCCAG(SEQ ID NO:8) E180Q-F:AAGCGGCCGCCTGCAGATGGATAGCAGTTGGC (SEQ ID NO:9) E180Q-R:GCCAACTGCTATCCATCTGCAGGCGGCCGCTT (SEQ ID NO:10) E209Q-F:CTCGACCCCGGCACAGGATTGGCAGTGTTTGTA(SEQ ID NO:11) E209Q-R:TACAAACACTGCCATCCTGTGCCGGGGTCGAG(SEQ ID NO:12) D201N-F:GGTGTTTTATAATCAAATCAATTATAGTGACTCGACCC(SEQ ID NO:13) D201N-R:GGGTCGAGTCACTATAATTGATTTGATTATAAACACC(SEQ ID NO:14) Table 1: PCR reaction system Example 2: Fermentation, separation, and purification of alginate lyase 1. Induced fermentation by alginate lyase Recombinant *E. coli* BL(DE3) containing the vector pET24a(+)-PpAly7A and the mutant was streaked onto a solid LB agar plate containing 30 μg / mL kanamycin. Single colonies were picked and transferred to 5 mL of liquid LB agar containing 30 μg / mL kanamycin and cultured overnight at 37°C with shaking. The bacterial culture was then transferred at a ratio of 1% (v:v) to 100 mL of liquid LB agar containing 30 μg / mL kanamycin and cultured at 37°C at 180 rpm until OD was reached. 600When the concentration was 0.4–0.6, isopropyl-β-disulfite dihexyphospholactone (IPTG) was added to a final concentration of 0.1 mM, and the mixture was induced at 18°C ​​for 20 hours. The fermentation broth was collected, centrifuged at 12,000 rpm for 10 min at 4°C, the supernatant was discarded, and the precipitate was resuspended in solution A (10 mL, 20 mM PB, 300 mM NaCl, pH = 7.3). The cells were then disrupted using a high-pressure grinder. The enzyme activity of the crude enzyme solution was determined using the A235 method, and the presence or absence of enzyme activity was used to determine whether fermentation was successful. The absorbance of the reaction system at 235 nm was measured, with an enzyme solution inactivated at 100°C for 10 min used as a control. Under these conditions, an increase of 0.1 mM in absorbance per minute was defined as one unit of enzyme activity (U).

[0029] 2. Isolation and purification of alginate lyase The obtained crude enzyme was isolated and purified as follows: A HisTrap HP (GE) nickel ion column was equilibrated with equilibration buffer A (20 mM PB, 500 mM NaCl, pH=7.3) for 5-10 column volumes at a flow rate of 1.0 mL / min. The crude enzyme solution, filtered through 0.45 μm and 0.22 μm microporous membranes, was loaded onto the column, and unbound contaminating proteins were eluted with equilibration buffer A. Elution was performed with equilibration buffer B (20 mM PB, 500 mM NaCl, pH=7.3, 500 mM imidazole) at different gradients. Contaminating proteins were removed with 5% and 10% equilibration buffers, and the target protein was eluted with 50% equilibration buffer B, and enzyme activity was measured. Other proteins were eluted with 100% equilibration buffer B, the column was rinsed with water, and finally, the column was stored in 20% ethanol. The collected target protein was dialyzed overnight in an ice-water bath to remove imidazole and some NaCl. The dialysate consisted of pH 7.3, 20 mM PB, and 100 mM NaCl. After dialyzing, the target protein was stored at -20°C. 20 μL of the dialyzed protein sample was mixed with 5 μL of 5× denaturing buffer (loading buffer), boiled for 10 min, and centrifuged at 12000 rpm for 10 min at room temperature. The supernatant was collected and subjected to SDS-PAGE electrophoresis. The stacking gel voltage was 80 V, and the separating gel voltage was 120 V. The electrophoretic gel was stained with Coomassie Brilliant Blue solution with shaking for 4 h. After destaining with destaining solution (methanol:acetic acid:water = 3:1:6), the gel was photographed and analyzed. The SDS-PAGE results are shown below. Figures 1-2 .

[0030] according to Figures 1-2 As a result, the molecular weight of the alginate lyase PpAly7A was approximately 25 kDa, consistent with the predicted molecular weight. Figure 1 The molecular weights of the salt bond mutants E25Q, E37Q, E81Q, E180Q, E209Q, and D201N are also approximately 25 kDa. Figure 2 ).

[0031] Example 3: Effect of temperature on alginate lyase 100 μL of purified PpAly7A alginate lyase of appropriate concentration was added to 900 μL of 0.2% alginate substrate (20 mM PB buffer, pH=6.6). The mixture was reacted for 10 min at different temperatures (0, 10, 20, 30, 40, 50, 60, 70 ℃). The A235 value was measured using a UV spectrophotometer. The highest enzyme activity was taken as 100%, and the relative enzyme activity of PpAly7A at different temperatures was calculated.

[0032] Purified PpAly7A alginate lyase at appropriate concentrations was incubated in water at different temperatures (0, 10, 20, 30, 40, 50, 60, 70 °C) for 1 h, followed immediately by an ice bath for 5 min. 100 μL of the enzyme solution was then mixed with 900 μL of substrate and reacted at 20 °C for 10 min. The A235 value was measured using a spectrophotometer. The remaining relative enzyme activity of PpAly7A after incubation at different temperatures was calculated, with the highest enzyme activity defined as 100%. The temperature stability of purified PpAly7A alginate lyase at appropriate concentrations was determined by incubating at 30, 40, and 50 °C for 0, 1, 2, 3, 4, 5, 6, and 7 days, following the same method.

[0033] To determine the half-life of the mutant at 50°C, appropriate concentrations of purified mutant enzyme were incubated at 50°C for 0, 1, 2, 3, 4, 5, 6, and 7 days, followed immediately by an ice bath for 5 min. 100 μL of the enzyme solution was then mixed with 900 μL of substrate and reacted at 20°C for 10 min. The A235 value was measured using a spectrophotometer. The highest enzyme activity was defined as 100%, and the remaining relative enzyme activity of each mutant after incubation for different times was calculated. The enzyme activity measured from the enzyme solution without any heat treatment was considered 100%.

[0034] 1. Effect of temperature on alginate lyase PpAly7A The optimal reaction temperature for PpAly7A is 20 °C, and it exhibits high activity within the 0-50 °C range. This indicates that PpAly7A possesses high catalytic activity at room temperature. Figure 3 (A). This enzyme retained 50% activity after incubation at 50℃ for 4.39 days, indicating that PpAly7A has good thermostability. Figure 3 (B)

[0035] 2. The effect of salt bonds on the thermal stability of PpAly7A See results Figure 4The half-lives of E25Q, E37Q, E81Q, E209Q, and D201N at 50°C were significantly reduced compared to the wild-type enzyme PpAly7 (4.39 d), decreasing to 0.2043 d, 2.371 d, 1.357 d, 2.587 d, and 0.789 d, respectively. E25Q lost all activity after 3 days of incubation at 50°C. The mutation at site 180 (E180Q) improved the thermostability of PpAly7A, increasing its half-life at 50°C to 7.246 d compared to the wild-type enzyme, making it a potential new target for further rational design.

[0036] Example 5: Degradation of biofilms by alginate lyase PpAly7A and its mutant 1.7A and substrate preference of mutants The substrate preference of 7A and its mutant was determined. 0.2% (w:v) alginate, poly M, and poly G were dissolved in 20 mM PB at pH 6.6 with 300 mM NaCl added. The enzyme activities of alginate lyase Pp7A and the thermostable mutant E180Q were measured at 20°C. The relative enzyme activities to other substrates were calculated, with the highest enzyme activity defined as 100%. Alginate, the main component of biofilms produced by *Pseudomonas aeruginosa*, is predominantly of the M configuration. 7A and its mutant preferentially degrade alginate. Determining the substrate preference of 7A and the mutant E180Q determined their degradation ability for poly M, providing a preliminary assessment of their biofilm degradation capabilities. Figure 5 The figure shows that E180Q has a preference for poly M up to 32.07%.

[0037] 2. Construction of Pseudomonas aeruginosa biofilm Pseudomonas aeruginosa PAO1 was inoculated onto LB agar plates and incubated upside down at 37 °C for 24 h. A single colony from the plate was picked and inoculated into a test tube containing 5 mL of LB broth, and incubated with shaking at 37 °C (180 rpm) for 12 h. The next day, the bacterial culture was transferred to culture medium at 1% (v / v) and incubated with shaking at 37 °C (180 rpm) until OD. 600 =0.5, then diluted with LB liquid culture medium to OD. 600 =0.05. The 96-well plates were labeled with experimental groups, and 100 μL of diluted bacterial culture was added to each well. 200 μL of culture medium was added to each well around the perimeter of the plate, and the plates were incubated at 37°C for approximately 24 h. The next day, the 96-well plates were removed and opened. 100 μL of solvent (20 mM PB) was added to the control group, and 100 μL (100 U / mL) of alginate lyase PpAly7A and the mutant were added to the experimental groups. The plates were then incubated at 37°C for 2 h.

[0038] 3. Determination of biofilm content After culturing, aspirate the bacterial culture from each well using a pipette. Add 150 μL of sterile physiological saline (0.9%, w / v) to each well and aspirate again. Repeat this process once to wash away any residual bacterial culture. Finally, aspirate the liquid from the well. Add 150 μL of methanol to the well for fixation for 15 min, then aspirate. Allow to air dry for 30 min, then add 150 μL of 0.02% (w / v) crystal violet solution. Incubate for staining for 30 min, then aspirate the crystal violet. Wash twice with physiological saline using the same method. After aspirating the liquid from the well, add 200 μL of 33% (v / v) glacial acetic acid solution to dissolve the crystal violet. Finally, measure the absorbance of each well at 590 nm using a microplate reader.

[0039] The biofilm content is calculated based on the absorbance in each well; the higher the absorbance, the higher the biofilm content. After removing the biofilm, the removal rate can be calculated by the amount remaining in the well. Biofilm removal rate (%) = (OD control group - OD experimental group) / OD control group × 100%.

[0040] Based on the aforementioned experiments, the mutant E180Q, which exhibits enhanced thermal stability compared to the wild-type enzyme, was selected and tested alongside the wild-type enzyme PpAly7A to determine its biofilm degradation capabilities, and the enzyme's biofilm scavenging effect was calculated. Results are shown below. Figure 6 The wild-type enzyme PpAly7A achieved a biofilm clearance rate of 24.86%, while the mutant E180Q showed a significant clearance effect on Pseudomonas aeruginosa biofilm, with a clearance rate of up to 64.0%, which is better than that of the wild-type enzyme PpAly7A.

[0041] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A mutant alginate lyase, characterized in that, The alginate lyase mutant described above has a mutation site of E180Q compared to alginate lyase PpAly7A. The amino acid sequence of alginate lyase PpAly7A is shown in SEQ ID NO:

2.

2. The gene encoding the alginate lyase mutant of claim 1.

3. A recombinant expression vector carrying the gene of claim 2.

4. The recombinant expression vector according to claim 3, characterized in that, pET series plasmids were used as expression vectors.

5. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria contain the gene described in claim 2 or the recombinant expression vector described in claim 3 or 4.

6. The genetically engineered bacterium according to claim 5, characterized in that, BL21 is used as the expression host.

7. The application of the alginate lyase mutant of claim 1, the gene of claim 2, the recombinant expression vector of claim 3 or 4, or the genetically engineered bacteria of claim 5 or 6 in the degradation of Pseudomonas aeruginosa biofilms.

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