A brown alga pectin lyase mutant e87q, a coding gene, a recombinant expression vector and a genetically engineered bacterium

Abstract

The application discloses a alginate lyase mutant E87Q, a coding gene, a recombinant expression vector and a genetically engineered bacterium, and belongs to the technical field of genetic engineering. The application takes alginate lyase PpAly7A as a starting template, and a mutant with improved relative specific activity is obtained through site-directed mutagenesis. Compared with the wild enzyme PpAly7A, the mutant E87Q has a relative specific activity increased to 133.82%, and the corresponding 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 E87Q, 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 low activity, 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 E87Q, 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 E87Q 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 alginate.

[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. This invention enriches the diversity of alginate lyases, promotes the understanding of their structure and function, and provides a theoretical basis for enzyme modification. The mutant E87Q exhibits a specific activity of 259.939 U / mg, representing a 133.82% increase in specific activity compared to the wild-type enzyme PpAly7A. This site can serve as a novel target for subsequent rational design. This invention provides a theoretical basis and a clear target for the molecular modification and industrial application of alginate lyases, possessing 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 2 The image shows an SDS-PAGE electrophoresis image of salt bond mutants. The samples tested were salt bond mutants E25Q, E37Q, E81Q, E87Q, E209Q, and D201N.

[0016] Figure 3 The graph shows the specific activity results of the salt bond mutant, where WT represents the alginate lyase PpAly7A. Detailed Implementation

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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 plecoglossicidaThe 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.

[0021] The optimized nucleotide sequence of the alginate lyase gene PpAly7A (SEQ ID NO: 1): ATGACCGTTAACATTAACAACCTGACCATTACCACCCCGGTGCCGACCAGCCCGACCAACCCAGTGGCCCTGGAACTGACCGGTGCCGAAGCCATTGCCCAGCTGCCGGAAGTTGTGAAAGTGCTGAGCGACGGCAGCATTCGCTTCTCAGCCCCTACCAAAGGCGCAAG CAGCAAAAGCACCCATCGTACCCGTTGCGAATGGAAAGAACCGGTTTACTGGAGCCTGGCAAGCGCAGATGAACACATAAATCTGCAGGAAATGACCCTGACCAAAGTGAATAGTGCACAAAAAGTGGTTATCAGCCAGCTGCATGTGAAGGATGATGACAGTCCGCCGG TTAAAGTTTTTTGGAGCAAAGGTAATATCACCTTAGGATTCCGCTCTACCTTCAATCAGGCGAGTCCCACCAATACCACCCTGTTAAAAGGTGTTCCATTAGGAGCAAAATTTAAAGTTACCATTCGCGCGTTAGCAAGTGGCGCCCTGACCGTTACCGCAGAATGTAAT GGCCACGCAGGTTCAAGCGGCCGCCTGGAAATGGATAGCAGTTGGCGGAGCAGTCTGCTGAATTCATGGTGGTGTTTATAATCAAATCGATTATAGTGACTCGACCCCGGCAGAAGATGGCAGTGTTTGTATTAGCAAACTGACGCTGACCCATAGTGACAGCAAT The amino acid sequence of the alginate lyase PpAly7A (SEQ ID NO: 2): MTVNINNLTITTPVPTSPTNPVALELTGAEAIAQLPEVVKVLSDGSIRFSAPTKGASSKSTHRTRCEWKEPVYWSLASADEHINLQEMTLTKVNSAQKVVISQLHVKDDDSPP VKVFWSKGNITLGFRSTFNQASPTNTTLLKGVPLGAKFKVTIRALASGALTVTAECNGHAGSSGRLEMDSSWRSSLLNFHGGVYNQIDYSDSTPAEDGSVCIISKLTLTHSSDN 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.

[0022] 2. Construction of mutants The present invention constructs six salt bond mutants, including E25Q, E37Q, E81Q, E87Q, E209Q, and D201N.

[0023] The Visual Molecular Dynamics (VMD) website predicted that PpAly7A has multiple potential salt bonds: Arg63-Asp201, GLU37-ARG48, GLU67-HIS193, GLU87-LYS150, GLU87-LYS218, 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, E87Q, E209Q, and D201N.

[0024] 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(+)-E87Q (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.

[0025] 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) E87Q-F:AACACATAAATCTGCAGCAGATGACCCTGACCAAAG(SEQ ID NO:9) E87Q-R:CTTTGGTCAGGGTCATCTGCTGCAGATTTATGTGTT(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).

[0026] 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 .

[0027] 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, E87Q, E209Q, and D201N are also approximately 25 kDa. Figure 2 ).

[0028] Example 3: Determination of specific activity of alginate lyase Protein concentration (mg / mL) was determined using a protein concentration assay kit (Sangon Biotech). The specific activity of alginate lyase was calculated based on the purified enzyme activity (U / mL), and the relative specific activity of the mutants was statistically analyzed. The results are shown in Table 2. Additionally, the specific activity graph of the salt bond mutants is shown below. Figure 3 .

[0029] Table 2 Comparison of specific activity of PpAly7A and mutants Compared with PpAly7A (specific activity 194.2367 U / mg), the mutant E87Q has a specific activity of 259.939 U / mg, and the relative specific activity is increased to 133.82%. It can be seen that the above mutation increases the specific activity of alginate lyase.

[0030] 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 E87Q 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 alginate.

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