A method for engineering salt adaptability of pl7 family alginate lyase

Abstract

The application discloses a method for improving salt adaptability of PL7 family alginate lyase. First, surface amino acid, enzyme catalytic site and conservative site are analyzed, then mutation sites are selected according to the analysis, so that positively charged amino acids in the target wild type PL7 family alginate lyase are mutated into negatively charged amino acids or non-charged amino acids; then mutation schemes of each mutation site are selected according to the analysis; if the conservative site analysis result shows that the mutation site exists negatively charged amino acid on other PL7 family alginate lyases, the mutation site is preferentially mutated into glutamic acid or aspartic acid with negative charge; if the site does not exist negatively charged amino acid on other PL7 family alginate lyases, the mutation site is mutated into negatively charged amino acid, and the mutation is preferentially performed into more appearing amino acid. Finally, the salt adaptability is regulated, and the alginate lyase suitable for low salt catalytic environment is obtained.

Description

Technical Field

[0001] This invention relates to the field of bioinformatics, and more particularly to a method for modifying the salt adaptability of PL7 family alginate lyase. Background Technology

[0002] Alginate is an important component of the cell walls of large brown algae such as kelp. Alginate lyase can break the β-1,4 glycosidic bonds of alginate polysaccharides through elimination reactions, which can effectively degumm and reduce the viscosity of brown algae, and produce a highly active seaweed extract rich in alginate oligosaccharides, which can be used as an animal feed additive or plant organic fertilizer.

[0003] Compared to physical and chemical degradation methods, enzymatic hydrolysis offers advantages such as mild reaction conditions, easy process control, high substrate specificity, high yield, and energy efficiency. Therefore, enzymatic hydrolysis, as a representative of biodegradation, will inevitably gradually replace traditional chemical degradation and gain a dominant position in future commercial production. Alginate lyases can break the β-1,4 glycosidic bonds of alginate polysaccharides through elimination reactions, forming alginic oligosaccharides with double bonds and various biological activities. Alginate lyases belong to the polysaccharide lyase (PL) family, mainly including families 5, 6, 7, 14, 15, 17, and 18, with the PL7 family being the most frequently reported.

[0004] Alginate lyase has great industrial potential, but high-salt environmental conditions limit its large-scale application. The salt adaptability of alginate lyase from different sources varies considerably, and there is a lack of systematic research on the rational modification of its salt adaptability. The development process is characterized by significant randomness and uncertainty, leading to substantial production costs for practical industrial applications and hindering the further development of alginate lyase in the brown algae processing industry. Summary of the Invention

[0005] The purpose of this invention is to provide a method for modifying the salt adaptability of PL7 family alginate lyases. This allows for the regulation of their salt adaptability, resulting in alginate lyases suitable for low-salt catalytic environments.

[0006] To achieve the above objective, a method for modifying the salt adaptability of PL7 family alginate lyases is characterized by comprising the following steps:

[0007] S1. Analysis prior to mutation design:

[0008] (1) Analysis of surface amino acids: The solvent accessibility surface area (SASA) value of each amino acid of wild-type PL7 family alginate lyase was calculated using VMD software. Then, the SASA value was divided by the theoretical maximum solvent accessibility area of ​​each amino acid to obtain the exposure ratio. If the exposure ratio is greater than 25%, the amino acid is considered to be a surface amino acid of the enzyme; otherwise, it is not a surface amino acid.

[0009] (2) Analysis of enzyme catalytic sites: Swiss-model and I-Tasser tools were used to search for templates of wild-type PL7 family alginate lyases to obtain information on PL7 family alginate lyases with similar structures and existing crystal structures. Catalytic site information of reference crystal structures was obtained through literature review. Pymol was used to superimpose and analyze the wild-type structure and the reference crystal structure to obtain catalytic site information of wild-type PL7 family alginate lyases.

[0010] (3) Analysis of conserved sites: BLAST was performed on the UniProt website using the amino acid sequence of the target PL7 family alginate lyase. 40-100 PL7 family alginate lyase sequences with similar length and 70-100% consistency were selected from the search results. Multiple sequence alignment was performed using the COBALT tool in the NCBI database. The degree of conservation of amino acids was determined based on the results.

[0011] S2. Based on the analysis prior to mutation design in S1, select mutation sites to mutate positively charged amino acids in the target wild-type PL7 family alginate lyases into negatively charged or uncharged amino acids: All positively charged amino acids in the wild-type PL7 family alginate lyases are the initial potential mutation targets. Inappropriate mutation points are eliminated one by one according to the following four principles to finally obtain rationally designed mutation sites:

[0012] (1) Select sites located on the protein surface, exclude sites inside the protein, and select surface amino acids that are in close contact with salt ions;

[0013] (2) Select sites far from the active site and exclude sites located at or near the active site to avoid damaging enzyme activity;

[0014] (3) Select non-conserved sites of enzymes and exclude highly conserved sites to avoid damaging the original function. At the same time, exclude highly conserved positively charged amino acid sites.

[0015] (4) Select sites that do not form salt bridges, exclude sites with weak interactions, and maintain the original hydrogen bonds, salt bridges and disulfide bonds.

[0016] S3. Based on the mutation sites obtained above, and combined with the degree of conservation of amino acids obtained in (3) of S1, select the mutation scheme for each mutation site: if the results of the conservation site analysis show that the mutation site has negatively charged amino acids on other PL7 family alginate lyases, then mutate it to glutamic acid or aspartic acid, which are negatively charged amino acids, preferentially; if the site of other PL7 family alginate lyases does not have negatively charged amino acids, then mutate it to glycine or alanine or valine or leucine or isoleucine or methionine or proline or tryptophan or serine or tyrosine or cysteine ​​or phenylalanine or asparagine or glutamine or threonine, preferentially mutating it to the more common amino acids.

[0017] The result is the mutated gene of the PL7 family alginate lyase.

[0018] The present invention also provides a PL7 family alginate lyase gene sequence with low salt adaptability, characterized in that it is a protein sequence obtained by mutation according to the above method.

[0019] The present invention also provides a recombinant plasmid of PL7 family alginate lyase with low salt adaptability, characterized in that it contains the gene sequence corresponding to the protein sequence described above.

[0020] Regarding highly conserved sites, if the consistency of a site is above 90%, then the site can be considered a highly conserved site.

[0021] Regarding the selection of sites far from the active site described in this invention, the distance between the site and the active site can be observed from the three-dimensional structure of the protein. Sites located at or near the active site (e.g., sites within 6 angstroms of the active site) are excluded to avoid damaging enzyme activity.

[0022] This invention modifies the existing alginate lyase Aly1 using a rational design method to regulate its salt adaptability, obtaining an alginate lyase suitable for low-salt catalytic environments, and simultaneously verifying this correlation. This provides a theoretical basis for the development and rational design of alginate lyases with specific salt adaptability, which can reduce the input cost of alginate lyase in practical industrial applications to a certain extent, promote the development of the deep processing industry of brown algae products, and broaden the application scenarios of alginate lyase. Attached Figure Description

[0023] Figure 1 : A schematic diagram showing the location of the 62 mutation sites selected in the first round of screening in the Aly1 structure.

[0024] Figure 2 The image shows the results of multiple sequence alignment using the Cobalt website on 67 sequences with a similarity of 70% to 100%.

[0025] Figure 3 Schematic diagram of the catalytic active sites of Aly1.

[0026] Figure 4 : Schematic diagram of the salt bridge that appears in Aly1 during the 10ns MD process.

[0027] Figure 5 Schematic diagram of the location of mutation sites K8D, K9D, K11E, R116Y, K157D, K184E, K189E, K170D, and K273E in the three-dimensional structure of the nine mutant enzyme M9A.

[0028] Figure 6 Schematic diagram of the location of mutation sites K157D and K184E in the three-dimensional structure of the second mutant enzyme M2A.

[0029] Figure 7 Schematic diagram of the location of mutation sites K8D and K184E in the three-dimensional structure of the second mutant enzyme M2B.

[0030] Figure 8 Schematic diagram of the location of mutation sites K9D and K157D in the three-dimensional structure of the M2C double mutant enzyme.

[0031] Figure 9 Schematic diagram of the location of mutation sites R116Y and K273E in the three-dimensional structure of the second mutant enzyme M2D.

[0032] Figure 10 Schematic diagram of the location of mutation sites R116Y and K170D in the three-dimensional structure of the second mutant enzyme M2E.

[0033] Figure 11 Schematic diagram of the location of mutation sites K170D and K273E in the three-dimensional structure of the second mutant enzyme M2F.

[0034] Figure 12 Schematic diagram of the location of mutation sites K11E, K157D, and K184E in the three-dimensional structure of the triple mutant enzyme M3A.

[0035] Figure 13 Schematic diagram of the location of mutation sites K157D, K184E, and K189E in the three-dimensional structure of the triple mutant enzyme M3B.

[0036] Figure 14 Schematic diagram of the location of mutation sites R116Y, K170D, and K273E in the three-dimensional structure of the triple mutant enzyme M3C.

[0037] Figure 15 Schematic diagram of the location of mutation sites K8D, K157D, K184E, K189E, and K170D in the three-dimensional structure of the five-mutant enzyme M5A. Detailed Implementation

[0038] The embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially. In the following embodiments, unless otherwise explicitly stated, "%" refers to weight percentage.

[0039] Example 1: Selection of mutation sites

[0040] Starting with the existing alginate lyase Aly1 in the laboratory, a mutant enzyme with reduced positively charged amino acids was rationally designed. The overall rational design process includes the following steps:

[0041] (1) The first round of screening involved positively charged amino acid analysis. Using PyMOL software or directly from the structure, all positively charged amino acids (H, K, R) of Aly1 were selected. Potential mutation sites required a series of screenings to ensure that the mutated enzyme protein maintained both structural stability and catalytic activity. After this round of analysis, 62 potential mutation sites were identified, such as... Figure 1 (Potential mutation sites are marked in red).

[0042] (2) The second round of screening involved amino acid conservation analysis, mainly including sequence collection, multiple sequence alignment, and conservation assessment. Conserved amino acids with mutation sites were excluded. Aly1 sequences (as shown in SEQ ID NO:1) were retrieved using NCBI and protein BLAST was performed. 67 sequences with similarity of 70%–100% were selected and subjected to multiple sequence alignment using the Cobalt website. The alignment results were viewed in Discovery Studio. Figure 2 Through multiple sequence comparison, the study focused on 62 basic amino acids, comparing the amino acid types of similar sequences at the basic amino acid positions of Aly1. If a position showed a relatively uniform amino acid type, it was considered a conserved site of the enzyme protein and should be avoided during mutation. The most frequent amino acid at each of the 62 non-conserved basic amino acid sites was then identified, and the corresponding basic amino acid at that site was mutated to the most frequent amino acid. This analysis eliminated 45 sites and identified 17 potential mutation sites.

[0043] (3) The third round of screening involved active site analysis. The catalytic active sites of alginate lyase Aly1 include Q141, H143, and Y257; mutations should avoid these active site regions as much as possible. This round of analysis (using software, such as...) Figure 3 (Confirmed) It was found that 17 potential mutation sites were all far away from the above-mentioned catalytic sites, which to some extent indicates that the active site region has good conservation. Therefore, there is no need to remove mutation sites in this round of screening.

[0044] (4) The fourth round of screening involved analyzing existing salt bridges based on molecular dynamics simulations, such as... Figure 4 By loading Aly1's 10ns molecular dynamics simulation data into VMD and calling the SaltBridges plugin of VMD software, information on salt bridges and their amino acid composition appearing in Aly1 during the 10ns MD process was obtained (e.g., the method for obtaining salt bridges: performing a 10ns simulation of the protein using molecular dynamics simulation software (NAMD, Gromacs, etc.) and then analyzing the results using the program's functions). Of the 17 potential mutation sites, 8 amino acids were involved in salt bridge formation; after discarding these, 9 potential mutation sites were finally identified, such as... Figure 5 .

[0045] Example 2: Construction and Salt Adaptation Characterization of a Two-Mutant Enzyme

[0046] (1) Mutation Site Combination. Amino acids that are far apart in the primary sequence of a protein may be close to each other in the three-dimensional structure, and neighboring amino acids may interact in the spatial structure. To avoid disrupting the interactions between amino acids and thus maintain enzyme stability, the mutation sites should be dispersed as much as possible when combining mutation sites. Based on the principle of mutation site dispersion, a mutation site combination scheme for two mutations was formulated for the selected 9 mutation sites. The positions of the mutation sites in the three-dimensional structure of the two-mutant enzyme are shown in the figure below. Figure 6-11 .

[0047] Table 1. Mutation sites of two mutant enzymes

[0048]

[0049] (2) Genome composition. Based on the designed mutant enzyme genome, codon optimization was performed (the optimized sequence is shown in SEQ ID NO:2), and the genome was synthesized and cloned by Shanghai Jereh Biotechnology Co., Ltd. The plasmid used was pET-28(a)+, the restriction enzyme sites were NdeI and XhoI, and the expression host was BL21(DE3).

[0050] (3) Expression and purification of the second mutant enzyme. The strain stored at -20℃ was taken out of the refrigerator and transferred to LB medium at an inoculation rate of 1%. It was placed in a shaker at 25℃ and 180 rpm for 20 h. An appropriate amount of bacterial cells was streaked onto a plate. After 24 h, single colonies with good growth were selected and inoculated into LB liquid medium for bacterial activation. Then, it was transferred to 200 mL of LB liquid medium containing 100 μg / mL kanamycin and incubated in a shaker at 37℃ and 180 rpm for about 3 h. When the OD600 reached 0.6-0.8, IPTG inducer was added to a final concentration of 0.05 mmol / L. It was then placed in a shaker at 16℃ and 180 rpm for 20 h. After centrifugation, the supernatant was removed, and the collected bacterial precipitate was resuspended in buffer and sonicated.

[0051] Purify the target protein according to the instructions of Seven Seas' Ni-NTA his·Bind Resin pre-packed column. The crude enzyme solution obtained by ultrasonic disruption was centrifuged and filtered through a 0.22 μm filter. The Tris-HCl buffer used for purification was also filtered for sterilization. The purification column was equilibrated with 10 column volumes of buffer 4. The crude enzyme solution was then loaded. After loading, the column was washed with 10 column volumes of loading buffer to remove any residual sample from the column walls. 10-20 column volumes of washing buffer were used to remove contaminating proteins from the purification column. After washing, the target protein was eluted with 4-10 column volumes of elution buffer. After collection, the column was washed with 8 M urea, followed by a large volume of distilled water, and then sealed for storage. Finally, the active fraction was dialyzed overnight, then concentrated by ultrafiltration and analyzed by SDS-PAGE electrophoresis to determine the enzyme purity and molecular weight.

[0052] (4) Characterization of salt adaptability of the double mutant enzyme. After ultrafiltration concentration and buffer replacement, the purified double mutant enzyme was used to measure enzyme activity at salt concentration gradients of 0 mM, 200 mM, 400 mM, 600 mM, 800 mM, and 1000 mM to study its salt adaptability. Each enzyme was reacted in a 500 μl reaction system with sodium alginate as substrate at 35 °C for 10 min. After DNS color development, OD540 was measured. The measured OD540 was substituted into the glucose concentration-absorbance standard curve to calculate the reducing sugar concentration. The relative enzyme activity at different salt concentrations was calculated using the highest concentration of reducing sugar as the baseline (enzyme activity 100%).

[0053] Table 2. Enzyme activity results for salt-adapted double mutant enzymes.

[0054]

[0055] The results of salt adaptability characterization show that the wild-type Aly1 exhibits maximum enzyme activity at a salt concentration of 0.6 M. The two mutants show significantly increased enzyme activity at lower salt concentrations (0-0.4 M) compared to the wild-type, with relative enzyme activity exceeding 80% at 0.4 M. M2E shows maximum enzyme activity at 0.4 M, and M2F's relative enzyme activity at 0.4 M is also close to 100%. This demonstrates that mutating positively charged (basic) amino acids to negatively charged (acidic) amino acids enhances the salt adaptability of alginate lyase Aly1 at low salt concentrations.

[0056] Example 3: Construction and Salt Adaptation Characterization of Triple Mutant Enzyme

[0057] (1) Mutation Site Combination. Amino acids that are far apart in the primary sequence of a protein may be close to each other in the three-dimensional structure, and neighboring amino acids may interact in the spatial structure. To avoid disrupting the interactions between amino acids and thus maintain enzyme stability, the mutation sites should be dispersed as much as possible when combining mutation sites. Based on the principle of mutation site dispersion, a three-mutation combination scheme was formulated for the nine selected mutation sites. The positions of the mutation sites in the three-mutant enzyme in the three-dimensional structure are shown in the figure below. Figure 12-14 .

[0058] Table 3. Mutation sites of triple mutant enzymes

[0059]

[0060] (2) Genome composition. Based on the designed mutant enzyme genome, codon optimization was performed online, and the genome was synthesized and cloned by Shanghai Jereh Biotechnology Co., Ltd. The plasmid used was pET-28(a)+, with NdeI and XhoI restriction sites, and the expression host was BL21(DE3).

[0061] (3) Expression and purification of the triple mutant enzyme. The strain stored at -20℃ was removed from the refrigerator and transferred to LB medium at a 1% inoculum. The medium was then shaken at 25℃ and 180 rpm for 20 h on a shaker. A suitable amount of bacterial cells was streaked onto a plate. After 24 h, single colonies with good growth were selected and inoculated into LB liquid medium for bacterial activation. The culture was then transferred to 200 mL of LB liquid medium containing 100 μg / mL kanamycin and incubated at 37℃ and 180 rpm for approximately 3 h on a shaker. When the OD600 reached 0.6-0.8, IPTG inducer was added to a final concentration of 0.05 mmol / L. The culture was then incubated again at 16℃ and 180 rpm for 20 h on a shaker. After centrifugation, the supernatant was removed, and the collected bacterial pellet was resuspended in buffer and sonicated.

[0062] The target protein was purified according to the instructions of Seven Seas' Ni-NTA his·Bind Resin pre-packed column. The crude enzyme solution obtained by ultrasonic disruption was centrifuged and filtered through a 0.22 μm filter. The Tris-HCl buffer used for purification was also filtered for sterilization. The purification column was equilibrated with 10 column volumes of buffer 4. The crude enzyme solution was then loaded. After loading, the column was washed with 10 column volumes of loading buffer to remove any residual sample from the column walls. 10-20 column volumes of washing buffer were used to remove contaminating proteins from the purification column. After washing, the target protein was eluted with 4-10 column volumes of elution buffer. After collection, the column was washed with 8 M urea, followed by a large volume of distilled water, and then sealed for storage. Finally, the active fraction was dialyzed overnight, then concentrated by ultrafiltration and analyzed by SDS-PAGE electrophoresis to determine the enzyme purity and molecular weight. The purification effect was good, and the molecular weight of the electrophoretic bands matched that of the target protein.

[0063] (4) Salt adaptability characterization of triple mutant enzymes. After ultrafiltration concentration and buffer replacement of the purified enzymes, enzyme activity was measured at salt concentration gradients of 0 mM, 200 mM, 400 mM, 600 mM, 800 mM, and 1000 mM. Each enzyme was reacted in a 500 μl reaction system with sodium alginate as substrate at 35 °C for 10 min, and OD540 was measured after DNS color development. The measured OD540 was substituted into the glucose concentration-absorbance standard curve to calculate the reducing sugar concentration. The relative enzyme activity at different salt concentrations was calculated using the highest concentration of reducing sugar as the baseline (enzyme activity 100%).

[0064] Table 4. Enzyme activity results for salt-adapted triple mutant enzymes.

[0065]

[0066]

[0067] The salt adaptability characterization results show that the wild-type Aly1 exhibits maximum enzyme activity at a salt concentration of 0.6 M. The triple mutants show significantly increased enzyme activity at lower salt concentrations (0-0.4 M) compared to the wild-type, with M3A and M3C showing nearly 100% relative activity at 0.2 M. This demonstrates that mutating positively charged (basic) amino acids to negatively charged (acidic) amino acids enhances the salt adaptability of alginate lyase Aly1 at low salt concentrations.

[0068] Example 4: Construction and Salt Adaptation Characterization of Penta-Mutase and Nine-Mutase

[0069] (1) Mutation Site Combination. Amino acids that are far apart in the primary sequence of a protein may be close to each other in the three-dimensional structure, and neighboring amino acids may interact in the spatial structure. To avoid disrupting the interactions between amino acids and thus maintain enzyme stability, the mutation sites should be dispersed as much as possible during mutation site combination. Based on the principle of mutation site dispersion, five-mutation and nine-mutation combination schemes were formulated for the selected nine mutation sites. The positions of the mutation sites in the three-dimensional structure of the five-mutant and nine-mutant enzymes are shown in the figure below. Figure 15 , Figure 5 .

[0070] Table 5. Mutation sites of pentamutase and nonamutase

[0071]

[0072] (2) Genome composition. Based on the designed mutant enzyme genome, codon optimization was performed online, and the genome was synthesized and cloned by Shanghai Jereh Biotechnology Co., Ltd. The plasmid used was pET-28(a)+, with NdeI and XhoI restriction sites, and the expression host was BL21(DE3).

[0073] (3) Expression and purification of pentamutase and quinamutase. The strain stored at -20℃ was removed from the refrigerator and transferred to LB medium at a 1% inoculum. The medium was then shaken at 25℃ and 180 rpm for 20 h on a shaker. A suitable amount of bacterial cells was streaked onto a plate. After 24 h, single colonies with good growth were selected and inoculated into LB liquid medium for bacterial activation. The culture was then transferred to 200 mL of LB liquid medium containing 100 μg / mL kanamycin and incubated at 37℃ and 180 rpm for approximately 3 h on a shaker. When the OD600 reached 0.6-0.8, IPTG inducer was added to a final concentration of 0.05 mmol / L. The culture was then incubated again at 16℃ and 180 rpm for 20 h on a shaker. After centrifugation, the supernatant was removed, and the collected bacterial pellet was resuspended in buffer and sonicated.

[0074] The target protein was purified according to the instructions of Seven Seas' Ni-NTA his·Bind Resin pre-packed column. The crude enzyme solution obtained by ultrasonic disruption was centrifuged and filtered through a 0.22 μm filter. The Tris-HCl buffer used for purification was also filtered for sterilization. The purification column was equilibrated with 10 column volumes of buffer 4. The crude enzyme solution was then loaded. After loading, the column was washed with 10 column volumes of loading buffer to remove any residual sample from the column walls. 10-20 column volumes of washing buffer were used to remove contaminating proteins from the purification column. After washing, the target protein was eluted with 4-10 column volumes of elution buffer. After collection, the column was washed with 8 M urea, followed by a large volume of distilled water, and then sealed for storage. Finally, the active fraction was dialyzed overnight, then concentrated by ultrafiltration and analyzed by SDS-PAGE electrophoresis to determine the enzyme purity and molecular weight. The purification effect was good, and the molecular weight of the electrophoretic bands matched that of the target protein.

[0075] (4) Salt adaptability characterization of pentamutase and quinamutase. After ultrafiltration concentration and buffer replacement of the purified enzymes, enzyme activity was measured at salt concentration gradients of 0 mM, 200 mM, 400 mM, 600 mM, 800 mM, and 1000 mM. Each enzyme was reacted in a 500 μl reaction system with sodium alginate as substrate at 35 °C for 10 min, and OD540 was measured after DNS color development. The measured OD540 was substituted into the glucose concentration-absorbance standard curve to calculate the reducing sugar concentration. The relative enzyme activity at different salt concentrations was calculated using the highest concentration of reducing sugar as the baseline (enzyme activity 100%).

[0076] Table 6. Enzyme activity results of salt-adaptive enzymes pentamutase and quinamutase. The characterization results of salt adaptability show that the wild-type Aly1 exhibits maximum enzyme activity at a salt concentration of 0.6 M. The five- and nine-mutant variants show significantly increased enzyme activities at lower salt concentrations compared to the wild-type. M5A achieves enzyme activities of 86.5% and 90.6% at 200 mM and 400 mM salt concentrations, respectively, while M9A achieves activities of 93.7% and 100% at 200 mM and 400 mM salt concentrations, respectively. This demonstrates that mutating positively charged (basic) amino acids to negatively charged (acidic) amino acids or uncharged amino acids enhances the salt adaptability of alginate lyase Aly1 at low salt concentrations.

[0077] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A PL7 family alginate lyase Aly1 with low-salt adaptability, characterized in that, The following double, triple, pentavalent, and nine mutations are performed based on the sequence SEQ ID NO:1; The double mutations are K157D, K184E; K8D, K184E; K9D, K157D; R116Y, K273E; R116Y, K170D; K170D, K273E; three mutations become K11E, K157D, K184E; K157D, K184E, K189E; R116Y, K170D, K273E; the five mutations are K8D, K157D, K184E, K189E, K170D, and the nine mutations are K8D, K9D, K11E, R116Y, K157D, K170D, K184E, K189E, K273E.

2. A recombinant plasmid containing the gene encoding the lyase Aly1 as described in claim 1.

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