CN116334055A - A method for modifying the salt adaptability of PL7 family alginate lyase - Google Patents

Abstract

The invention discloses a method for improving the adaptability of alginate lyase salt of PL7 family. Firstly, analyzing surface amino acid, enzyme catalytic site and conserved site, and then selecting mutation site according to analysis so as to make the positively charged amino acid of target wild type PL7 family algin lyase be mutated into negatively charged amino acid or uncharged amino acid: the mutation scheme for each mutation site was then selected based on these analyses: if the conservation site analysis result shows that mutation sites have negatively charged amino acids on other PL7 family alginate lyase, the mutation sites are preferentially mutated into glutamic acid or aspartic acid with the negatively charged amino acids; if the site of other PL7 family algin lyase does not have negatively charged amino acid, the algin lyase is mutated to negatively charged amino acid, and preferably mutated to more occurring amino acid. Finally, the salt adaptability of the algin lyase is regulated and controlled, and the algin lyase suitable for a low-salt catalytic environment is obtained.

Description

A method for modifying the salt adaptability of PL7 family alginate lyase

technical field

The invention relates to the field of bioinformatics, in particular to a method for modifying the salt adaptability of PL7 family alginate lyase.

Background technique

Alginate is an important component of the cell wall of large brown algae such as kelp. Alginate lyase can break the β-1,4 glycosidic bond of alginate polysaccharide through elimination reaction, effectively degumming and reducing viscosity of brown algae, and produce rich alginate. Highly active seaweed extract with sugar, used as animal feed additive or plant organic fertilizer.

Compared with physical and chemical degradation methods, enzymatic hydrolysis has the advantages of mild reaction conditions, easy process control, strong substrate specificity, high yield, energy saving and environmental protection, etc. Therefore, biodegradation represented by enzymatic hydrolysis will gradually replace traditional biodegradation. chemical degradation, which will occupy an advantageous position in future commercial production. Alginate lyase can break the β-1,4 glycosidic bond of alginate polysaccharide based on the elimination reaction to form fucoidan oligosaccharides with double bonds and various biological activities. Alginate lyase belongs to the Polysaccharide Lyase (PL) family of polysaccharide degrading enzymes, mainly including the 5th, 6th, 7th, 14th, 15th, 17th and 18th families. Currently, the most reported alginate is the PL7 family Lyase.

Alginate lyase has great potential in industry, but environmental conditions such as high salinity restrict its large-scale application. The salt adaptability of alginate lyase from different sources is quite different. At present, there is a lack of systematic research on the rational transformation of its salt adaptability. The high production cost restricts the further development of alginate lyase in brown algae processing industry.

Contents of the invention

The purpose of the present invention is to provide a method for modifying the salt adaptability of PL7 family alginate lyase. In this way, the regulation of its salt adaptability can be realized, and the alginate lyase suitable for low-salt catalytic environment can be obtained.

In order to achieve the above object, a method for transforming the salt adaptability of PL7 family alginate lyase is characterized in that it comprises the following steps:

S1. Analysis before mutation design:

(1) Analysis of surface amino acids: use VMD software to calculate the solvent-accessible surface area value of each amino acid of the wild-type PL7 family alginate lyase, that is, the SASA value, and then divide the SASA value by the theoretical maximum solvent-accessible area of each amino acid Obtain the exposure ratio, if the exposure ratio is greater than 25%, the amino acid is considered to be the surface amino acid of the enzyme, otherwise it does not belong to the surface amino acid;

(2) Analysis of enzyme catalytic sites: use Swiss-model and I-Tasser tools to search for templates of wild-type PL7 family alginate lyases, and obtain information on PL7 family alginate lyases with similar structures and reported crystal structures , Obtain the catalytic site information of the reference crystal structure through literature research, superimpose and analyze the structure of the wild-type structure and the reference crystal structure through Pymol, and obtain the catalytic site information of the wild-type PL7 family alginate lyase;

(3) Analysis of conserved sites: Blast the amino acid sequence of the target PL7 family alginate lyase on the UniProt website, and select 40-100 PL7 family alginates with similar length and 70-100% identity from the search results For the lyase sequence, use the COBALT tool of the NCBI database to perform multiple sequence alignments, and know the degree of amino acid conservation according to the results;

S2. Select the mutation site according to the analysis before the mutation design in S1 to mutate the positively charged amino acid in the target wild-type PL7 family alginate lyase into a negatively charged amino acid or an uncharged amino acid: wild-type PL7 family alginate cleavage All positively charged amino acids in the enzyme are the initial potential mutation targets, and the inappropriate mutation points are eliminated one by one through the following four principles, and finally the rationally designed mutation sites are obtained:

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

(2) Select a site away from the active center, exclude sites located at or near the active center, and avoid damaging the enzyme activity;

(3) Select the non-conserved sites of the enzyme, exclude highly conserved sites, avoid destroying the original function, and exclude highly conserved positively charged amino acid sites;

(4) Select sites that do not form salt bridges, exclude related sites with weak forces, and maintain the original hydrogen bonds, salt bridges and disulfide bonds;

S3. According to the mutation sites obtained above, combined with the degree of conservation of the amino acids obtained in (3) of S1, select the mutation scheme for each mutation site: if the conservative site analysis results show that the mutation site is in other PL7 family alginate lyases If there is a negatively charged amino acid at this site, it is preferentially mutated to glutamic acid or aspartic acid, which is a negatively charged amino acid; if there is no negatively charged amino acid at this site of other PL7 family alginate lyases, it is mutated to uncharged amino acids 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 mutated to the more frequently occurring amino acid;

The obtained gene is the mutated PL7 family alginate lyase gene.

The present invention also provides a PL7 family alginate lyase gene sequence with low-salt adaptability, which is characterized in that the protein sequence is mutated according to the above method.

The present invention also provides a recombinant plasmid of PL7 family alginate lyase with low-salt adaptability, which is characterized in that it contains the gene sequence corresponding to the above-mentioned protein sequence.

With regard to highly conserved sites, it can be considered that if the identity of a certain site is above 90%, it can be determined that the site is a highly conserved site.

Regarding the selection of the site far away from the active center described in the present invention, the distance between it and the active center can be observed from the three-dimensional structure of the protein, and the sites located at or near the active center are excluded (for example, the distance from the active center is 6 sites within angstroms) to avoid damage to enzyme activity.

The present invention transforms the existing alginate lyase Aly1 through a rational design method, realizes the regulation and control of its salt adaptability, obtains the alginate lyase suitable for a low-salt catalytic environment, and verifies the correlation at the same time. It provides a theoretical basis for the development and rational design of alginate lyase with specific salt adaptability, which can reduce the input cost of alginate lyase in practical industrial application to a certain extent, promote the development of deep processing industry of brown algae products, and broaden the range of alginate cracking. Enzyme application scenarios.

Description of drawings

Figure 1: Schematic diagram of the positions of the 62 mutation sites in the first round of screening in the Aly1 structure.

Figure 2: 67 sequences with a similarity of 70% to 100% were selected for multiple sequence alignment through the Cobalt website.

Figure 3: Schematic diagram of the catalytic active site of Aly1.

Figure 4: Schematic diagram of the salt bridge that Aly1 appears during 10ns MD.

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

Figure 6: Schematic diagram of the positions of mutation sites K157D and K184E in the three-dimensional structure of the double mutant enzyme M2A.

Figure 7: Schematic diagram of the positions of mutation sites K8D and K184E in the three-dimensional structure of the double mutant enzyme M2B.

Figure 8: Schematic diagram of the positions of mutation sites K9D and K157D in the three-dimensional structure of the double mutant enzyme M2C.

Figure 9: Schematic diagram of the positions of the mutation sites R116Y and K273E in the three-dimensional structure of the double mutant enzyme M2D.

Figure 10: Schematic diagram of the positions of the mutation sites R116Y and K170D in the three-dimensional structure of the double mutant enzyme M2E.

Figure 11: Schematic diagram of the positions of mutation sites K170D and K273E in the three-dimensional structure of the double mutant enzyme M2F.

Figure 12: Schematic diagram of the positions of mutation sites K11E, K157D, and K184E in the three-dimensional structure of the triple mutant enzyme M3A.

Figure 13: Schematic diagram of the positions of mutation sites K157D, K184E, and K189E in the three-dimensional structure of the triple mutant enzyme M3B.

Figure 14: Schematic diagram of the positions of the mutation sites R116Y, K170D, and K273E in the three-dimensional structure of the triple mutant enzyme M3C.

Figure 15: Schematic diagram of the positions of mutation sites K8D, K157D, K184E, K189E, and K170D in the three-dimensional structure of the five mutant enzymes M5A.

Detailed ways

Embodiments of the present invention are described in detail below, examples of which are shown in the drawings, wherein the same or similar reference numerals designate the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by referring to the figures are exemplary and are intended to explain the present invention and should not be construed as limiting the present invention. If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products. In the following examples, if not explicitly stated, "%" refers to percentage by weight.

Embodiment 1: selection of mutation site

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

(1) The first round of screening is the analysis of positively charged amino acids. Use PyMOL software or directly select all positively charged amino acids (H, K, R) of Aly1 from the structure. Potential mutation points need to go through a series of screening, so that the mutated enzyme protein can maintain both structural stability and certain catalytic activity. active. After this round of analysis, 62 potential mutation points were screened out as shown in Figure 1 (potential mutation sites marked in red).

(2) The second round of screening is amino acid conservation analysis, which mainly includes sequence collection, multiple sequence alignment and conservation assessment. Such conservative amino acids will be removed when selecting mutation sites. Retrieve the Aly1 sequence (as shown in SEQ ID NO: 1) through NCBI and perform protein BLAST, select 67 sequences with a similarity of 70% to 100% for multiple sequence alignment through the Cobalt website, and view the comparison results in Discovery studio as follows figure 2. Through multiple sequence comparison, focus on analyzing 62 basic amino acids, and longitudinally compare the amino acid types of similar sequences at the basic amino acid positions of Aly1. If the amino acid types at a certain position are found to be relatively uniform, it is considered that this position is a conserved site of the enzyme protein, and the mutation need to avoid. The amino acids with the highest frequency in the 62 basic amino acid non-conserved positions were counted, and then the basic amino acids at the corresponding positions were mutated into the above-mentioned amino acids with the highest frequency. After this round of analysis, a total of 45 sites were eliminated, and 17 potential mutation points were screened out.

(3) The third round of screening is active site analysis. The catalytic active sites of alginate lyase Aly1 include Q141, H143 and Y257, which should be avoided as much as possible during mutation. After this round of analysis (with the help of software, for example, it can be confirmed with the help of Figure 3), it was found that 17 potential mutation points are far away from the above-mentioned catalytic site, which indicates to a certain extent that the active site region is well conserved, so there is no need to eliminate mutations in this round of screening site.

(4) The fourth round of screening is the analysis of existing salt bridges based on molecular dynamics simulation, as shown in Figure 4. Load the MD simulation data of Aly110ns through VMD, call the SaltBridges plug-in of VMD software, obtain the salt bridge and its amino acid composition information (such as the acquisition method of salt bridge: use molecular dynamics simulation software (NAMD, Gromacs, etc.) perform 10ns simulation of the protein, and use the program function analysis to obtain). Among the 17 potential mutation points, 8 amino acids were involved in the formation of salt bridges, and 9 potential mutation points were finally determined after being discarded, as shown in Figure 5.

Example 2: Construction and salt adaptability characterization of double mutant enzymes

(1) Combination of mutation points. Amino acids that are far apart in the protein primary sequence may be close to each other in the three-dimensional structure, and there may be interactions between adjacent amino acids in the spatial structure. In order to avoid destroying the interaction between amino acids and maintain the stability of the enzyme, it is necessary to combine mutation points. Spread out the mutation points as much as possible. According to the principle of dispersion of mutation points, a two-mutation mutation point combination scheme was formulated for the selected 9 mutation sites. The position of the mutation site in the three-dimensional structure of the two mutant enzymes is shown in Figure 6-11.

Table 1 Mutation site table of two mutant enzymes

(2) Whole gene composition. According to the designed mutant enzyme whole gene, after codon optimization (the optimized sequence is shown in SEQ ID NO: 2), it was synthesized and cloned by Shanghai Jierui Bioengineering Co., Ltd. The plasmid used is pET-28(a)+, NdeI and XhoI are used for restriction sites, and the expression host is BL21(DE3).

(3) Expression and purification of the double mutant enzyme. Take the strain stored at -20°C out of the refrigerator, transfer it to LB medium according to the inoculum amount of 1%, place it in a shaker at 25°C, and shake at 180r/min for 20h, take an appropriate amount of bacteria for streaking on the plate, and select after 24h A single colony with good growth was inoculated into LB liquid medium for strain activation, and then transferred to 200 mL LB liquid medium containing 100 μg/mL kalamycin, placed in a shaker at 37 ° C, 180 rpm for about 3 hours, until the OD600 reached At 0.6-0.8, add IPTG inducer to a final concentration of 0.05mmol/L, place it again in a shaker at a low temperature of 16°C, and incubate at 180rpm for 20h. Centrifuge the cultured bacterial liquid and remove the supernatant, resuspend the collected bacterial cell pellet with buffer, and perform ultrasonic disruption.

Purify the target protein according to the instruction manual of Ni-NTA his·Bind Resin prepacked column from Qihai Company. After centrifuging the crude enzyme solution prepared by ultrasonic breaking, pass through a 0.22 μm filter membrane. Pass the crude enzyme solution through a 0.22 μm filter membrane, and the Tris-HCl buffer used for purification also needs to be sterilized by filtration; first balance the purification column, load 10 times the column volume to sample 4 buffer balance; carry out loading crude enzyme solution; load the sample After completion, wash the column with loading buffer for 10 column volumes, and wash the residual sample on the side wall of the purification column; use 10-20 times the column volume of impurity buffer to wash the impurity protein from the purification column; wash After the impurity is completed, the target protein is eluted with 4-10 times the column volume of elution buffer; after the collection is completed, the column is washed with 8M urea column volume, and then washed with a large amount of distilled water, and sealed for storage. Finally, the active part was dialyzed overnight, and concentrated by ultrafiltration for SDS-PAGE electrophoresis detection to determine the purity and molecular weight of the enzyme.

(4) The characterization of the salt adaptability of the double mutant enzyme. After the purified double mutant enzyme was concentrated by ultrafiltration and the buffer was replaced, the salt concentration gradient of 0mM, 200mM, 400mM, 600mM, 800mM, and 1000mM was set to measure the enzyme activity to study the salt adaptability of the double mutant enzyme. Each enzyme was reacted at 35°C for 10 min in 500 μl reaction system with sodium alginate as substrate, and OD540 was measured after DNS color development. Substitute the measured OD540 into the glucose concentration-absorbance standard curve to calculate the reducing sugar concentration, and use the highest concentration of reducing sugar as a benchmark (enzyme activity 100%) to calculate the relative enzyme activity under different salt concentrations.

Table 2 Enzyme activity result table of two mutant enzyme salt adaptability

According to the characterization results of salt adaptability, it can be seen that the wild-type Aly1 has the maximum enzyme activity at a salt concentration of 0.6M, and the enzyme activity of the two mutants at a lower salt concentration of 0-0.4M is significantly higher than that of the wild type , the relative enzyme activity at 0.4M salt concentration has reached more than 80%, M2E has the maximum enzyme activity at 0.4M salt concentration, and the relative enzyme activity of M2F at 0.4M salt concentration is also close to 100%. It can be seen that mutating positively charged (basic) amino acids into negatively charged (acidic) amino acids can enhance the salt adaptability of alginate lyase Aly1 at low salt concentrations.

Example 3: Construction of triple mutant enzyme and characterization of salt adaptability

(1) Combination of mutation points. Amino acids that are far apart in the protein primary sequence may be close to each other in the three-dimensional structure, and there may be interactions between adjacent amino acids in the spatial structure. In order to avoid destroying the interaction between amino acids and maintain the stability of the enzyme, it is necessary to combine mutation points. Spread out the mutation points as much as possible. Based on the principle of dispersion of mutation points, a three-mutation mutation point combination scheme was formulated for the selected nine mutation sites. The positions of the mutation sites in the three-dimensional structure of the three mutant enzymes are shown in Figures 12-14.

Table 3 Mutation site table of three mutant enzymes

(2) Whole gene composition. According to the design of the whole gene of the mutant enzyme, after online codon optimization, it was synthesized and cloned by Shanghai Jierui Bioengineering Co., Ltd. The plasmid used is pET-28(a)+, NdeI and XhoI are used for restriction sites, and the expression host is BL21(DE3).

(3) Expression and purification of the triple mutant enzyme. Take the strain stored at -20°C out of the refrigerator, transfer it to LB medium according to the inoculum amount of 1%, place it in a shaker at 25°C, and shake at 180r/min for 20h, take an appropriate amount of bacteria for streaking on the plate, and select after 24h A single colony with good growth was inoculated into LB liquid medium for strain activation, and then transferred to 200 mL LB liquid medium containing 100 μg/mL kalinomycin, placed in a shaker at 37 ° C, 180 rpm for about 3 hours, until the OD600 reached At 0.6-0.8, add IPTG inducer to a final concentration of 0.05mmol/L, place it again in a shaker at a low temperature of 16°C, and incubate at 180rpm for 20h. Centrifuge the cultured bacterial liquid and remove the supernatant, resuspend the collected bacterial cell pellet with buffer, and perform ultrasonic disruption.

Purify the target protein according to the instruction manual of Ni-NTA his·Bind Resin prepacked column from Qihai Company. After centrifuging the crude enzyme solution prepared by ultrasonic breaking, pass through a 0.22 μm filter membrane. Pass the crude enzyme solution through a 0.22 μm filter membrane, and the Tris-HCl buffer used for purification also needs to be sterilized by filtration; first balance the purification column, load 10 times the column volume to sample 4 buffer balance; carry out loading crude enzyme solution; load the sample After completion, wash the column with loading buffer for 10 column volumes, and wash the residual sample on the side wall of the purification column; use 10-20 times the column volume of impurity buffer to wash the impurity protein from the purification column; wash After the impurity is completed, the target protein is eluted with 4-10 times the column volume of elution buffer; after the collection is completed, the column is washed with 8M urea column volume, and then washed with a large amount of distilled water, and sealed for storage. Finally, the active part was dialyzed overnight, and concentrated by ultrafiltration for SDS-PAGE electrophoresis detection to determine the purity and molecular weight of the enzyme. The purification effect was good, and the molecular weight of the electrophoresis band was consistent with the target protein.

(4) Salt adaptability characterization of triple mutant enzymes. After the purified enzyme was concentrated by ultrafiltration and the buffer was replaced, a salt concentration gradient of 0 mM, 200 mM, 400 mM, 600 mM, 800 mM, and 1000 mM was set to determine the enzyme activity. Each enzyme was reacted at 35°C for 10 min in 500 μl reaction system with sodium alginate as substrate, and OD540 was measured after DNS color development. Substitute the measured OD540 into the glucose concentration-absorbance standard curve to calculate the reducing sugar concentration, and use the highest concentration of reducing sugar as a benchmark (enzyme activity 100%) to calculate the relative enzyme activity under different salt concentrations.

Table 4 Enzyme activity result table of triple mutant enzyme salt adaptability

According to the characterization results of salt adaptability, it can be seen that the wild-type Aly1 has the maximum enzyme activity at a salt concentration of 0.6M, and the enzyme activity of the three mutants at a lower salt concentration of 0-0.4M is significantly higher than that of the wild type , the relative enzyme activity of M3A and M3C at 0.2M salt concentration was close to 100%. It can be seen that mutating positively charged (basic) amino acids into negatively charged (acidic) amino acids can enhance the salt adaptability of alginate lyase Aly1 at low salt concentrations.

Example 4: Construction of five mutant enzymes and nine mutant enzymes and characterization of salt adaptability

(1) Combination of mutation points. Amino acids that are far apart in the protein primary sequence may be close to each other in the three-dimensional structure, and there may be interactions between adjacent amino acids in the spatial structure. In order to avoid destroying the interaction between amino acids and maintain the stability of the enzyme, it is necessary to combine mutation points. Spread out the mutation points as much as possible. Based on the principle of dispersion of mutation points, a five-mutation and nine-mutation mutation point combination scheme was formulated for the selected nine mutation sites. The positions of the mutation sites in the three-dimensional structure of the five mutant enzymes and nine mutant enzymes are shown in Figure 15 and Figure 5 .

Table 5 Five mutant enzymes, the mutation site table of nine mutant enzymes

(2) Whole gene composition. According to the design of the whole gene of the mutant enzyme, after online codon optimization, it was synthesized and cloned by Shanghai Jierui Bioengineering Co., Ltd. The plasmid used is pET-28(a)+, NdeI and XhoI are used for restriction sites, and the expression host is BL21(DE3).

(3) Expression and purification of five mutant enzymes and nine mutant enzymes. Take the strain stored at -20°C out of the refrigerator, transfer it to LB medium according to the inoculum amount of 1%, place it in a shaker at 25°C, and shake at 180r/min for 20h, take an appropriate amount of bacteria for streaking on the plate, and select after 24h A single colony with good growth was inoculated into LB liquid medium for strain activation, and then transferred to 200 mL LB liquid medium containing 100 μg/mL kalinomycin, placed in a shaker at 37 ° C, 180 rpm for about 3 hours, until the OD600 reached At 0.6-0.8, add IPTG inducer to a final concentration of 0.05mmol/L, place it again in a shaker at a low temperature of 16°C, and incubate at 180rpm for 20h. Centrifuge the cultured bacterial liquid and remove the supernatant, resuspend the collected bacterial cell pellet with buffer, and perform ultrasonic disruption.

Purify the target protein according to the instruction manual of Ni-NTA his·Bind Resin prepacked column from Qihai Company. After centrifuging the crude enzyme solution prepared by ultrasonic breaking, pass through a 0.22 μm filter membrane. Pass the crude enzyme solution through a 0.22 μm filter membrane, and the Tris-HCl buffer used for purification also needs to be sterilized by filtration; first balance the purification column, load 10 times the column volume to sample 4 buffer balance; carry out loading crude enzyme solution; load the sample After completion, wash the column with loading buffer for 10 column volumes, and wash the residual sample on the side wall of the purification column; use 10-20 times the column volume of impurity buffer to wash the impurity protein from the purification column; wash After the impurity is completed, the target protein is eluted with 4-10 times the column volume of elution buffer; after the collection is completed, the column is washed with 8M urea column volume, and then washed with a large amount of distilled water, and sealed for storage. Finally, the active part was dialyzed overnight, and concentrated by ultrafiltration for SDS-PAGE electrophoresis detection to determine the purity and molecular weight of the enzyme. The purification effect was good, and the molecular weight of the electrophoresis band was consistent with the target protein.

(4) Salt adaptability characterization of five mutant enzymes and nine mutant enzymes. After the purified enzyme was concentrated by ultrafiltration and the buffer was replaced, a salt concentration gradient of 0 mM, 200 mM, 400 mM, 600 mM, 800 mM, and 1000 mM was set to determine the enzyme activity. Each enzyme was reacted at 35°C for 10 min in 500 μl reaction system with sodium alginate as substrate, and OD540 was measured after DNS color development. Substitute the measured OD540 into the glucose concentration-absorbance standard curve to calculate the reducing sugar concentration, and use the highest concentration of reducing sugar as a benchmark (enzyme activity 100%) to calculate the relative enzyme activity under different salt concentrations.

The enzymatic activity result table of the salt adaptability of table 6 five mutant enzymes and nine mutant enzymes

According to the characterization results of salt adaptability, it can be seen that the wild-type Aly1 has the maximum enzyme activity at a salt concentration of 0.6M, and the enzyme activities of the five mutants and nine mutants at lower salt concentrations are significantly improved compared with the wild type. The enzyme activity of M5A reaches 86.5% and 90.6% under the conditions of 200mM and 400mM salt concentration, and the enzyme activity of M9A reaches 93.7% and 100% under the condition of 200mM and 400mM salt concentration. It can be seen that mutating positively charged (basic) amino acids to negatively charged (acidic) amino acids or uncharged amino acids can enhance the salt adaptability of alginate lyase Aly1 at low salt concentrations.

Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be construed as limitations to the present invention. Variations, modifications, substitutions, and modifications to the above-described embodiments are possible within the scope of the present invention.

Claims (3)

1. A method for modifying the adaptability of alginate lyase salt of PL7 family, which is characterized by comprising the following steps:

s1, analysis before mutation design:

(1) Analysis of surface amino acids: calculating solvent accessibility surface area values (SASA values) of all amino acids of the wild PL7 family algin lyase by utilizing VMD software, dividing the SASA values by the theoretical maximum solvent accessibility area of each amino acid to obtain exposure proportion, and if the exposure proportion is greater than 25%, considering the amino acid as the surface amino acid of the enzyme, otherwise, not belonging to the surface amino acid;

(2) Analysis of enzyme catalytic sites: template searching is carried out on the wild type PL7 family algin lyase by utilizing a Swiss-model and I-Tasser tool to obtain PL7 family algin lyase information with similar structures and reported by existing crystal structures, catalytic site information of a reference crystal structure is obtained through literature investigation, and catalytic site information of the wild type PL7 family algin lyase is obtained through superposition and structural analysis of the wild type structure and the reference crystal structure by Pymol;

(3) Analysis of conserved sites: performing blast on a UniProt website by using the amino acid sequence of target PL7 family algin lyase, selecting 40-100 PL7 family algin lyase sequences with similar lengths and 70-100% consistency from search results, performing multi-sequence comparison by using COBALT tools of NCBI database, and knowing the conservation degree of amino acid according to the results;

s2, according to analysis before mutation design in S1, mutation sites are selected to enable positively charged amino acids in target wild type PL7 family algin lyase to be mutated into negatively charged amino acids or uncharged amino acids: all positively charged amino acids in the wild type PL7 family algin lyase are the initial potential mutation targets, inappropriate mutation points are eliminated one by one through the following 4 principles, and finally, rationally designed mutation sites are obtained:

(1) Selecting a site positioned on the surface of the protein, excluding the site inside the protein, and selecting surface amino acid in close contact with salt ions;

(2) Selecting a site far away from an active center, excluding the site positioned at or near the active center, and avoiding damaging enzyme activity;

(3) Selecting non-conservative sites of enzyme, excluding highly conservative sites, avoiding destroying the original functions, and excluding highly conserved positively charged amino acid sites;

(4) Selecting a site which does not form a salt bridge, eliminating a relevant site with weak acting force, and maintaining the original hydrogen bond, salt bridge and disulfide bond;

s3, selecting mutation schemes of all mutation sites according to the conservation degree of the amino acid obtained in the step (3) of the combined S1: if the conservation site analysis result shows that mutation sites have negatively charged amino acids on other PL7 family alginate lyase, the mutation sites are preferentially mutated into glutamic acid or aspartic acid with the negatively charged amino acids; if the other PL7 family algin lyase does not have negatively charged amino acid at the site, mutating 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 which are not charged amino acids, preferentially mutating it to more occurring amino acids;

the obtained gene is the mutated PL7 family algin lyase gene.

2. A PL7 family alginate lyase gene sequence having low salt adaptation, characterized by a protein sequence obtained by mutation according to the method of claim 1.

3. A recombinant plasmid of PL7 family algin lyase with low salt adaptability, which is characterized by comprising a gene sequence corresponding to the protein sequence of claim 2.

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