CN116334055A - Method for improving adaptability of PL7 family algin lyase salt - 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

Method for improving adaptability of PL7 family algin lyase salt

Technical Field

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

Background

Algin is an important component of the cell walls of large brown algae such as kelp, and the like, and the algin lyase can break beta-1, 4 glycosidic bonds of the algin polysaccharide through elimination reaction, can effectively degum and reduce the viscosity of brown algae, and can produce high-activity seaweed extract rich in brown algae oligosaccharides, which is used as an animal feed additive or a plant organic fertilizer.

Compared with the physical degradation method and the chemical degradation method, the enzymolysis method has the advantages of mild reaction conditions, easy control of the process, strong substrate specificity, high yield, energy conservation, environmental protection and the like, so that biodegradation represented by the enzymolysis method is necessary to gradually replace the traditional chemical degradation, and the biodegradation is dominant in future commercial production. The alginate lyase can break beta-1, 4 glycosidic bond of alginate polysaccharide based on elimination reaction to form alginate oligosaccharide with double bond and multiple biological activities. Algin lyase belongs to the family of polysaccharide degrading enzymes Polysaccharide Lyase (PL), mainly including the 5 th, 6 th, 7 th, 14 th, 15 th, 17 th and 18 th families, with the most recently reported algin lyase being the PL7 family.

Algin lyase has great potential in industry, but environmental conditions such as high salt restrict the large-scale application. The salt adaptability of the algin lyase from different sources has larger difference, and the system research on the salt adaptability rational transformation of the algin lyase is lacking at present, and the development process has larger randomness and blindness, so that larger production cost is required to be input in the actual industrial application, and the further development of the algin lyase in the brown algae processing industry is restricted.

Disclosure of Invention

The invention aims to provide a method for modifying the adaptability of alginate lyase salt of PL7 family. Thereby realizing the regulation and control of the salt adaptability thereof and obtaining the algin lyase suitable for the low-salt catalytic environment.

To achieve the above object, a method for modifying the adaptability of alginate lyase salt of PL7 family, comprising the steps of:

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.

The invention also provides a PL7 family algin lyase gene sequence with low salt adaptability, which is characterized in that the protein sequence obtained by mutation according to the method.

The invention also provides a recombinant plasmid of the PL7 family algin lyase with low salt adaptability, which is characterized by comprising a gene sequence corresponding to the protein sequence.

Regarding the highly conserved site, it is considered that if the identity of a certain site is 90% or more, the site is considered to belong to the highly conserved site.

Regarding the selection of sites far from the active center according to the present invention, the distance from the active center can be observed from the three-dimensional structure of the protein, excluding sites located at or near the active center (e.g., excluding sites within 6 angstroms from the active center), avoiding damage to the enzyme activity.

According to the invention, the existing algin lyase Aly1 is modified by a rational design method, so that the salt adaptability of the algin lyase is regulated and controlled, the algin lyase suitable for a low-salt catalytic environment is obtained, and the correlation is verified. The method provides a theoretical basis for developing and rationally designing the algin lyase with specific salt adaptability, can reduce the input cost of the algin lyase in practical industrial application to a certain extent, promotes the development of the deep processing industry of algin products, and widens the application scene of the algin lyase.

Drawings

Fig. 1: the position of the 62 mutation sites in the Aly structure for the first round of screening is schematically shown.

Fig. 2: 67 sequences with 70% -100% similarity are selected to carry out multi-sequence comparison result graphs through the Cobalt website.

Fig. 3: aly1 schematic of the catalytically active sites.

Fig. 4: aly1 a schematic of salt bridges occurring during a 10ns MD process.

Fig. 5: the position of mutation site K8D, K9D, K11E, R116Y, K157D, K184E, K189E, K170D, K273E in the three-dimensional structure of nine mutant enzyme M9A is schematically shown.

Fig. 6: schematic representation of the position of mutation site K157D, K E in the three-dimensional structure of the two-mutant enzyme M2A.

Fig. 7: schematic representation of the position of mutation site K8D, K184E in the three-dimensional structure of the two-mutant enzyme M2B.

Fig. 8: schematic representation of the position of mutation site K9D, K D in the three-dimensional structure of the two-mutant enzyme M2C.

Fig. 9: schematic representation of the position of mutation site R116Y, K273E in the M2D three-dimensional structure of the two-mutant enzyme.

Fig. 10: schematic representation of the position of mutation site R116Y, K D in the three-dimensional structure of the two-mutant enzyme M2E.

Fig. 11: schematic representation of the position of mutation site K170D, K273E in the three-dimensional structure of the two-mutant enzyme M2F.

Fig. 12: schematic representation of the position of mutation site K11E, K157D, K184E in the three-dimensional structure of the triple mutant enzyme M3A.

Fig. 13: schematic representation of the position of mutation site K157D, K184E, K189E in the three-dimensional structure of the triple mutant enzyme M3B.

Fig. 14: schematic representation of the position of mutation site R116Y, K170D, K273E in the three-dimensional structure of the triple mutant enzyme M3C.

Fig. 15: the position of mutation site K8D, K157D, K184E, K189E, K D in the three-dimensional structure of five mutant enzyme M5A is schematically shown.

Detailed Description

Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. In the examples below, "%" refers to weight percent, unless explicitly stated otherwise.

Example 1: selection of mutation sites

Starting from the prior alginate lyase Aly1 in the laboratory, the mutant enzyme with reduced positive charge amino acid is rationally designed, and the overall rationally design process comprises the following steps:

(1) The first round of screening was positive charge amino acid analysis. All positive charge amino acids Aly1 (H, K, R) are selected from the structure by using PyMOL software or directly, and potential mutation points need to be subjected to a series of screening to ensure that the mutated enzyme protein not only maintains structural stability but also has certain catalytic activity. Through this round of analysis, 62 potential mutation points were screened out as shown in FIG. 1 (red marks are potential mutation points).

(2) The second round of screening is a conservation analysis of amino acids, and mainly comprises sequence collection, multiple sequence alignment and conservation assessment. Such conserved amino acids will be deleted when the mutation site is selected. The Aly sequence (shown as SEQ ID NO: 1) was retrieved through NCBI and protein BLAST was performed, 67 sequences with a similarity of 70% -100% were selected for multiple sequence alignment through the Cobalt website, and the comparison results were viewed in Discovery studio as shown in FIG. 2. Through multiple sequence comparison, the amino acid types of similar sequences at the positions of 62 basic amino acids are analyzed in an important way, the amino acid types of similar sequences at the positions of Aly basic amino acids are compared longitudinally, and if the amino acid types at a certain position are found to be uniform, the position is considered to be a conserved site of the enzyme protein, and the mutation needs to be avoided. The amino acid with highest occurrence frequency in the non-conservative sites of 62 basic amino acids is counted, and then the basic amino acid of the corresponding site is mutated into the amino acid with the highest occurrence frequency. Through this round of analysis, 45 sites were removed altogether, and 17 potential mutation points were screened out.

(3) The third round of screening was active site analysis. The catalytic active site of alginate lyase Aly1 comprises Q141, H143 and Y257, and the active site region should be avoided as much as possible during mutation. Through this round of analysis (by means of software, such as can be confirmed by means of fig. 3), 17 potential mutation points are found to be far away from the above-mentioned catalytic sites, which indicates to a certain extent that the active site region has better conservation, so that the mutation points do not need to be removed in this round of screening.

(4) The fourth round of screening is an existing salt bridge analysis based on molecular dynamics simulation as shown in figure 4. Loading Aly 10ns MD simulation data by a VMD, calling a SaltBuridge plug-in of VMD software, and obtaining Aly salt bridge and amino acid composition information thereof in the 10ns MD process (for example, a salt bridge obtaining method comprises the steps of simulating protein for 10ns by using molecular dynamics simulation software (NAMD, gromacs and the like) and obtaining the salt bridge by using program function analysis). 8 amino acids out of 17 potential mutation points involved in salt bridge formation, and 9 potential mutation points were finally determined after truncation as shown in FIG. 5.

Example 2: construction of the two mutant enzymes and salt-adaptation characterization

(1) Mutation point combination. Amino acids whose primary sequences are far apart may be close to each other in a three-dimensional structure, while adjacent amino acids in a spatial structure may have interactions, so that the stability of the enzyme is maintained in order to avoid disrupting the interactions between amino acids, and mutation points should be dispersed as much as possible when mutation point combination is performed. And (3) formulating a mutation point combination scheme of two mutations for the 9 selected mutation points according to a mutation point dispersion principle. The positions of the mutation sites in the two mutant enzymes in the three-dimensional structure are shown in FIGS. 6-11.

TABLE 1 mutation site Table of two mutant enzymes

(2) Complete gene composition. According to the designed mutant enzyme whole gene, the sequence after codon optimization (the optimized sequence is shown as SEQ ID NO: 2) is synthesized and cloned by Shanghai JieRui bioengineering Co. The plasmid used was pET-28 (a) +, ndeI and XhoI were used for the cleavage site, and BL21 (DE 3) was used as the expression host.

(3) Expression and purification of the two mutant enzymes. Taking out the strain stored at-20deg.C from refrigerator, transferring into LB culture medium according to 1% inoculum size, placing into shaking table at 25deg.C, shaking at 180r/min for 20 hr, taking appropriate amount of thallus, performing plate streaking, selecting single colony with good growth vigor after 24 hr, inoculating into LB liquid culture medium, activating strain, transferring into 200mLLB liquid culture medium containing 100 μg/mL of caliamycin, placing into shaking table, culturing at 37 deg.C at 180rpm for 3 hr, when OD600 reaches 0.6-0.8, adding IPTG inducer to final concentration of 0.05mmol/L, placing into shaking table again at low temperature of 16 deg.C at 180rpm for 20 hr. Centrifuging the cultured bacterial liquid, removing the supernatant, re-suspending the collected bacterial precipitate by using a buffer solution, and performing ultrasonic crushing.

The target protein was purified by reference to the Ni-NTA his/Bind Resin pre-cartridge of Qihai Co. Centrifuging the crude enzyme solution prepared by ultrasonic wall breaking, and filtering with 0.22 μm filter membrane. Filtering the crude enzyme solution with a 0.22 μm filter membrane, and filtering and sterilizing the Tris-HCl buffer solution used for purification; firstly, balancing a purification column, and loading a sample 4 buffer solution in a volume which is 10 times that of the column; loading a crude enzyme solution; after loading, washing the column for 10 column volumes by using a loading buffer solution, and washing the residual sample on the side wall of the purification column; washing the purified protein from the purification column with 10-20 column volumes of wash buffer; eluting the target protein by using 4-10 times of column volume elution buffer after the impurity washing is finished; after the collection is completed, the column is washed by 8M urea column volume, and then is washed by a large amount of distilled water, and the column is stored in a closed manner. Finally, the active fraction was dialyzed overnight, concentrated by ultrafiltration and subjected to SDS-PAGE electrophoresis to determine the purity and molecular weight of the enzyme.

(4) Characterization of the salt adaptation of the two mutant enzymes. After ultrafiltration concentration and buffer replacement of the purified dimutase, the enzyme activity was measured by a salt concentration gradient of 0mM, 200mM, 400mM, 600mM, 800mM, 1000mM, to examine the salt adaptability of the dimutase. Each enzyme was reacted at 35℃for 10min in a reaction system of 500. Mu.l using sodium alginate as a substrate, and after color development of DNS, OD540 was measured. Substituting the measured OD540 into a glucose concentration-absorbance standard curve to calculate the concentration of the reducing sugar, and calculating the relative enzyme activities under different salt concentrations by taking the highest concentration of the reducing sugar as a reference (enzyme activity 100%).

Table 2 table of enzyme activity results for two mutant enzyme salt adaptations

According to the characterization result of salt adaptability, the wild type Aly1 has the maximum enzyme activity at the salt concentration of 0.6M, the enzyme activity of the two mutants at the salt concentration of 0-0.4M is obviously improved compared with that of the wild type, the relative enzyme activity at the salt concentration of 0.4M is more than 80%, the M2E has the maximum enzyme activity at the salt concentration of 0.4M, and the relative enzyme activity of the M2F at the salt concentration of 0.4M is close to 100%. It can be seen that mutating a positively charged (basic) amino acid to a negatively charged (acidic) amino acid can enhance the salt adaptation of algin lyase Aly at low salt concentrations.

Example 3: construction of the Trimutant enzyme and salt adaptability characterization

(1) Mutation point combination. Amino acids whose primary sequences are far apart may be close to each other in a three-dimensional structure, while adjacent amino acids in a spatial structure may have interactions, so that the stability of the enzyme is maintained in order to avoid disrupting the interactions between amino acids, and mutation points should be dispersed as much as possible when mutation point combination is performed. And (3) formulating a mutation point combination scheme of three mutations for the 9 selected mutation points according to a mutation point dispersion principle. The position of the mutation site in the three-dimensional structure of the triple mutant enzyme is shown in FIGS. 12-14.

TABLE 3 mutation site Table of Trimutant enzymes

(2) Complete gene composition. And (3) carrying out on-line codon optimization according to the designed mutant enzyme whole gene, and then synthesizing and cloning by Shanghai Jieli bioengineering Co. The plasmid used was pET-28 (a) +, ndeI and XhoI were used for the cleavage site, and BL21 (DE 3) was used as the expression host.

(3) Expression and purification of the triple mutant enzyme. Taking out the strain stored at-20deg.C from refrigerator, transferring into LB culture medium according to 1% inoculum size, placing into shaking table at 25deg.C, shaking at 180r/min for 20 hr, taking appropriate amount of thallus, streaking, selecting single colony with good growth vigor after 24 hr, inoculating into LB liquid culture medium for strain activation, transferring into 200mLLB liquid culture medium containing 100 μg/mL of caliamycin, placing into shaking table at 37deg.C, culturing at 180rpm for 3 hr, adding IPTG inducer to final concentration of 0.05mmol/L when OD600 reaches 0.6-0.8, placing into shaking table at low temperature of 16deg.C, culturing at 180rpm for 20 hr. Centrifuging the cultured bacterial liquid, removing the supernatant, re-suspending the collected bacterial precipitate by using a buffer solution, and performing ultrasonic crushing.

The target protein was purified by reference to the Ni-NTA his/Bind Resin pre-cartridge of Qihai Co. Centrifuging the crude enzyme solution prepared by ultrasonic wall breaking, and filtering with 0.22 μm filter membrane. Filtering the crude enzyme solution with a 0.22 μm filter membrane, and filtering and sterilizing the Tris-HCl buffer solution used for purification; firstly, balancing a purification column, and loading a sample 4 buffer solution in a volume which is 10 times that of the column; loading a crude enzyme solution; after loading, washing the column for 10 column volumes by using a loading buffer solution, and washing the residual sample on the side wall of the purification column; washing the purified protein from the purification column with 10-20 column volumes of wash buffer; eluting the target protein by using 4-10 times of column volume elution buffer after the impurity washing is finished; after the collection is completed, the column is washed by 8M urea column volume, and then is washed by a large amount of distilled water, and the column is stored in a closed manner. Finally, the active fraction was dialyzed overnight, concentrated by ultrafiltration and subjected to SDS-PAGE electrophoresis to determine the purity and molecular weight of the enzyme. The purification effect is good, and the molecular weight of the electrophoresis band is consistent with that of the target protein.

(4) Salt adaptation characterization of the triple mutant enzyme. After ultrafiltration concentration and buffer replacement of the purified enzyme, the enzyme activity was measured by setting a salt concentration gradient of 0mM, 200mM, 400mM, 600mM, 800mM, and 1000 mM. Each enzyme was reacted at 35℃for 10min in a reaction system of 500. Mu.l using sodium alginate as a substrate, and after color development of DNS, OD540 was measured. Substituting the measured OD540 into a glucose concentration-absorbance standard curve to calculate the concentration of the reducing sugar, and calculating the relative enzyme activities under different salt concentrations by taking the highest concentration of the reducing sugar as a reference (enzyme activity 100%).

Table 4 table of enzyme activity results for three mutant enzyme salt adaptation

According to the characterization result of salt adaptability, the wild type Aly1 has the maximum enzyme activity at the salt concentration of 0.6M, the enzyme activity of the three mutants at the salt concentration of 0-0.4M is obviously improved compared with the wild type, and the relative enzyme activities of M3A and M3C at the salt concentration of 0.2M are nearly 100%. It can be seen that mutating a positively charged (basic) amino acid to a negatively charged (acidic) amino acid can enhance the salt adaptation of algin lyase Aly at low salt concentrations.

Example 4: construction of five-mutant enzyme and nine-mutant enzyme and salt adaptability characterization

(1) Mutation point combination. Amino acids whose primary sequences are far apart may be close to each other in a three-dimensional structure, while adjacent amino acids in a spatial structure may have interactions, so that the stability of the enzyme is maintained in order to avoid disrupting the interactions between amino acids, and mutation points should be dispersed as much as possible when mutation point combination is performed. And (3) formulating mutation point combination schemes of five mutations and nine mutations for the 9 selected mutation points according to a mutation point dispersion principle. The positions of mutation sites in the five-mutant enzyme and the nine-mutant enzyme in the three-dimensional structures are shown in fig. 15 and 5.

TABLE 5 mutation site Table of five-mutant enzyme and nine-mutant enzyme

(2) Complete gene composition. And (3) carrying out on-line codon optimization according to the designed mutant enzyme whole gene, and then synthesizing and cloning by Shanghai Jieli bioengineering Co. The plasmid used was pET-28 (a) +, ndeI and XhoI were used for the cleavage site, and BL21 (DE 3) was used as the expression host.

(3) And (5) purifying the expression of the five-mutant enzyme and the nine-mutant enzyme. Taking out the strain stored at-20deg.C from refrigerator, transferring into LB culture medium according to 1% inoculum size, placing into shaking table at 25deg.C, shaking at 180r/min for 20 hr, taking appropriate amount of thallus, streaking, selecting single colony with good growth vigor after 24 hr, inoculating into LB liquid culture medium for strain activation, transferring into 200mLLB liquid culture medium containing 100 μg/mL of caliamycin, placing into shaking table at 37deg.C, culturing at 180rpm for 3 hr, adding IPTG inducer to final concentration of 0.05mmol/L when OD600 reaches 0.6-0.8, placing into shaking table at low temperature of 16deg.C, culturing at 180rpm for 20 hr. Centrifuging the cultured bacterial liquid, removing the supernatant, re-suspending the collected bacterial precipitate by using a buffer solution, and performing ultrasonic crushing.

The target protein was purified by reference to the Ni-NTA his/Bind Resin pre-cartridge of Qihai Co. Centrifuging the crude enzyme solution prepared by ultrasonic wall breaking, and filtering with 0.22 μm filter membrane. Filtering the crude enzyme solution with a 0.22 μm filter membrane, and filtering and sterilizing the Tris-HCl buffer solution used for purification; firstly, balancing a purification column, and loading a sample 4 buffer solution in a volume which is 10 times that of the column; loading a crude enzyme solution; after loading, washing the column for 10 column volumes by using a loading buffer solution, and washing the residual sample on the side wall of the purification column; washing the purified protein from the purification column with 10-20 column volumes of wash buffer; eluting the target protein by using 4-10 times of column volume elution buffer after the impurity washing is finished; after the collection is completed, the column is washed by 8M urea column volume, and then is washed by a large amount of distilled water, and the column is stored in a closed manner. Finally, the active fraction was dialyzed overnight, concentrated by ultrafiltration and subjected to SDS-PAGE electrophoresis to determine the purity and molecular weight of the enzyme. The purification effect is good, and the molecular weight of the electrophoresis band is consistent with that of the target protein.

(4) Salt adaptability characterization of five-mutant enzyme and nine-mutant enzyme. After ultrafiltration concentration and buffer replacement of the purified enzyme, the enzyme activity was measured by setting a salt concentration gradient of 0mM, 200mM, 400mM, 600mM, 800mM, and 1000 mM. Each enzyme was reacted at 35℃for 10min in a reaction system of 500. Mu.l using sodium alginate as a substrate, and after color development of DNS, OD540 was measured. Substituting the measured OD540 into a glucose concentration-absorbance standard curve to calculate the concentration of the reducing sugar, and calculating the relative enzyme activities under different salt concentrations by taking the highest concentration of the reducing sugar as a reference (enzyme activity 100%).

TABLE 6 Table of enzyme Activity results of salt adaptability of five-mutant enzyme and nine-mutant enzyme

According to the characterization result of salt adaptability, the wild type Aly1 has the maximum enzyme activity at the salt concentration of 0.6M, the enzyme activities of the five mutant and the nine mutant at the lower salt concentration are obviously improved compared with the wild type, the enzyme activities of M5A at the salt concentrations of 200mM and 400mM reach 86.5% and 90.6%, and the enzyme activities of M9A at the salt concentrations of 200mM and 400mM reach 93.7% and 100%. It can be seen that mutation of positively charged (basic) amino acids to negatively charged (acidic) amino acids or uncharged amino acids can enhance the salt adaptation of algin lyase Aly1 at low salt concentrations.

Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives, and variations may be made in the above embodiments by those skilled in the art without departing from the spirit and principles of the 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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