CN112921020A - Algin lyase mutant for relieving divalent metal ion dependence and application thereof - Google Patents

Abstract

The invention relates to the technical field of protein engineering, in particular to an alginate lyase mutant for relieving the dependence of divalent metal ions and application thereof. The mutant is alginate lyase amino acid sequence which takes polyG as a specific substrate when carrying out enzymolysis in PL7 family alginate lyase, at least in the same three-dimensional structure position with aspartic acid D146 in AlgAT5, or the amino acid at D146 position is mutated into amino acid with positive charge, glycine, alanine, valine, leucine or isoleucine based on the multi-sequence alignment of the structure. The beneficial mutations can provide theoretical and technical support for the industrial development and application of alginate lyase and promote the green and efficient preparation of alginate oligosaccharides.

Description

Algin lyase mutant for relieving divalent metal ion dependence and application thereof

Technical Field

The invention relates to the technical field of protein engineering, in particular to an alginate lyase mutant for relieving the dependence of divalent metal ions and application thereof.

Background

The phaeophyta is the most important component of marine macroalgae, has important significance in the aspects of ecological values of marine carbon fixation, marine ecological environment and the like, and also has important application value as a biomass resource. There are many macroalgae belonging to the phylum Phaeophyta, such as Laminaria japonica (Laminaria hyperborean), Ecklonia cava (Macrocystis pyrifera), Laminaria digitata (Lamimiria digita), Ascophyllum nodosum (Ascophyllum nodosum), Laminaria japonica (Laminaria japonica), Macrocystis japonica (Lessonia nigrescens), and Antarctic algae (Durvillea antarctica). The main component of the brown algae is algin, the content can reach 40 percent at most according to different growing positions and seasons, the algin has similar specific functions and is regarded as structural substance cellulose in terrestrial plants, and the main biological function of the algin in the macroalgae is a structural support function and a support and stabilization function. Due to the unique physicochemical properties such as hydrophilicity, gel formation, viscosity, biocompatibility and the like, the chitosan hydrogel has been widely applied to various fields such as food, medicine, chemical industry and the like for years, has gel characteristics, and is widely applied to industries such as food, textile, printing and dyeing, biology, medicine and the like as a stabilizer, a thickening agent and a plasticizer. For example, the dietary fiber has the functions of controlling blood sugar and blood fat, delaying senility, resisting tumor, enhancing the immunity of the organism and other ecological effects and health care functions; due to good biocompatibility, the algin can also be used as a matrix material to wrap the medicine which needs to be released from a special site, so that the medicine slow release application is realized, and in addition, the algin can also be used as a biological scaffold material for medical application.

The structure of algin is a linear macromolecule which is polymerized by two sugar units of beta-D-mannuronic acid (PolyM) and alpha-L-guluronic acid (PolyG) through 1,4 glycosidic bonds. Brown algae polysaccharide and its degradation product brown algae oligosaccharide have been widely used in pharmaceutical, food, chemical and other fields. The alginate lyase can cleave the 1,4 glycosidic bond of the alginate through a beta-elimination reaction to generate an unsaturated uronic acid with a C4,5 double bond at a non-reducing end. The high viscosity and gelling property of algin are widely used as food modifiers in the food industry. The alginate lyase breaks 1,4 glycosidic bond of alginate through beta elimination reaction, and generates 4-deoxy-L-erythro-hex-4-enol pyranouronic acid with C4,5 double bond at non-reducing end. The brown algae oligosaccharide has been proved to have a plurality of application fields such as stimulation of the growth of human endothelial cells, promotion of macrophage secretion of cell factors and the like, and is widely applied to a plurality of fields such as pharmacy, food, chemical industry, plant protection, animal protection and the like. Algin is converted into unsaturated monosaccharide by endo-and exo-alginases, so that the alginases can be used as biocatalysts for producing renewable chemicals and biofuel. Algin exists as a long chain macromolecule, usually in the form of alginate, and often forms alginate with metal ions, such as sodium alginate, calcium alginate, and the like. Two of the PolyG chains form an "egg-box" (egg box) structure with a divalent metal ion, usually a calcium ion. This form results in a more rigid and less susceptible to degradation.

The alginate-derived oligosaccharide produced by degrading algin with the biological enzyme method has the characteristics of greenness, high efficiency, strong specificity and the like. However, many alginate lyase enzymes reported to have obvious divalent metal ion-dependent tolerance, and the catalytic efficiency of the enzyme is very significantly reduced after the metal chelating agent existing in the environment removes the divalent metal ions in the reaction system, for example, AlyPG is taken as an example, Zn is added²⁺And Mn²⁺Compared with the AlyPG wild type, the enzyme activity is improved to 131% and 175%, but the enzyme activity is reduced to 17% of the AlyPG wild type after EDTA chelate is added and divalent metal ions are removed. Further, AlyA and AlyA1 have the same characteristics. This divalent metal dependence necessitates the presence of metal ions in the environment to allow the catalytic reaction to take place, otherwise the reaction cannot proceed. This disadvantage severely limits the widespread use of alginate lyase.

Disclosure of Invention

The invention aims to provide an alginate lyase mutant for relieving divalent metal ion dependence and application thereof.

In order to achieve the purpose, the invention adopts the technical scheme that:

a divalent metal ion-dependent releasing alginate lyase mutant is characterized in that the mutant is in an amino acid sequence of alginate lyase which takes polyG as a specific substrate during enzymolysis in PL7 family alginate lyase, at least the amino acid sequence is positioned on the same three-dimensional structure position with aspartic acid D146 in AlgAT5, or the amino acid at the D146 position is mutated into amino acid with positive charge, glycine, alanine, valine, leucine or isoleucine based on structural multiple sequence alignment.

The algin lyase is AlgAT5, alyPG, Alg _ M3, Alg, Alyl1, AlyA, AlyPI, Algb or AlyA1, AlyQ, AlgB, Alg2A or AlyVGI.

The algin lyase is AlgAT5, alyPG, Alyl1, AlyA, AlyPI and AlyQ. The amino acids in the lyase amino acid sequence that are at the same three-dimensional structural position as aspartic acid D146 in AlgAT5, or that are structurally based on multiple sequence alignments at position D146 are aspartic acid D or glutamic acid E mutated to a positively charged amino acid (lysine, histidine or arginine), as well as glycine, alanine, valine, leucine, isoleucine.

Preferably mutated to a positively charged amino acid (lysine, histidine or arginine)

The lyase AlgAT5 also comprises a mutation from alanine at position 229 to cysteine.

The lyase AlgAT5 mutant is D146H/A229C.

An expression vector containing any one of the mutants.

A genetically engineered bacterium contains the expression vector.

The application of a mutant in preparing alginate oligosaccharides by removing divalent metal ion dependent catalytic algin.

A method for preparing alginate oligosaccharides by catalyzing algin for relieving divalent metal ion dependence is characterized in that: adding the mutant of claim 1 to alginate in 0.2M NaAC-HAC buffer at pH5.8, 0.1-0.3M NaCl, 0.5-2mM CaCl₂At 50-80 deg.C, using mutant to relieve divalent metal ion dependenceThe alginate oligosaccharide is prepared by the catalytic alginate reaction.

The invention has the advantages that:

according to the invention, alginate lyase mutants D146K, D146H and D146R which are subjected to divalent metal ion dependence removal are obtained by methods such as structural analysis, rational design and site-directed mutagenesis, and a combined mutant D146H/A229C is further constructed by introducing a pair of disulfide bonds, so that the optimal reaction temperature is increased to 80 ℃, the thermal stability of the alginate lyase is remarkably improved, the dependence on divalent metal ions can be removed, and the green and efficient preparation of alginate oligosaccharides is promoted.

Drawings

Fig. 1 is a graph illustrating the effect of EDTA on AlgAT5 of the metal ion and divalent metal ion chelating agents provided in the examples of the present invention. Wherein A is the change of enzyme activity after 1mM of different metal ions and 1mM of divalent metal ion chelating agent EDTA are added, and B is whether 1mM of Ca is added or not²⁺Then reducing the viscosity of algin by AlgAT5, wherein the effect on catalytic activity is already achieved (the change of the absorbance of OD235 nm), C is the effect of different concentrations of divalent metal ion chelating agent EDTA on AlgAT5, D is the effect of no enzyme in the enzyme reaction kinetic process of AlgAT5, then AlgAT5 is added to start the reaction, 1mM EDTA is added to stop the reaction, and 2mM Ca is added to stop the reaction²⁺The reaction can continue again.

FIG. 2 is a graph showing the effect of different metal ions on the enzymatic activity of AlgAT5 according to an embodiment of the present invention.

FIG. 3 is a diagram of analysis of key amino acids in catalytic channels of different specificities provided by the embodiments of the present invention.

FIG. 4 is a graph showing the effect of EDTA on the Asp146 point mutation of AlgAT5 according to the present invention.

FIG. 5 shows Ca provided in the examples of the present invention²⁺Influence of the Asp146 point mutation on AlgAT 5.

Fig. 6 is a diagram showing the alignment results of the structures based on the sequence alignment of AlgAT5 and alyPG and the alignment results based on the crystal structures provided in the examples of the present invention, wherein a is the alignment results of AlgAT5 and alyPG, and B is the alignment results of aspartic acid D146 of AlgAT5 and aspartic acid D124 of alyPG at positions in the three-dimensional structures.

FIG. 7 is a graph showing the effect of EDTA on Asp124 point mutation of alyPG according to the present invention.

FIG. 8 shows Ca provided in the examples of the present invention²⁺Influence of Asp124 point mutation on alyPG.

FIG. 9 is a graph of the positions of cysteines in AlgAT5 and disulfide bonds detected by DTT method, wherein A is the position of three free cysteines in AlgAT5, and B is the graph of disulfide bonds detected by DTT method.

FIG. 10 is a diagram of amino acids interacting with three cysteines provided by an example of the present invention.

FIG. 11 is a graph showing the detection of disulfide bonds formed between A229C and C82 and the measurement of reaction temperature, wherein A is the positions of A229 and C82 before mutagenesis, B is the state of disulfide bonds formed between C229 and C82 after mutagenesis, C is the comparison of the optimum temperature of A229C with the wild type, and D is the DTT method for detecting the generation of disulfide bonds.

FIG. 12 is a diagram illustrating the optimum temperature of D146H/A229C and the effect of EDTA on it according to an embodiment of the present invention, wherein A is D146H/A229C, and B is a diagram illustrating the effect of EDTA on D146H/A229C.

Detailed Description

The principles and features of this invention are further described below in the examples which are set forth to illustrate the invention and are not intended to limit the scope of the invention. In addition, the experimental methods used in the following examples are all conventional in the art unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 analysis of the Metal ion-dependent alginate lyase of the PL7 family based on structural and catalytic Properties

After analyzing alginate lyase in PL7 family, we found that most enzymes with substrate specificity of polyG have obvious divalent metal ion activation effect, the enzyme activity is obviously improved after adding the divalent metal ions, and the enzyme activity is obviously reduced after adding a metal ion chelating agent such as EDTA or EGTA to remove the divalent metal ions in the reaction system.

Taking the alyPG as an example, Zn is added²⁺And Mn²⁺Compared with the wild type, the enzyme activity is increased to 131% and 175%, however, after the metal ion chelating agent EDTA is added, the enzyme activity is reduced to 17% of the wild type. For Alg _ M3, Ca²⁺ Mn²⁺、Co^{2 +}、Mg²⁺The enzyme activity can be obviously improved; for Alg, Ca²⁺、Mn²⁺The enzyme activity can be obviously improved; for Alyl1, Mg²⁺And Zn²⁺The enzyme activity can be obviously improved; for AlyA, Ba²⁺And Ca²⁺The enzyme activity can be obviously improved, and the enzyme activity is reduced to 48.3 percent of that of a wild type after the metal ion chelating agent EDTA is added; for AlyPI, Fe²⁺And Ca²⁺The enzyme activity can be obviously improved, and the enzyme activity is reduced to 56 percent of that of a wild type after the metal ion chelating agent EDTA is added; for Algb, Fe²⁺And Ca²⁺The enzyme activity can be obviously improved; for AlyA1, after the metal ion chelating agent EDTA is added, the enzyme activity is reduced to 10% of that of the wild type, and after the metal ion chelating agent EGTA is added, the enzyme activity is reduced to 5% of that of the wild type; for AlyQ, the enzyme activity is reduced to 37.1% of that of the wild type after 1mM of metal ion chelating agent EDTA is added, and the enzyme activity is reduced to 24.7% of that of the wild type after 5mM of metal ion chelating agent EDTA is added. For AlgAT5, Co²⁺、Ca²⁺、Mn²⁺、Fe²⁺The enzyme activity can be obviously improved, and the enzyme activity is reduced to 10 percent of that of a wild type after the metal ion chelating agent EDTA is added. See table 1 for results.

TABLE 1 Effect of Metal ions and Metal ion chelators on PolyG substrate-preferred enzymes.

However, most of the enzymes with substrate specificity of PolyM show completely different results, the enzyme activity is not obviously changed after the divalent metal ions are added, and the enzyme activity is not obviously inhibited after the divalent metal ions in the reaction system are removed by adding a metal ion chelating agent such as EDTA or EGTA. By FlalyFor example, the addition of divalent metal ions has no obvious influence, and after the addition of metal ion chelating agents EDTA and EGTA, the relative enzyme activity is respectively changed to 95.4 percent and 106.4 percent compared with the wild type, and the relative enzyme activity also has no obvious change; for AlgMsp, the activity of AlgMsp was independent of the presence of divalent cations, there was no significant difference in the metal ion chelator EDTA pre-treatment and untreated AlgMsp activity; for NitAly, the activity was independent of the presence of divalent cations, there was no significant difference in the activity of the metal ion chelator EDTA pre-treatment and untreated; for AlxM, the activity is independent of the presence of divalent cations; for Alg7D, divalent metal ion Ca⁺、Zn^{2 +}、Fe²⁺All are inhibitors, and can perform functions without participation of divalent metal ions; for a 1-II', the divalent metal ion had no effect on the enzyme activity, and there was no significant difference in the activity of the metal ion chelator, EDTA pre-treatment and no treatment; for AlyDW11, divalent metal ions have no obvious influence on the enzyme activity, and after the metal ion chelating agent EDTA is added, the enzyme activity is reduced a little, and 75% of the activity of the wild type is still kept; for A9mT, Ca²⁺、Mn²⁺、Mg²⁺The enzyme activity is not influenced, the enzyme activity is changed into 101% of the wild type after EDTA is added, and the enzyme activity is changed into 103% of the wild type after EGTA is added. See table 2 for results.

Table 2. effect of metal ions and metal ion chelators on PolyM substrate-preferred enzymes.

Example 2 inducible expression of AlgAT5 in E.coli

The pET-30a-AlgAT5 expression plasmid (Lefuli, Suhang, Jishiqi, Luming. algin lyase coding gene and the application thereof, which is preserved in a refrigerator at the temperature of-80 ℃): chinese, 201810862414.3[ P ].2018-08-01.] BL21 protein-expressing strain, 5. mu.L was taken out and inoculated in 5mL LB plus kanamycin liquid medium at 200rpm and cultured overnight at 37 ℃ as seeds. The next day, the activated cells were transferred to 500mL LB liquid medium containing kanamycin, cultured at 200rpm at 37 ℃ until OD600 was 0.5-0.8, added with 1mM IPTG, and shake-cultured in a 22-25 ℃ constant temperature shaker at 200rpm for 16-18h to induce protein expression. The alginate lyase AlgAT5 was purified by Ni-NTA Resin (available from TransGen Biotech). After the purification is completed, the purification condition of the recombinant alginate lyase AlgAT5 is detected by polyacrylamide gel electrophoresis. The recombinant alginate lyase AlgAT5 sample with the protein purity of more than 98 percent detected by polyacrylamide gel electrophoresis is loaded into an ultrafiltration tube with the minimum molecular cut-off of 10kD, AlgAT5 is concentrated at 4000rpm and 4 ℃, and the buffer solution is replaced by 100mM Tris-HCl with the pH value of 8.0. To prepare the recombinant alginate lyase AlgAT5 enzyme solution.

The enzyme activity determination method comprises purifying above to obtain alginate lyase AlgAT5 protein, adding 50 μ l of 2 μ g/mL enzyme into 0.2% alginate, 0.2M NaAC-HAC buffer solution with pH of 5.8, 0.2M NaCl, 1mM CaCl₂The change in OD235nm was measured at 70 ℃ in an ultraviolet spectrophotometer with cyclic heating in a water bath. One unit of enzyme activity was defined as the change in OD235nm value per minute by 0.1 value. Specific enzyme activity definition: the ratio of enzyme activity to the amount of the corresponding protein. Wherein, the reaction system contains 0.2M NaAC-HAC buffer solution with pH of 5.8, 0.2M NaCl and 1mM CaCl2 in 0.2% of algin.

The recombinant alginate lyase AlgAT5 obtained above is added into a reaction system containing different metal ions. Wherein the metal ion is Cu²⁺、Ba²⁺、Co²⁺、Zn²⁺、Ca²⁺、Mg²⁺、Mn²⁺、Fe²⁺、Ni⁺、K⁺、Fe³⁺And a divalent metal ion chelating agent EDTA, and then reacting for 1min under optimal conditions, and measuring the activity of the enzyme at OD235nm according to the aforementioned spectrophotometry. The activity in the control was determined without addition of metal ions (set to 100%). Wherein, when metal ions are added into a reaction system (2g/L sodium alginate, 200mM NaCl, 200mM acetic acid-sodium acetate buffer solution, pH 5.8), the final concentration of the ions is 1mM, and the final concentration of the metal ion chelating agent EDTA is also 1mM (see figures 1 and 2). Is composed ofFurther defining the effect of the concentration of EDTA on the enzymatic activity of AlgAT5, we further tested the enzymatic activity of AlgAT5 at various concentrations of EDTA (0, 0.05, 0.1, 0.2, 0.25, 0.5, 1mM), setting the enzymatic activity at 0mM to 100%. Viscosity change measurement during sodium alginate degradation, to measure viscosity change during sodium alginate degradation, 1% (w/v) substrate concentration was used, the volume was 15mL, AlgAT5 was added at 0.01mg/mL using a BROOKFIELD VISCOMETER VISCOMETER, set at 6.0RPM, and the measurement time was 300s, taking one point every 10 s. The reaction was carried out with the temperature of a 70 ℃ water bath maintained. The OD235 change during the reaction was also measured by using an ultraviolet spectrophotometer capable of continuously measuring OD235nm, and the measurement time was also set at 300s, and samples were taken every 10 s. The measurement was carried out with and without the addition of 1mM calcium ion, respectively.

Results from metal ions and chemicals shown in FIG. 1A for recombinant alginate lyase AlgAT5, at a concentration of 1 mM:

(1) metal ion chelating agents EDTA, Cu²⁺、Ba²⁺Exhibits an inhibitory effect on the activity of AlgAT 5;

(2)Co²⁺、Ca²⁺、Mn²⁺、Fe²⁺the divalent metal examples have the promotion effect on the enzyme activity, the enzyme activity is improved by more than two times compared with the wild type, and the rest divalent and trivalent metal ions have certain promotion effect on the enzyme activity;

EDTA with different concentrations is respectively added into the reaction system to determine the influence of the EDTA on the alginate lyase AlgAT5, the result is shown in figure 1C, D, the enzyme activity curve is shown after 1mM of EDTA is added during the determination of the enzyme activity, and then 1mM of Ca is supplemented²⁺And (4) ions, and measuring the influence of the ions on enzyme activity. It was thus found that the enzyme activity of AlgAT5 decreased to 60% when 0.1mM EDTA was added, that the enzyme activity of AlgAT5 decreased to 20% when 0.2mM EDTA was added, and that the enzyme activity of AlgAT5 decreased to 90% when 1mM EDTA was added, as shown in FIG. 1C, D.

As long-chain macromolecules, the viscosity of a high-concentration sodium alginate aqueous solution is high, and in order to detect the viscosity change of the alginate lyase AlgAT5 in the process of degrading sodium alginate, the viscosity change of the alginate lyase AlgAT5 is detectedThe viscosity reduction process of 1% (w/v) sodium alginate in the reaction process of 300s at 70 ℃ is continuously monitored by a viscometer, and as can be seen from fig. 1B, the viscosity is rapidly reduced along with the enzymolysis reaction, the viscosity can be reduced to 55% of the initial value by 30s and the viscosity can be reduced to 32% of the initial value by 60s, which accords with the characteristics of the incision type alginate lyase, and shows that AlgAT5 is an incision type alginate lyase. Simultaneously comparing the addition of Ca in the reaction system²⁺And no Ca is added²⁺From the difference, it can be seen that when we add 1mMCa to the reaction system²⁺Then the viscosity is reduced to 25 percent of the initial value after 30s of reaction and 19 percent after 60s of reaction, and the viscosity reduction effect is obviously better than that without Ca²⁺The condition of (2). Further description is given of Ca²⁺The reaction proceeds with acceleration of the divalent metal ions as represented. The mechanism therein deserves our thought. The change in OD235nm as the reaction proceeded was also measured by an ultraviolet spectrophotometer. It can also be seen that 1mMCa was added²⁺After entering the reaction system, the catalytic efficiency of AlgAT5 was increased by nearly 2 times.

In order to further research the effect of metal ions on alginate lyase, the enzyme activity curve is obtained after 1mM EDTA is added during the enzyme activity determination, and then 1mM Ca is supplemented²⁺And (4) ions, and measuring the influence of the ions on enzyme activity. Further, the specific procedure for determining the activity of different metal ions on the enzyme is as follows. In the first stage, only 2g/L sodium alginate substrate was used without enzyme as reference. In the second stage, the enzyme is added to start the reaction and there may be an optical absorption value of OD 235. In the third stage, EDTA was added to chelate the metal ions and inhibit the reaction from proceeding, and it was observed that the light absorption of OD235 did not increase. And in the fourth stage, different metal ions are added to observe whether the reaction can be continued.

As is clear from the results in FIG. 2, 1mM of Co was contained²⁺、Ca²⁺、Mn²⁺、Fe²⁺The divalent metal ions have obvious promotion effect on enzyme activity, are improved by more than two times compared with wild type, and the rest divalent metal ions Zn²⁺And Mg²⁺Has certain promotion effect on enzyme activity, but monovalent ions such as Na⁺And K⁺For enzymesThe improvement of the activity has no obvious effect.

Therefore, it can be shown that the metal ions play an important role in the reaction process of the AlgAT5 for degrading algin, and the metal ions cannot play a role to catalyze the reaction without the presence of the divalent metal ions. Many alginate lyase enzymes reported at present have obvious divalent metal ion-dependent tolerance, and the catalytic efficiency of the enzyme is very obviously reduced after the metal chelating agent exists in the environment to remove the divalent metal ions in the reaction system. This metal dependence also limits the widespread use of alginate lyase. The results are shown in FIG. 2.

Example 3, construction of D146E, D146H, D146K, D146R, D146A and D146P mutants and enzyme activity determination.

Based on the analysis of example 2, which performed site-directed mutagenesis on the amino acid at position 146, and further analyzed the amino acids near the substrate binding region in the PL7 family, we found Asp146 to be the key amino acid for determining the substrate specificity of PolyG and PolyM, in combination with the results of multiple sequence alignments of different substrate specificities, and speculated that Asp146 also plays an important role in binding to metal ions.

Since aspartic acid D is known to be an amino acid with a negatively charged side chain, and glutamic acid E is also known to have this property, it is preferred to mutate it to glutamic acid E which is also negatively charged but has a slightly longer side chain length. And further selecting histidine H, arginine R and lysine K which are mutated into amino acids with positive charges, and observing the influence of charge reversal on the metal ion dependence effect. Further selection was made to subject the amino acid mutation to saturation mutagenesis, which was changed to alanine and a rigid amino acid proline. Thus, a series of mutations D146E, D146H, D146K, D146R, D146A, D146P were made to Asp 146.

The primers required are shown below

D146P

Forward:5'-CAGATGATCCTGTTATAATGATTCGTTTAGAAGGAAATC-3'

Reverse:5'-AACAGGATCATCTGAATCATGAATTTGTCCC-3'

D146A

Forward:5'-CAGATGATGCTGTTATAATGATTCGTTTAGAAGGAAAT-3'

Reverse:5'-TAACAGCATCATCTGAATCATGAATTTGTCC-3'

D146E

Forward:5'-CAGATGATGAAGTTATAATGATTCGTTTAGAAGGAAATC-3'

Reverse:5'-TATAACTTCATCATCTGAATCATGAATTTGTCC-3'

D146K

Forward:5'-CAGATGATAAGGTTATAATGATTCGTTTAGAAGGAAATC-3'

Reverse:5'-TATAACCTTATCATCTGAATCATGAATTTGTCCC-3'

D146H

Forward:5'-CAGATGATCATGTTATAATGATTCGTTTAGAAGGAAA-3'

Reverse:5'-TATAACATGATCATCTGAATCATGAATTTGTCCC-3'

D146R

Forward:5'-CAGATGATCGTGTTATAATGATTCGTTTAGAAGGAAAT-3'

Reverse:5'-TTATAACACGATCATCTGAATCATGAATTTGTCCC-3'

The original gene sequence of the alginate lyase AlgAT5 is shown in SEQ ID NO. 1.

SEQ ID NO.1

ATGAAGGGAAGATTAAAAAAATGGTGTAGTGGCTTTCTAATTGCTATGTTAGTATCTACACCAACAGGAATGGTTAATGCAGCAAGTTTGCTTCCATCAGACATTTTAGATTTGACTAATTGGAAACTTACATTACCTATTAATGATGCAGAAGAAATTACGCAACCAGAATTAGATAGTTATGAACATAGTGAGTACTTTCATGTAAATGATGATGGAGATGCAGTCGTATTTAAAGCACACTGTGGAGGAGATACTACAGAGGGTTCTTCGTATCCAAGATGTGAACTTAGAGAAATGACAAATGATGGACAAGATAAGGCTAGTTGGTCTACTACATCTGGAACACATACTATGATAATTGATCAAAAAATCACACATCTTCCCGAAGTAAAAGACCATGTTGTTGTGGGACAAATTCATGATTCAGATGATGATGTTATAATGATTCGTTTAGAAGGAAATCATTTATTTGTAGAAGGGGATGGAGAGGAACTTGCAGATTTAGATACAGATTATGAATTAGGAACAAGATTTACTGTAAAGATAGTGGCATCCGGAGGTAAAATTAAAGTATATTATAATGGAGATTTAAAATTAACTTATAATAAGAGTGTTTCAGGATGTTATTTTAAAGCAGGTATGTATACTCAATCTAACACCAGCAAAGGTGATAGTGAGGATGCATATGGGGAAAATGAAATTTATAATCTAGTAGTAACCCATAGT

(a) Sequence characteristics:

length: 729

Type: gene sequences

Chain type: single strand

Topology: linearity

(b) Molecular type: DNA

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: defluvitala phaphyphia sp

The original amino acid sequence of the algin lyase AlgAT5 is shown in SEQ ID NO.2

SEQ ID NO.2:

MKGRLKKWCSGFLIAMLVSTPTGMVNAASLLPSDILDLTNWKLTLPINDAEEITQPELDSYEHSEYFHVNDDGDAVVFKAHCGGDTTEGSSYPRCELREMTNDGQDKASWSTTSGTHTMIIDQKITHLPEVKDHVVVGQIHDSDDDVIMIRLEGNHLFVEGDGEELADLDTDYELGTRFTVKIVASGGKIKVYYNGDLKLTYNKSVSGCYFKAGMYTQSNTSKGDSEDAYGENEIYNLVVTHS

(a) Sequence characteristics:

length: 243

Type: amino acid sequence

Chain type: single strand

Topology: linearity

(b) Molecular type: protein

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: defluvitala phaphyphia sp

The mutant D146P is obtained by mutating aspartic acid D at the 146 th site of the alginate lyase AlgAT5 amino acid sequence into proline P, and the amino acid is shown as the following SEQ ID NO. 3.

SEQ ID NO.3:

MKGRLKKWCSGFLIAMLVSTPTGMVNAASLLPSDILDLTNWKLTLPINDAEEITQPELDSYEHSEYFHVNDDGDAVVFKAHCGGDTTEGSSYPRCELREMTNDGQDKASWSTTSGTHTMIIDQKITHLPEVKDHVVVGQIHDSDDPVIMIRLEGNHLFVEGDGEELADLDTDYELGTRFTVKIVASGGKIKVYYNGDLKLTYNKSVSGCYFKAGMYTQSNTSKGDSEDAYGENEIYNLVVTHS

(a) Sequence characteristics:

length: 243

Type: amino acid sequence

Chain type: single strand

Topology: linearity

(b) Molecular type: protein

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: defluvitala phaphyphia sp

The mutant D146A is obtained by mutating aspartic acid D at the 146 th site of the alginate lyase AlgAT5 amino acid sequence into proline P, and the amino acid is shown as the following SEQ ID NO. 4.

SEQ ID NO.4:

MKGRLKKWCSGFLIAMLVSTPTGMVNAASLLPSDILDLTNWKLTLPINDAEEITQPELDSYEHSEYFHVNDDGDAVVFKAHCGGDTTEGSSYPRCELREMTNDGQDKASWSTTSGTHTMIIDQKITHLPEVKDHVVVGQIHDSDDAVIMIRLEGNHLFVEGDGEELADLDTDYELGTRFTVKIVASGGKIKVYYNGDLKLTYNKSVSGCYFKAGMYTQSNTSKGDSEDAYGENEIYNLVVTHS

(a) Sequence characteristics:

length: 243

Type: amino acid sequence

Chain type: single strand

Topology: linearity

(b) Molecular type: protein

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: defluvitala phaphyphia sp

The mutant D146E is obtained by mutating aspartic acid D at the 146 th site of the alginate lyase AlgAT5 amino acid sequence to glutamic acid E, wherein the amino acid is shown as the following SEQ ID NO. 5.

SEQ ID NO.5:

MKGRLKKWCSGFLIAMLVSTPTGMVNAASLLPSDILDLTNWKLTLPINDAEEITQPELDSYEHSEYFHVNDDGDAVVFKAHCGGDTTEGSSYPRCELREMTNDGQDKASWSTTSGTHTMIIDQKITHLPEVKDHVVVGQIHDSDDEVIMIRLEGNHLFVEGDGEELADLDTDYELGTRFTVKIVASGGKIKVYYNGDLKLTYNKSVSGCYFKAGMYTQSNTSKGDSEDAYGENEIYNLVVTHS

(a) Sequence characteristics:

length: 243

Type: amino acid sequence

Chain type: single strand

Topology: linearity

(b) Molecular type: protein

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: defluvitala phaphyphia sp

The mutant D146K is obtained by mutating aspartic acid D at the 146 th site of the alginate lyase AlgAT5 amino acid sequence into lysine K, and the amino acid is shown as the following SEQ ID NO. 6.

SEQ ID NO.6:

MKGRLKKWCSGFLIAMLVSTPTGMVNAASLLPSDILDLTNWKLTLPINDAEEITQPELDSYEHSEYFHVNDDGDAVVFKAHCGGDTTEGSSYPRCELREMTNDGQDKASWSTTSGTHTMIIDQKITHLPEVKDHVVVGQIHDSDDKVIMIRLEGNHLFVEGDGEELADLDTDYELGTRFTVKIVASGGKIKVYYNGDLKLTYNKSVSGCYFKAGMYTQSNTSKGDSEDAYGENEIYNLVVTHS

(a) Sequence characteristics:

length: 243

Type: amino acid sequence

Chain type: single strand

Topology: linearity

(b) Molecular type: protein

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: defluvitala phaphyphia sp

Mutant D146H is obtained by mutating aspartic acid D at position 146 of alginate lyase AlgAT5 amino acid sequence to histidine H, the amino acid is shown as SEQ ID NO. 7.

SEQ ID NO.7:

MKGRLKKWCSGFLIAMLVSTPTGMVNAASLLPSDILDLTNWKLTLPINDAEEITQPELDSYEHSEYFHVNDDGDAVVFKAHCGGDTTEGSSYPRCELREMTNDGQDKASWSTTSGTHTMIIDQKITHLPEVKDHVVVGQIHDSDDHVIMIRLEGNHLFVEGDGEELADLDTDYELGTRFTVKIVASGGKIKVYYNGDLKLTYNKSVSGCYFKAGMYTQSNTSKGDSEDAYGENEIYNLVVTHS

(a) Sequence characteristics:

length: 243

Type: amino acid sequence

Chain type: single strand

Topology: linearity

(b) Molecular type: protein

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: defluvitala phaphyphia sp

The mutant D146R is obtained by mutating aspartic acid D at the 146 th site of the alginate lyase AlgAT5 amino acid sequence into arginine R, wherein the amino acid is shown as the following SEQ ID NO. 8.

SEQ ID NO.8:

MKGRLKKWCSGFLIAMLVSTPTGMVNAASLLPSDILDLTNWKLTLPINDAEEITQPELDSYEHSEYFHVNDDGDAVVFKAHCGGDTTEGSSYPRCELREMTNDGQDKASWSTTSGTHTMIIDQKITHLPEVKDHVVVGQIHDSDDRVIMIRLEGNHLFVEGDGEELADLDTDYELGTRFTVKIVASGGKIKVYYNGDLKLTYNKSVSGCYFKAGMYTQSNTSKGDSEDAYGENEIYNLVVTHS

(a) Sequence characteristics:

length: 243

Type: amino acid sequence

Chain type: single strand

Topology: linearity

(b) Molecular type: protein

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: defluvitala phaphyphia sp

After obtaining corresponding plasmids through mutation and sending the plasmids to a sequencing company (Pongoni sequencing company) for sequencing verification, mutant plasmids pET-30a-D146E, pET-30a-D146H, pET-30a-D146K, pET-30a-D146R, pET-30a-D146A and pET-30a-D146P are respectively transformed into an expression strain BL21(DE3, Beijing Quanyujin company) for inducible expression. The relevant procedure was as in example 2. After obtaining the related purified mutant, detecting.

Enzymes of either polyM or polyG specificity in the PL7 family are very similar at positions +1, +2, +3, +4 in the substrate binding region, and show significant identity both from the conservation of amino acid residues and from the distribution of surface electrostatic charges. The most important differences are at-1, -2, -3 positions, where polyG-specific enzymes are negatively charged, while polyM-and polyMG-specific enzymes are positively charged, as viewed by the surface static charge distribution. Analysis of the key amino acids revealed that Asp146 was either conserved in all enzymes specific for PolyG or was replaced by the similar amino acid Glu. However, the same positions are all conserved to Pro in both PolyM and PolyMG specific enzymes, as shown in FIG. 3.

Further analysis of amino acids near the substrate binding region in the PL7 family, combined with multiple sequence alignments of different substrate specificities, found that Asp146 is the key amino acid determining the substrate specificity of PolyG and PolyM, and speculated that Asp146 also plays an important role in binding to metal ions. After mutating Asp146 to Glu146 with the same negative charge, D146E was also sensitive to EDTA, and the enzyme activity was 27.5% when 1mM EDTA was added compared to no EDTA. However, after mutating Asp146 to His146, Lys146 and Arg146, which are positively charged, these several mutants all appeared to be insensitive to EDTA. In the case of D146K, the enzyme activity was 97.5% when EDTA was added at 1mM, compared with that when EDTA was not added, and there was no significant difference. The same D146H and D149R showed almost no change in enzyme activity after addition of 1mM EDTA compared to the initial state. Further analysis of the same sites in PolyM were all proline (Pro), so we found that 1mM Ca was added to D146P after mutating Asp146 to Pro146²⁺After EDTA, the enzyme activity was 138.2% and 89.6% compared to the initial state without metal ions and EDTA, which is also no longer sensitive to metal ions and metal ion chelators. Similar to D146P, D146A is also no longer sensitive to metal ions and metal ion chelators. Based on the above-mentioned phenomenon, it is preliminarily presumed that the negatively charged amino acid Asp146 plays a role in assisting the binding of the metal ion to the substrate during the reaction catalyzed by the divalent metal ion, and the negatively charged Asp146 in the reaction accelerates the reaction by neutralizing the negative charge on the polysaccharide chain with the divalent metal ion. Asp146 mutated to a positively charged amino acidThe dependence of the divalent metal ions was clearly removed later, and even in the presence of the metal ion chelating agent, D146H, D146K, and D146R all appeared to be consistent with the case where no metal chelating agent was added. See, fig. 4 and 5 for results. The mutants enable the catalytic process of the enzyme to be independent of divalent metal ions, can independently drive functions, and greatly broadens the application range of the enzyme.

Example 4, construction of the mutant of alyPG-D124E, alyPG-D124H, alyPG-D124K, alyPG-D124R, alyPG-D124A and alyPG-D124P, and determination of enzyme activity.

To further clarify the role of the mutant Asp146 in AlgAT5 in releasing divalent metal ions, we further performed site-directed mutagenesis using another alginate lyase alyPG with substrate specificity of the PL7 family, PolyG type, and examined the release of metal ion dependence. From the results of the alignment of the sequences of structure-based AlgAT5 and alopg and the alignment based on the crystal structure, it was found that D124 is present in the same three-dimensional structure position in alopg as Asp146 in AlgAT 5. We therefore performed point mutation verification on the D124 site of the enzyme, alyPG, see FIGS. 6A and 6B

The synthetic sequence of the alyPG is shown in SEQ ID NO.9 by the synthetic sequence of the Wuxi Qinglan company, and the gene is cloned on a clone vector PMV plasmid and named as PMV-alyPG. pEasy-BluntE1 (Beijing Quanyu Co.) vector was used as the expression of alyPG.

The gene sequence of the algin lyase alyPG is shown as SEQ ID NO. 9.

SEQ ID NO.9：

GCCGAGCCCTGCGACTACCCGGCCCAGCAGCTCGACCTGACCGACTGGAAGGTCACACTGCCGATCGGCTCGTCCGGAAAGCCCTCCGAGATCGAGCAGCCGGCCCTCGACACGTTCGCCACCGCTCCGTGGTTCCAGGTCAACGCCAAGTGCACGGGCGTGCAGTTCCGGGCCGCCGTGAACGGCGTGACGACGTCCGGCTCGGGCTACCCCCGGTCGGAGCTCCGGGAGATGACGGACGGGGGCGAGGAGAAGGCGTCCTGGTCCGCGACGTCGGGCACCCACACGATGGTCTTCCGGGAGGCCTTCAACCACCTCCCCGAGGTCAAGCCCCACCTGGTCGGTGCGCAGATCCACGACGGCGACGACGACGTCACGGTCTTCCGCCTGGAGGGCACCAGCCTCTACATCACCAAGGGCGACGACACGCACCACAAGCTGGTGACGAGCGACTACAAGCTCAACACCGTCTTCGAGGGCAAGTTCGTCGTGAGCGGGGGCAAGATCAAGGTCTACTACAACGGCGTCCTGCAGACCACGATCAGCCACACCAGCTCGGGCAACTACTTCAAGGCGGGCGCGTACACGCAGGCCAACTGCAGCAACTCCTCGCCCTGCAGCAGCTCGAACTACGGCCAGGTGTCCCTGTACAAGCTGCAGGTGACACACTCC

(a) Sequence characteristics:

length: 672

Type: gene sequences

Chain type: single strand

Topology: linearity

(b) Molecular type: DNA

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: corynebacterium sp

The amino acid sequence of the algin lyase alyPG is shown in SEQ ID NO. 10. SEQ ID NO. 10:

AEPCDYPAQQLDLTDWKVTLPIGSSGKPSEIEQPALDTFATAPWFQVNAKCTGVQFRAAVNGVTTSGSGYPRSELREMTDGGEEKASWSATSGTHTMVFREAFNHLPEVKPHLVGAQIHDGDDDVTVFRLEGTSLYITKGDDTHHKLVTSDYKLNTVFEGKFVVSGGKIKVYYNGVLQTTISHTSSGNYFKAGAYTQANCSNSSPCSSSNYGQVSLYKLQVTHS

(a) sequence characteristics:

length: 224

Type: gene sequences

Chain type: single strand

Topology: linearity

(b) Molecular type: protein

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: corynebacterium sp

The cloning primers were designed as follows:

F：GCCGAGCCCTGCGACTACCCGGCCCAGC

R：GGAGTGTGTCACCTGCAGCTTGTA

blunt-ended ligation with pEasy-BluntE1 was performed to obtain a pEasy-BluntE1-alyPG protein expression plasmid.

The primers for constructing the mutant of the alyPG-D124E, the alyPG-D124H, the alyPG-D124K, the alyPG-D124R, the alyPG-D124A and the alyPG-D124P are as follows:

alyPG-D124E

Forward:5'-CGAGGTCACGGTCTTCCGCC-3'

Reverse:5'-CCGTGACCTCGTCGTCGCCGTCG-3'

alyPG-D124H

Forward:5'-CGACGACCACGTCACGGTCTTCCGCC-3'

Reverse:5'-CGTGGTCGTCGCCGTCGTGG-3'

alyPG-D124K

Forward:5'-CGACGACAAAGTCACGGTCTTCCGC-3'

Reverse:5'-TGACTTTGTCGTCGCCGTCGTG-3'

alyPG-D124R

Forward:5'-CCGCGTCACGGTCTTCCGCC-3'

Reverse:5'-CGTGACGCGGTCGTCGCCGTCGTGG-3'

alyPG-D124A

Forward:5'-CGACGCCGTCACGGTCTTCCGCC-3'

Forward:5'-CGACGCCGTCACGGTCTTCCGCC-3'

alyPG-D124P

Forward:5'-GACGACCCCGTCACGGTCTTCCGCC-3'

Reverse:5'-ACGGGGTCGTCGCCGTCGTGG-3'

the mutant alyPG-D124E of the invention mutates aspartic acid D at the 124 th site of an amino acid sequence of alginate lyase alyPG into glutamic acid E, and the amino acid is shown as the following SEQ ID NO. 11.

SEQ ID NO.11:

AEPCDYPAQQLDLTDWKVTLPIGSSGKPSEIEQPALDTFATAPWFQVNAKCTGVQFRAAVNGVTTSGSGYPRSELREMTDGGEEKASWSATSGTHTMVFREAFNHLPEVKPHLVGAQIHDGDDEVTVFRLEGTSLYITKGDDTHHKLVTSDYKLNTVFEGKFVVSGGKIKVYYNGVLQTTISHTSSGNYFKAGAYTQANCSNSSPCSSSNYGQVSLYKLQVTHS

(a) Sequence characteristics:

length: 224

Type: gene sequences

Chain type: single strand

Topology: linearity

(b) Molecular type: protein

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: corynebacterium sp

The mutant alyPG-D124H of the invention mutates aspartic acid D at the 124 th site of an amino acid sequence of alginate lyase into histidine H, and the amino acid is shown as the following SEQ ID NO. 12.

SEQ ID NO.12:

AEPCDYPAQQLDLTDWKVTLPIGSSGKPSEIEQPALDTFATAPWFQVNAKCTGVQFRAAVNGVTTSGSGYPRSELREMTDGGEEKASWSATSGTHTMVFREAFNHLPEVKPHLVGAQIHDGDDHVTVFRLEGTSLYITKGDDTHHKLVTSDYKLNTVFEGKFVVSGGKIKVYYNGVLQTTISHTSSGNYFKAGAYTQANCSNSSPCSSSNYGQVSLYKLQVTHS

(a) Sequence characteristics:

length: 224

Type: gene sequences

Chain type: single strand

Topology: linearity

(b) Molecular type: protein

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: corynebacterium sp

The mutant alyPG-D124K of the invention mutates aspartic acid D at the 124 th site of an algin lyase alyPG amino acid sequence into lysine K, and the amino acid is shown as the following SEQ ID NO. 13.

SEQ ID NO.13:

AEPCDYPAQQLDLTDWKVTLPIGSSGKPSEIEQPALDTFATAPWFQVNAKCTGVQFRAAVNGVTTSGSGYPRSELREMTDGGEEKASWSATSGTHTMVFREAFNHLPEVKPHLVGAQIHDGDDKVTVFRLEGTSLYITKGDDTHHKLVTSDYKLNTVFEGKFVVSGGKIKVYYNGVLQTTISHTSSGNYFKAGAYTQANCSNSSPCSSSNYGQVSLYKLQVTHS

(a) Sequence characteristics:

length: 224

Type: gene sequences

Chain type: single strand

Topology: linearity

(b) Molecular type: protein

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: corynebacterium sp

The mutant alyPG-D124R of the invention mutates aspartic acid D at the 124 th site of an amino acid sequence of alginate lyase alyPG into arginine R, and the amino acid is shown as the following SEQ ID NO: 14.

SEQ ID NO.14:

AEPCDYPAQQLDLTDWKVTLPIGSSGKPSEIEQPALDTFATAPWFQVNAKCTGVQFRAAVNGVTTSGSGYPRSELREMTDGGEEKASWSATSGTHTMVFREAFNHLPEVKPHLVGAQIHDGDDRVTVFRLEGTSLYITKGDDTHHKLVTSDYKLNTVFEGKFVVSGGKIKVYYNGVLQTTISHTSSGNYFKAGAYTQANCSNSSPCSSSNYGQVSLYKLQVTHS

(a) Sequence characteristics:

length: 224

Type: gene sequences

Chain type: single strand

Topology: linearity

(b) Molecular type: protein

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: corynebacterium sp

The mutant alyPG-D124A of the invention changes aspartic acid D at the 124 th site of the algin lyase alyPG amino acid sequence into alanine A, and the amino acid is shown as the following SEQ ID NO. 15.

SEQ ID NO.15:

AEPCDYPAQQLDLTDWKVTLPIGSSGKPSEIEQPALDTFATAPWFQVNAKCTGVQFRAAVNGVTTSGSGYPRSELREMTDGGEEKASWSATSGTHTMVFREAFNHLPEVKPHLVGAQIHDGDDAVTVFRLEGTSLYITKGDDTHHKLVTSDYKLNTVFEGKFVVSGGKIKVYYNGVLQTTISHTSSGNYFKAGAYTQANCSNSSPCSSSNYGQVSLYKLQVTHS

(a) Sequence characteristics:

length: 224

Type: gene sequences

Chain type: single strand

Topology: linearity

(b) Molecular type: protein

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: corynebacterium sp

The mutant alyPG-D124P of the invention mutates aspartic acid D at the 124 th site of an amino acid sequence of alginate lyase alyPG into proline P, and the amino acid is shown as the following SEQ ID NO: 16.

SEQ ID NO.16:

AEPCDYPAQQLDLTDWKVTLPIGSSGKPSEIEQPALDTFATAPWFQVNAKCTGVQFRAAVNGVTTSGSGYPRSELREMTDGGEEKASWSATSGTHTMVFREAFNHLPEVKPHLVGAQIHDGDDPVTVFRLEGTSLYITKGDDTHHKLVTSDYKLNTVFEGKFVVSGGKIKVYYNGVLQTTISHTSSGNYFKAGAYTQANCSNSSPCSSSNYGQVSLYKLQVTHS

(a) Sequence characteristics:

length: 224

Type: gene sequences

Chain type: single strand

Topology: linearity

(b) Molecular type: protein

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: corynebacterium sp

Each constructed mutant plasmid is transformed into an expression strain BL21 (holo-type gold company) for protein induction expression and purification, and relevant steps are consistent with those in example 2. After obtaining the relevant well-purified mutant,

they were each tested in 0.2M NaAC-HAC buffer, 0.2M NaCl, pH5.8, temperature 70 ℃ with 1mM Ca added²⁺And 1mM EDTA, and the change of enzyme activity. After the wild type is added with EDTA, the enzyme activity is changed into 17 percent of the wild type, and after the aspartic acid D with negative charge is mutated into the glutamic acid E with the same negative charge, the enzyme activity is changed into 20 percent of the wild type after the alyPG-D124E, the enzyme activity is still obviously reduced, and the interaction of metal ions is not released. When mutated to histidine H with a positive charge, the enzyme activity became 96% of wild type. When mutated to a positively charged lysineAfter the amino acid K, the enzyme activity is changed to 99 percent of the wild type. When mutated to arginine R with positive charge, the enzyme activity becomes 98% of that of wild type. When the mutant is mutated into alanine A, the enzyme activity is changed to 91 percent of that of the wild type. When the mutant is changed into proline P, the enzyme activity is changed into 80% of wild type. See fig. 7.

Wild type added 1mM Ca²⁺Then the enzyme activity is changed into 158% of the wild type, when aspartic acid D with negative charge is mutated into glutamic acid E with the same negative charge, and after the amyPG-D124E, the enzyme activity is changed into 188% of the wild type, which is still obviously improved, and the metal ions can still obviously improve the enzyme activity. When the mutant is mutated into histidine H with positive charge, the enzyme activity is changed into 108 percent of the wild type. When the mutant is mutated into lysine K with positive charge, the enzyme activity is changed into 119% of that of the wild type. When the mutant is mutated into arginine R with positive charge, the enzyme activity is changed into 110 percent of that of the wild type. When the mutant is alanine A, the enzyme activity is changed to 218 percent of the wild type. When the mutant is changed into proline P, the enzyme activity is changed into 168 percent of the wild type. See fig. 8.

Consistent with AlgAT5, after mutating the negatively charged amino acid aspartic acid D to a positively charged amino acid, divalent metal ion Ca was added²⁺The enzyme activity is not obviously increased and is not obviously reduced after EDTA is added, which shows that the divalent metal ion dependence of the enzyme can be relieved after the amino acid at the position is changed into the amino acid with positive charge, the application limit of the enzyme is reduced, and the use scene of the enzyme is expanded.

Example 5 construction of mutant A229C with increased catalytic temperature

The reaction temperature rise has the advantages of accelerating the reaction rate, shortening the production time, accelerating the dissolution of the substrate algin, preventing bacterial contamination and the like, but according to the structure, the AlgAT5 has no disulfide bond in the structure. But with three non-conserved cysteines in the nearest distance

Much greater than the distance that disulfide bonds can form. Adding different concentrations simultaneouslyWhen the native-SDS gel is run after DTT (reducing agent), the molecular weights of the proteins are not different, which indicates that the wild type protein does not form disulfide bonds. See fig. 9 and 10. In order to further increase the reaction temperature of the enzyme, a pair of disulfide bonds is rationally designed by means of homologous modeling and the like. Alanine at position 229 was mutated to cysteine, together with Cys82, since the distance between these two amino acids is less than

And the orientations are consistent, and disulfide bonds are easily formed.

The algin lyase mutant A229C obtained after the purification is measured under an ultraviolet spectrophotometer with water bath circulation heating to determine the change value of OD235 nm. One unit of enzyme activity was defined as the change in OD235nm value per minute by 0.1 value.

Determination of optimum reaction temperature for alginate lyase mutant A229C

The enzyme activities in different temperature ranges (30 deg.C, 40 deg.C, 50 deg.C, 60 deg.C, 65 deg.C, 70 deg.C, 75 deg.C, 80 deg.C, 85 deg.C, 90 deg.C) were measured under the condition of pH5.8, and the optimum reaction temperature was determined.

The mutant A229C of the present invention is prepared by mutating alanine A at the 222 th site of the amino acid sequence of alginate lyase AlgAT5 to cysteine C, wherein the amino acid is shown as SEQ ID NO: 17.

SEQ ID NO.17:

MKGRLKKWCSGFLIAMLVSTPTGMVNAASLLPSDILDLTNWKLTLPINDAEEITQPELDSYEHSEYFHVNDDGDAVVFKAHCGGDTTEGSSYPRCELREMTNDGQDKASWSTTSGTHTMIIDQKITHLPEVKDHVVVGQIHDSDDDVIMIRLEGNHLFVEGDGEELADLDTDYELGTRFTVKIVASGGKIKVYYNGDLKLTYNKSVSGCYFKAGMYTQSNTSKGDSEDCYGENEIYNLVVTHS

(a) Sequence characteristics:

length: 243

Type: amino acid sequence

Chain type: single strand

Topology: linearity

(b) Molecular type: protein

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: defluvitala phaphyphia sp

The primers are shown below

A229C

Forward:5'-GATTGCTATGGGGAAAATGAAATTTATAATCTAG-3'

Reverse:5'-CCCCATAGCAATCCTCACTATCACCTTTGC-3'

Through mutation, the mutant A229C has the broken disulfide bond in the presence of DTT and the migration rate is slow, so the molecular weight is slightly larger than that of the band without DTT in the buffer, which proves that the disulfide bond is really formed. According to the determination of the optimal reaction temperature, the optimal reaction temperature of the mutant A229C is increased from 70 ℃ of the wild type to 80 ℃, the reaction temperature of the enzyme is obviously increased, and the application scene of the enzyme is further expanded. See fig. 11.

Example 6 construction of mutant D146H/A229C to relieve divalent Metal ion dependence while increasing catalytic temperature

The mutant D146H/A229C of the invention is the amino acid sequence of alginate lyase AlgAT5, wherein the aspartic acid D at the 146 th site is mutated into histidine H, and the alanine A at the 222 th site is mutated into cysteine C, and the amino acid is shown as SEQ ID NO: 18.

SEQ ID NO.18:

MKGRLKKWCSGFLIAMLVSTPTGMVNAASLLPSDILDLTNWKLTLPINDAEEITQPELDSYEHSEYFHVNDDGDAVVFKAHCGGDTTEGSSYPRCELREMTNDGQDKASWSTTSGTHTMIIDQKITHLPEVKDHVVVGQIHDSDDHVIMIRLEGNHLFVEGDGEELADLDTDYELGTRFTVKIVASGGKIKVYYNGDLKLTYNKSVSGCYFKAGMYTQSNTSKGDSEDCYGENEIYNLVVTHS

(a) Sequence characteristics:

length: 243

Type: amino acid sequence

Chain type: single strand

Topology: linearity

(b) Molecular type: protein

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: defluvitala phaphyphia sp

The primers are shown below

D146H

Forward:5'-CAGATGATCATGTTATAATGATTCGTTTAGAAGGAAA-3'

Reverse:5'-TATAACATGATCATCTGAATCATGAATTTGTCCC-3'

A229C

Forward:5'-GATTGCTATGGGGAAAATGAAATTTATAATCTAG-3'

Reverse:5'-CCCCATAGCAATCCTCACTATCACCTTTGC-3'

Through mutation, the optimum reaction temperature of the mutant D146H/A229C is increased to 80 ℃, the reaction temperature of the enzyme is obviously increased, meanwhile, the enzyme activity of the enzyme is not obviously reduced after EDTA is added, the dependence of divalent metal ions can be removed, and the application scene of the enzyme is further expanded, as shown in figure 12.

Sequence listing

<110> institute of bioenergy and Process in Qingdao, China academy of sciences

<120> alginate lyase mutant for relieving divalent metal ion dependence and application thereof

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 729

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgaagggaa gattaaaaaa atggtgtagt ggctttctaa ttgctatgtt agtatctaca 60

ccaacaggaa tggttaatgc agcaagtttg cttccatcag acattttaga tttgactaat 120

tggaaactta cattacctat taatgatgca gaagaaatta cgcaaccaga attagatagt 180

tatgaacata gtgagtactt tcatgtaaat gatgatggag atgcagtcgt atttaaagca 240

cactgtggag gagatactac agagggttct tcgtatccaa gatgtgaact tagagaaatg 300

acaaatgatg gacaagataa ggctagttgg tctactacat ctggaacaca tactatgata 360

attgatcaaa aaatcacaca tcttcccgaa gtaaaagacc atgttgttgt gggacaaatt 420

catgattcag atgatgatgt tataatgatt cgtttagaag gaaatcattt atttgtagaa 480

ggggatggag aggaacttgc agatttagat acagattatg aattaggaac aagatttact 540

gtaaagatag tggcatccgg aggtaaaatt aaagtatatt ataatggaga tttaaaatta 600

acttataata agagtgtttc aggatgttat tttaaagcag gtatgtatac tcaatctaac 660

accagcaaag gtgatagtga ggatgcatat ggggaaaatg aaatttataa tctagtagta 720

acccatagt 729

<210> 2

<211> 243

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Lys Gly Arg Leu Lys Lys Trp Cys Ser Gly Phe Leu Ile Ala Met

1 5 10 15

Leu Val Ser Thr Pro Thr Gly Met Val Asn Ala Ala Ser Leu Leu Pro

20 25 30

Ser Asp Ile Leu Asp Leu Thr Asn Trp Lys Leu Thr Leu Pro Ile Asn

35 40 45

Asp Ala Glu Glu Ile Thr Gln Pro Glu Leu Asp Ser Tyr Glu His Ser

50 55 60

Glu Tyr Phe His Val Asn Asp Asp Gly Asp Ala Val Val Phe Lys Ala

65 70 75 80

His Cys Gly Gly Asp Thr Thr Glu Gly Ser Ser Tyr Pro Arg Cys Glu

85 90 95

Leu Arg Glu Met Thr Asn Asp Gly Gln Asp Lys Ala Ser Trp Ser Thr

100 105 110

Thr Ser Gly Thr His Thr Met Ile Ile Asp Gln Lys Ile Thr His Leu

115 120 125

Pro Glu Val Lys Asp His Val Val Val Gly Gln Ile His Asp Ser Asp

130 135 140

Asp Asp Val Ile Met Ile Arg Leu Glu Gly Asn His Leu Phe Val Glu

145 150 155 160

Gly Asp Gly Glu Glu Leu Ala Asp Leu Asp Thr Asp Tyr Glu Leu Gly

165 170 175

Thr Arg Phe Thr Val Lys Ile Val Ala Ser Gly Gly Lys Ile Lys Val

180 185 190

Tyr Tyr Asn Gly Asp Leu Lys Leu Thr Tyr Asn Lys Ser Val Ser Gly

195 200 205

Cys Tyr Phe Lys Ala Gly Met Tyr Thr Gln Ser Asn Thr Ser Lys Gly

210 215 220

Asp Ser Glu Asp Ala Tyr Gly Glu Asn Glu Ile Tyr Asn Leu Val Val

225 230 235 240

Thr His Ser

<210> 3

<211> 243

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Lys Gly Arg Leu Lys Lys Trp Cys Ser Gly Phe Leu Ile Ala Met

1 5 10 15

Leu Val Ser Thr Pro Thr Gly Met Val Asn Ala Ala Ser Leu Leu Pro

20 25 30

Ser Asp Ile Leu Asp Leu Thr Asn Trp Lys Leu Thr Leu Pro Ile Asn

35 40 45

Asp Ala Glu Glu Ile Thr Gln Pro Glu Leu Asp Ser Tyr Glu His Ser

50 55 60

Glu Tyr Phe His Val Asn Asp Asp Gly Asp Ala Val Val Phe Lys Ala

65 70 75 80

His Cys Gly Gly Asp Thr Thr Glu Gly Ser Ser Tyr Pro Arg Cys Glu

85 90 95

Leu Arg Glu Met Thr Asn Asp Gly Gln Asp Lys Ala Ser Trp Ser Thr

100 105 110

Thr Ser Gly Thr His Thr Met Ile Ile Asp Gln Lys Ile Thr His Leu

115 120 125

Pro Glu Val Lys Asp His Val Val Val Gly Gln Ile His Asp Ser Asp

130 135 140

Asp Pro Val Ile Met Ile Arg Leu Glu Gly Asn His Leu Phe Val Glu

145 150 155 160

Gly Asp Gly Glu Glu Leu Ala Asp Leu Asp Thr Asp Tyr Glu Leu Gly

165 170 175

Thr Arg Phe Thr Val Lys Ile Val Ala Ser Gly Gly Lys Ile Lys Val

180 185 190

Tyr Tyr Asn Gly Asp Leu Lys Leu Thr Tyr Asn Lys Ser Val Ser Gly

195 200 205

Cys Tyr Phe Lys Ala Gly Met Tyr Thr Gln Ser Asn Thr Ser Lys Gly

210 215 220

Asp Ser Glu Asp Ala Tyr Gly Glu Asn Glu Ile Tyr Asn Leu Val Val

225 230 235 240

Thr His Ser

<210> 4

<211> 243

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Lys Gly Arg Leu Lys Lys Trp Cys Ser Gly Phe Leu Ile Ala Met

1 5 10 15

Leu Val Ser Thr Pro Thr Gly Met Val Asn Ala Ala Ser Leu Leu Pro

20 25 30

Ser Asp Ile Leu Asp Leu Thr Asn Trp Lys Leu Thr Leu Pro Ile Asn

35 40 45

Asp Ala Glu Glu Ile Thr Gln Pro Glu Leu Asp Ser Tyr Glu His Ser

50 55 60

Glu Tyr Phe His Val Asn Asp Asp Gly Asp Ala Val Val Phe Lys Ala

65 70 75 80

His Cys Gly Gly Asp Thr Thr Glu Gly Ser Ser Tyr Pro Arg Cys Glu

85 90 95

Leu Arg Glu Met Thr Asn Asp Gly Gln Asp Lys Ala Ser Trp Ser Thr

100 105 110

Thr Ser Gly Thr His Thr Met Ile Ile Asp Gln Lys Ile Thr His Leu

115 120 125

Pro Glu Val Lys Asp His Val Val Val Gly Gln Ile His Asp Ser Asp

130 135 140

Asp Ala Val Ile Met Ile Arg Leu Glu Gly Asn His Leu Phe Val Glu

145 150 155 160

Gly Asp Gly Glu Glu Leu Ala Asp Leu Asp Thr Asp Tyr Glu Leu Gly

165 170 175

Thr Arg Phe Thr Val Lys Ile Val Ala Ser Gly Gly Lys Ile Lys Val

180 185 190

Tyr Tyr Asn Gly Asp Leu Lys Leu Thr Tyr Asn Lys Ser Val Ser Gly

195 200 205

Cys Tyr Phe Lys Ala Gly Met Tyr Thr Gln Ser Asn Thr Ser Lys Gly

210 215 220

Asp Ser Glu Asp Ala Tyr Gly Glu Asn Glu Ile Tyr Asn Leu Val Val

225 230 235 240

Thr His Ser

<210> 5

<211> 243

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Met Lys Gly Arg Leu Lys Lys Trp Cys Ser Gly Phe Leu Ile Ala Met

1 5 10 15

Leu Val Ser Thr Pro Thr Gly Met Val Asn Ala Ala Ser Leu Leu Pro

20 25 30

Ser Asp Ile Leu Asp Leu Thr Asn Trp Lys Leu Thr Leu Pro Ile Asn

35 40 45

Asp Ala Glu Glu Ile Thr Gln Pro Glu Leu Asp Ser Tyr Glu His Ser

50 55 60

Glu Tyr Phe His Val Asn Asp Asp Gly Asp Ala Val Val Phe Lys Ala

65 70 75 80

His Cys Gly Gly Asp Thr Thr Glu Gly Ser Ser Tyr Pro Arg Cys Glu

85 90 95

Leu Arg Glu Met Thr Asn Asp Gly Gln Asp Lys Ala Ser Trp Ser Thr

100 105 110

Thr Ser Gly Thr His Thr Met Ile Ile Asp Gln Lys Ile Thr His Leu

115 120 125

Pro Glu Val Lys Asp His Val Val Val Gly Gln Ile His Asp Ser Asp

130 135 140

Asp Glu Val Ile Met Ile Arg Leu Glu Gly Asn His Leu Phe Val Glu

145 150 155 160

Gly Asp Gly Glu Glu Leu Ala Asp Leu Asp Thr Asp Tyr Glu Leu Gly

165 170 175

Thr Arg Phe Thr Val Lys Ile Val Ala Ser Gly Gly Lys Ile Lys Val

180 185 190

Tyr Tyr Asn Gly Asp Leu Lys Leu Thr Tyr Asn Lys Ser Val Ser Gly

195 200 205

Cys Tyr Phe Lys Ala Gly Met Tyr Thr Gln Ser Asn Thr Ser Lys Gly

210 215 220

Asp Ser Glu Asp Ala Tyr Gly Glu Asn Glu Ile Tyr Asn Leu Val Val

225 230 235 240

Thr His Ser

<210> 6

<211> 243

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Lys Gly Arg Leu Lys Lys Trp Cys Ser Gly Phe Leu Ile Ala Met

1 5 10 15

Leu Val Ser Thr Pro Thr Gly Met Val Asn Ala Ala Ser Leu Leu Pro

20 25 30

Ser Asp Ile Leu Asp Leu Thr Asn Trp Lys Leu Thr Leu Pro Ile Asn

35 40 45

Asp Ala Glu Glu Ile Thr Gln Pro Glu Leu Asp Ser Tyr Glu His Ser

50 55 60

Glu Tyr Phe His Val Asn Asp Asp Gly Asp Ala Val Val Phe Lys Ala

65 70 75 80

His Cys Gly Gly Asp Thr Thr Glu Gly Ser Ser Tyr Pro Arg Cys Glu

85 90 95

Leu Arg Glu Met Thr Asn Asp Gly Gln Asp Lys Ala Ser Trp Ser Thr

100 105 110

Thr Ser Gly Thr His Thr Met Ile Ile Asp Gln Lys Ile Thr His Leu

115 120 125

Pro Glu Val Lys Asp His Val Val Val Gly Gln Ile His Asp Ser Asp

130 135 140

Asp Lys Val Ile Met Ile Arg Leu Glu Gly Asn His Leu Phe Val Glu

145 150 155 160

Gly Asp Gly Glu Glu Leu Ala Asp Leu Asp Thr Asp Tyr Glu Leu Gly

165 170 175

Thr Arg Phe Thr Val Lys Ile Val Ala Ser Gly Gly Lys Ile Lys Val

180 185 190

Tyr Tyr Asn Gly Asp Leu Lys Leu Thr Tyr Asn Lys Ser Val Ser Gly

195 200 205

Cys Tyr Phe Lys Ala Gly Met Tyr Thr Gln Ser Asn Thr Ser Lys Gly

210 215 220

Asp Ser Glu Asp Ala Tyr Gly Glu Asn Glu Ile Tyr Asn Leu Val Val

225 230 235 240

Thr His Ser

<210> 7

<211> 243

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Met Lys Gly Arg Leu Lys Lys Trp Cys Ser Gly Phe Leu Ile Ala Met

1 5 10 15

Leu Val Ser Thr Pro Thr Gly Met Val Asn Ala Ala Ser Leu Leu Pro

20 25 30

Ser Asp Ile Leu Asp Leu Thr Asn Trp Lys Leu Thr Leu Pro Ile Asn

35 40 45

Asp Ala Glu Glu Ile Thr Gln Pro Glu Leu Asp Ser Tyr Glu His Ser

50 55 60

Glu Tyr Phe His Val Asn Asp Asp Gly Asp Ala Val Val Phe Lys Ala

65 70 75 80

His Cys Gly Gly Asp Thr Thr Glu Gly Ser Ser Tyr Pro Arg Cys Glu

85 90 95

Leu Arg Glu Met Thr Asn Asp Gly Gln Asp Lys Ala Ser Trp Ser Thr

100 105 110

Thr Ser Gly Thr His Thr Met Ile Ile Asp Gln Lys Ile Thr His Leu

115 120 125

Pro Glu Val Lys Asp His Val Val Val Gly Gln Ile His Asp Ser Asp

130 135 140

Asp His Val Ile Met Ile Arg Leu Glu Gly Asn His Leu Phe Val Glu

145 150 155 160

Gly Asp Gly Glu Glu Leu Ala Asp Leu Asp Thr Asp Tyr Glu Leu Gly

165 170 175

Thr Arg Phe Thr Val Lys Ile Val Ala Ser Gly Gly Lys Ile Lys Val

180 185 190

Tyr Tyr Asn Gly Asp Leu Lys Leu Thr Tyr Asn Lys Ser Val Ser Gly

195 200 205

Cys Tyr Phe Lys Ala Gly Met Tyr Thr Gln Ser Asn Thr Ser Lys Gly

210 215 220

Asp Ser Glu Asp Ala Tyr Gly Glu Asn Glu Ile Tyr Asn Leu Val Val

225 230 235 240

Thr His Ser

<210> 8

<211> 243

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Met Lys Gly Arg Leu Lys Lys Trp Cys Ser Gly Phe Leu Ile Ala Met

1 5 10 15

Leu Val Ser Thr Pro Thr Gly Met Val Asn Ala Ala Ser Leu Leu Pro

20 25 30

Ser Asp Ile Leu Asp Leu Thr Asn Trp Lys Leu Thr Leu Pro Ile Asn

35 40 45

Asp Ala Glu Glu Ile Thr Gln Pro Glu Leu Asp Ser Tyr Glu His Ser

50 55 60

Glu Tyr Phe His Val Asn Asp Asp Gly Asp Ala Val Val Phe Lys Ala

65 70 75 80

His Cys Gly Gly Asp Thr Thr Glu Gly Ser Ser Tyr Pro Arg Cys Glu

85 90 95

Leu Arg Glu Met Thr Asn Asp Gly Gln Asp Lys Ala Ser Trp Ser Thr

100 105 110

Thr Ser Gly Thr His Thr Met Ile Ile Asp Gln Lys Ile Thr His Leu

115 120 125

Pro Glu Val Lys Asp His Val Val Val Gly Gln Ile His Asp Ser Asp

130 135 140

Asp Arg Val Ile Met Ile Arg Leu Glu Gly Asn His Leu Phe Val Glu

145 150 155 160

Gly Asp Gly Glu Glu Leu Ala Asp Leu Asp Thr Asp Tyr Glu Leu Gly

165 170 175

Thr Arg Phe Thr Val Lys Ile Val Ala Ser Gly Gly Lys Ile Lys Val

180 185 190

Tyr Tyr Asn Gly Asp Leu Lys Leu Thr Tyr Asn Lys Ser Val Ser Gly

195 200 205

Cys Tyr Phe Lys Ala Gly Met Tyr Thr Gln Ser Asn Thr Ser Lys Gly

210 215 220

Asp Ser Glu Asp Ala Tyr Gly Glu Asn Glu Ile Tyr Asn Leu Val Val

225 230 235 240

Thr His Ser

<210> 9

<211> 672

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gccgagccct gcgactaccc ggcccagcag ctcgacctga ccgactggaa ggtcacactg 60

ccgatcggct cgtccggaaa gccctccgag atcgagcagc cggccctcga cacgttcgcc 120

accgctccgt ggttccaggt caacgccaag tgcacgggcg tgcagttccg ggccgccgtg 180

aacggcgtga cgacgtccgg ctcgggctac ccccggtcgg agctccggga gatgacggac 240

gggggcgagg agaaggcgtc ctggtccgcg acgtcgggca cccacacgat ggtcttccgg 300

gaggccttca accacctccc cgaggtcaag ccccacctgg tcggtgcgca gatccacgac 360

ggcgacgacg acgtcacggt cttccgcctg gagggcacca gcctctacat caccaagggc 420

gacgacacgc accacaagct ggtgacgagc gactacaagc tcaacaccgt cttcgagggc 480

aagttcgtcg tgagcggggg caagatcaag gtctactaca acggcgtcct gcagaccacg 540

atcagccaca ccagctcggg caactacttc aaggcgggcg cgtacacgca ggccaactgc 600

agcaactcct cgccctgcag cagctcgaac tacggccagg tgtccctgta caagctgcag 660

gtgacacact cc 672

<210> 10

<211> 224

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Ala Glu Pro Cys Asp Tyr Pro Ala Gln Gln Leu Asp Leu Thr Asp Trp

1 5 10 15

Lys Val Thr Leu Pro Ile Gly Ser Ser Gly Lys Pro Ser Glu Ile Glu

20 25 30

Gln Pro Ala Leu Asp Thr Phe Ala Thr Ala Pro Trp Phe Gln Val Asn

35 40 45

Ala Lys Cys Thr Gly Val Gln Phe Arg Ala Ala Val Asn Gly Val Thr

50 55 60

Thr Ser Gly Ser Gly Tyr Pro Arg Ser Glu Leu Arg Glu Met Thr Asp

65 70 75 80

Gly Gly Glu Glu Lys Ala Ser Trp Ser Ala Thr Ser Gly Thr His Thr

85 90 95

Met Val Phe Arg Glu Ala Phe Asn His Leu Pro Glu Val Lys Pro His

100 105 110

Leu Val Gly Ala Gln Ile His Asp Gly Asp Asp Asp Val Thr Val Phe

115 120 125

Arg Leu Glu Gly Thr Ser Leu Tyr Ile Thr Lys Gly Asp Asp Thr His

130 135 140

His Lys Leu Val Thr Ser Asp Tyr Lys Leu Asn Thr Val Phe Glu Gly

145 150 155 160

Lys Phe Val Val Ser Gly Gly Lys Ile Lys Val Tyr Tyr Asn Gly Val

165 170 175

Leu Gln Thr Thr Ile Ser His Thr Ser Ser Gly Asn Tyr Phe Lys Ala

180 185 190

Gly Ala Tyr Thr Gln Ala Asn Cys Ser Asn Ser Ser Pro Cys Ser Ser

195 200 205

Ser Asn Tyr Gly Gln Val Ser Leu Tyr Lys Leu Gln Val Thr His Ser

210 215 220

<210> 11

<211> 224

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Ala Glu Pro Cys Asp Tyr Pro Ala Gln Gln Leu Asp Leu Thr Asp Trp

1 5 10 15

Lys Val Thr Leu Pro Ile Gly Ser Ser Gly Lys Pro Ser Glu Ile Glu

20 25 30

Gln Pro Ala Leu Asp Thr Phe Ala Thr Ala Pro Trp Phe Gln Val Asn

35 40 45

Ala Lys Cys Thr Gly Val Gln Phe Arg Ala Ala Val Asn Gly Val Thr

50 55 60

Thr Ser Gly Ser Gly Tyr Pro Arg Ser Glu Leu Arg Glu Met Thr Asp

65 70 75 80

Gly Gly Glu Glu Lys Ala Ser Trp Ser Ala Thr Ser Gly Thr His Thr

85 90 95

Met Val Phe Arg Glu Ala Phe Asn His Leu Pro Glu Val Lys Pro His

100 105 110

Leu Val Gly Ala Gln Ile His Asp Gly Asp Asp Glu Val Thr Val Phe

115 120 125

Arg Leu Glu Gly Thr Ser Leu Tyr Ile Thr Lys Gly Asp Asp Thr His

130 135 140

His Lys Leu Val Thr Ser Asp Tyr Lys Leu Asn Thr Val Phe Glu Gly

145 150 155 160

Lys Phe Val Val Ser Gly Gly Lys Ile Lys Val Tyr Tyr Asn Gly Val

165 170 175

Leu Gln Thr Thr Ile Ser His Thr Ser Ser Gly Asn Tyr Phe Lys Ala

180 185 190

Gly Ala Tyr Thr Gln Ala Asn Cys Ser Asn Ser Ser Pro Cys Ser Ser

195 200 205

Ser Asn Tyr Gly Gln Val Ser Leu Tyr Lys Leu Gln Val Thr His Ser

210 215 220

<210> 12

<211> 224

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Ala Glu Pro Cys Asp Tyr Pro Ala Gln Gln Leu Asp Leu Thr Asp Trp

1 5 10 15

Lys Val Thr Leu Pro Ile Gly Ser Ser Gly Lys Pro Ser Glu Ile Glu

20 25 30

Gln Pro Ala Leu Asp Thr Phe Ala Thr Ala Pro Trp Phe Gln Val Asn

35 40 45

Ala Lys Cys Thr Gly Val Gln Phe Arg Ala Ala Val Asn Gly Val Thr

50 55 60

Thr Ser Gly Ser Gly Tyr Pro Arg Ser Glu Leu Arg Glu Met Thr Asp

65 70 75 80

Gly Gly Glu Glu Lys Ala Ser Trp Ser Ala Thr Ser Gly Thr His Thr

85 90 95

Met Val Phe Arg Glu Ala Phe Asn His Leu Pro Glu Val Lys Pro His

100 105 110

Leu Val Gly Ala Gln Ile His Asp Gly Asp Asp His Val Thr Val Phe

115 120 125

Arg Leu Glu Gly Thr Ser Leu Tyr Ile Thr Lys Gly Asp Asp Thr His

130 135 140

His Lys Leu Val Thr Ser Asp Tyr Lys Leu Asn Thr Val Phe Glu Gly

145 150 155 160

Lys Phe Val Val Ser Gly Gly Lys Ile Lys Val Tyr Tyr Asn Gly Val

165 170 175

Leu Gln Thr Thr Ile Ser His Thr Ser Ser Gly Asn Tyr Phe Lys Ala

180 185 190

Gly Ala Tyr Thr Gln Ala Asn Cys Ser Asn Ser Ser Pro Cys Ser Ser

195 200 205

Ser Asn Tyr Gly Gln Val Ser Leu Tyr Lys Leu Gln Val Thr His Ser

210 215 220

<210> 13

<211> 224

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Ala Glu Pro Cys Asp Tyr Pro Ala Gln Gln Leu Asp Leu Thr Asp Trp

1 5 10 15

Lys Val Thr Leu Pro Ile Gly Ser Ser Gly Lys Pro Ser Glu Ile Glu

20 25 30

Gln Pro Ala Leu Asp Thr Phe Ala Thr Ala Pro Trp Phe Gln Val Asn

35 40 45

Ala Lys Cys Thr Gly Val Gln Phe Arg Ala Ala Val Asn Gly Val Thr

50 55 60

Thr Ser Gly Ser Gly Tyr Pro Arg Ser Glu Leu Arg Glu Met Thr Asp

65 70 75 80

Gly Gly Glu Glu Lys Ala Ser Trp Ser Ala Thr Ser Gly Thr His Thr

85 90 95

Met Val Phe Arg Glu Ala Phe Asn His Leu Pro Glu Val Lys Pro His

100 105 110

Leu Val Gly Ala Gln Ile His Asp Gly Asp Asp Lys Val Thr Val Phe

115 120 125

Arg Leu Glu Gly Thr Ser Leu Tyr Ile Thr Lys Gly Asp Asp Thr His

130 135 140

His Lys Leu Val Thr Ser Asp Tyr Lys Leu Asn Thr Val Phe Glu Gly

145 150 155 160

Lys Phe Val Val Ser Gly Gly Lys Ile Lys Val Tyr Tyr Asn Gly Val

165 170 175

Leu Gln Thr Thr Ile Ser His Thr Ser Ser Gly Asn Tyr Phe Lys Ala

180 185 190

Gly Ala Tyr Thr Gln Ala Asn Cys Ser Asn Ser Ser Pro Cys Ser Ser

195 200 205

Ser Asn Tyr Gly Gln Val Ser Leu Tyr Lys Leu Gln Val Thr His Ser

210 215 220

<210> 14

<211> 224

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Ala Glu Pro Cys Asp Tyr Pro Ala Gln Gln Leu Asp Leu Thr Asp Trp

1 5 10 15

Lys Val Thr Leu Pro Ile Gly Ser Ser Gly Lys Pro Ser Glu Ile Glu

20 25 30

Gln Pro Ala Leu Asp Thr Phe Ala Thr Ala Pro Trp Phe Gln Val Asn

35 40 45

Ala Lys Cys Thr Gly Val Gln Phe Arg Ala Ala Val Asn Gly Val Thr

50 55 60

Thr Ser Gly Ser Gly Tyr Pro Arg Ser Glu Leu Arg Glu Met Thr Asp

65 70 75 80

Gly Gly Glu Glu Lys Ala Ser Trp Ser Ala Thr Ser Gly Thr His Thr

85 90 95

Met Val Phe Arg Glu Ala Phe Asn His Leu Pro Glu Val Lys Pro His

100 105 110

Leu Val Gly Ala Gln Ile His Asp Gly Asp Asp Arg Val Thr Val Phe

115 120 125

Arg Leu Glu Gly Thr Ser Leu Tyr Ile Thr Lys Gly Asp Asp Thr His

130 135 140

His Lys Leu Val Thr Ser Asp Tyr Lys Leu Asn Thr Val Phe Glu Gly

145 150 155 160

Lys Phe Val Val Ser Gly Gly Lys Ile Lys Val Tyr Tyr Asn Gly Val

165 170 175

Leu Gln Thr Thr Ile Ser His Thr Ser Ser Gly Asn Tyr Phe Lys Ala

180 185 190

Gly Ala Tyr Thr Gln Ala Asn Cys Ser Asn Ser Ser Pro Cys Ser Ser

195 200 205

Ser Asn Tyr Gly Gln Val Ser Leu Tyr Lys Leu Gln Val Thr His Ser

210 215 220

<210> 15

<211> 224

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Ala Glu Pro Cys Asp Tyr Pro Ala Gln Gln Leu Asp Leu Thr Asp Trp

1 5 10 15

Lys Val Thr Leu Pro Ile Gly Ser Ser Gly Lys Pro Ser Glu Ile Glu

20 25 30

Gln Pro Ala Leu Asp Thr Phe Ala Thr Ala Pro Trp Phe Gln Val Asn

35 40 45

Ala Lys Cys Thr Gly Val Gln Phe Arg Ala Ala Val Asn Gly Val Thr

50 55 60

Thr Ser Gly Ser Gly Tyr Pro Arg Ser Glu Leu Arg Glu Met Thr Asp

65 70 75 80

Gly Gly Glu Glu Lys Ala Ser Trp Ser Ala Thr Ser Gly Thr His Thr

85 90 95

Met Val Phe Arg Glu Ala Phe Asn His Leu Pro Glu Val Lys Pro His

100 105 110

Leu Val Gly Ala Gln Ile His Asp Gly Asp Asp Ala Val Thr Val Phe

115 120 125

Arg Leu Glu Gly Thr Ser Leu Tyr Ile Thr Lys Gly Asp Asp Thr His

130 135 140

His Lys Leu Val Thr Ser Asp Tyr Lys Leu Asn Thr Val Phe Glu Gly

145 150 155 160

Lys Phe Val Val Ser Gly Gly Lys Ile Lys Val Tyr Tyr Asn Gly Val

165 170 175

Leu Gln Thr Thr Ile Ser His Thr Ser Ser Gly Asn Tyr Phe Lys Ala

180 185 190

Gly Ala Tyr Thr Gln Ala Asn Cys Ser Asn Ser Ser Pro Cys Ser Ser

195 200 205

Ser Asn Tyr Gly Gln Val Ser Leu Tyr Lys Leu Gln Val Thr His Ser

210 215 220

<210> 16

<211> 224

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Ala Glu Pro Cys Asp Tyr Pro Ala Gln Gln Leu Asp Leu Thr Asp Trp

1 5 10 15

Lys Val Thr Leu Pro Ile Gly Ser Ser Gly Lys Pro Ser Glu Ile Glu

20 25 30

Gln Pro Ala Leu Asp Thr Phe Ala Thr Ala Pro Trp Phe Gln Val Asn

35 40 45

Ala Lys Cys Thr Gly Val Gln Phe Arg Ala Ala Val Asn Gly Val Thr

50 55 60

Thr Ser Gly Ser Gly Tyr Pro Arg Ser Glu Leu Arg Glu Met Thr Asp

65 70 75 80

Gly Gly Glu Glu Lys Ala Ser Trp Ser Ala Thr Ser Gly Thr His Thr

85 90 95

Met Val Phe Arg Glu Ala Phe Asn His Leu Pro Glu Val Lys Pro His

100 105 110

Leu Val Gly Ala Gln Ile His Asp Gly Asp Asp Pro Val Thr Val Phe

115 120 125

Arg Leu Glu Gly Thr Ser Leu Tyr Ile Thr Lys Gly Asp Asp Thr His

130 135 140

His Lys Leu Val Thr Ser Asp Tyr Lys Leu Asn Thr Val Phe Glu Gly

145 150 155 160

Lys Phe Val Val Ser Gly Gly Lys Ile Lys Val Tyr Tyr Asn Gly Val

165 170 175

Leu Gln Thr Thr Ile Ser His Thr Ser Ser Gly Asn Tyr Phe Lys Ala

180 185 190

Gly Ala Tyr Thr Gln Ala Asn Cys Ser Asn Ser Ser Pro Cys Ser Ser

195 200 205

Ser Asn Tyr Gly Gln Val Ser Leu Tyr Lys Leu Gln Val Thr His Ser

210 215 220

<210> 17

<211> 243

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Met Lys Gly Arg Leu Lys Lys Trp Cys Ser Gly Phe Leu Ile Ala Met

1 5 10 15

Leu Val Ser Thr Pro Thr Gly Met Val Asn Ala Ala Ser Leu Leu Pro

20 25 30

Ser Asp Ile Leu Asp Leu Thr Asn Trp Lys Leu Thr Leu Pro Ile Asn

35 40 45

Asp Ala Glu Glu Ile Thr Gln Pro Glu Leu Asp Ser Tyr Glu His Ser

50 55 60

Glu Tyr Phe His Val Asn Asp Asp Gly Asp Ala Val Val Phe Lys Ala

65 70 75 80

His Cys Gly Gly Asp Thr Thr Glu Gly Ser Ser Tyr Pro Arg Cys Glu

85 90 95

Leu Arg Glu Met Thr Asn Asp Gly Gln Asp Lys Ala Ser Trp Ser Thr

100 105 110

Thr Ser Gly Thr His Thr Met Ile Ile Asp Gln Lys Ile Thr His Leu

115 120 125

Pro Glu Val Lys Asp His Val Val Val Gly Gln Ile His Asp Ser Asp

130 135 140

Asp Asp Val Ile Met Ile Arg Leu Glu Gly Asn His Leu Phe Val Glu

145 150 155 160

Gly Asp Gly Glu Glu Leu Ala Asp Leu Asp Thr Asp Tyr Glu Leu Gly

165 170 175

Thr Arg Phe Thr Val Lys Ile Val Ala Ser Gly Gly Lys Ile Lys Val

180 185 190

Tyr Tyr Asn Gly Asp Leu Lys Leu Thr Tyr Asn Lys Ser Val Ser Gly

195 200 205

Cys Tyr Phe Lys Ala Gly Met Tyr Thr Gln Ser Asn Thr Ser Lys Gly

210 215 220

Asp Ser Glu Asp Cys Tyr Gly Glu Asn Glu Ile Tyr Asn Leu Val Val

225 230 235 240

Thr His Ser

<210> 18

<211> 243

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Met Lys Gly Arg Leu Lys Lys Trp Cys Ser Gly Phe Leu Ile Ala Met

1 5 10 15

Leu Val Ser Thr Pro Thr Gly Met Val Asn Ala Ala Ser Leu Leu Pro

20 25 30

Ser Asp Ile Leu Asp Leu Thr Asn Trp Lys Leu Thr Leu Pro Ile Asn

35 40 45

Asp Ala Glu Glu Ile Thr Gln Pro Glu Leu Asp Ser Tyr Glu His Ser

50 55 60

Glu Tyr Phe His Val Asn Asp Asp Gly Asp Ala Val Val Phe Lys Ala

65 70 75 80

His Cys Gly Gly Asp Thr Thr Glu Gly Ser Ser Tyr Pro Arg Cys Glu

85 90 95

Leu Arg Glu Met Thr Asn Asp Gly Gln Asp Lys Ala Ser Trp Ser Thr

100 105 110

Thr Ser Gly Thr His Thr Met Ile Ile Asp Gln Lys Ile Thr His Leu

115 120 125

Pro Glu Val Lys Asp His Val Val Val Gly Gln Ile His Asp Ser Asp

130 135 140

Asp His Val Ile Met Ile Arg Leu Glu Gly Asn His Leu Phe Val Glu

145 150 155 160

Gly Asp Gly Glu Glu Leu Ala Asp Leu Asp Thr Asp Tyr Glu Leu Gly

165 170 175

Thr Arg Phe Thr Val Lys Ile Val Ala Ser Gly Gly Lys Ile Lys Val

180 185 190

Tyr Tyr Asn Gly Asp Leu Lys Leu Thr Tyr Asn Lys Ser Val Ser Gly

195 200 205

Cys Tyr Phe Lys Ala Gly Met Tyr Thr Gln Ser Asn Thr Ser Lys Gly

210 215 220

Asp Ser Glu Asp Cys Tyr Gly Glu Asn Glu Ile Tyr Asn Leu Val Val

225 230 235 240

Thr His Ser

Claims (8)

1. A mutant alginate lyase for relieving divalent metal ion dependence, which is characterized in that: the mutant is alginate lyase amino acid sequence which takes polyG as a specific substrate when carrying out enzymolysis in PL7 family alginate lyase, at least in the same three-dimensional structure position with aspartic acid D146 in AlgAT5, or the amino acid at D146 position is mutated into amino acid with positive charge, glycine, alanine, valine, leucine or isoleucine based on the multi-sequence alignment of the structure.

2. The mutant alginate lyase released from the dependence of divalent metal ion as set forth in claim 1, wherein: the algin lyase is AlgAT5, alyPG, Alg _ M3, Alg, Alyl1, AlyA, AlyPI, Algb or AlyA1, AlyQ, AlgB, Alg2A or AlyVGI.

3. The mutant alginate lyase released from the dependence of divalent metal ions as set forth in claim 2, wherein: the algin lyase is AlgAT5, alyPG, Alyl1, AlyA, AlyPI and AlyQ.

4. The divalent metal ion-dependent alginate lyase mutant according to any one of claims 1 to 3, wherein: the amino acids in the lyase amino acid sequence that are at the same three-dimensional structural position as aspartic acid D146 in AlgAT5, or that are structurally based on multiple sequence alignments at position D146 are aspartic acid D or glutamic acid E mutated to a positively charged amino acid (lysine, histidine or arginine), as well as glycine, alanine, valine, leucine, isoleucine.

5. An expression vector, characterized in that: an expression vector comprising any one of the mutants of claim 1.

6. A genetically engineered bacterium, which is characterized in that: a genetically engineered bacterium containing the expression vector according to claim 5.

7. Use of a mutant according to claim 1, wherein: the use of the mutant of any one of claims 1-4 for the preparation of alginate oligosaccharides by removing divalent metal ion dependent catalytic algin.

8. A method for preparing alginate oligosaccharides by catalyzing algin for relieving divalent metal ion dependence is characterized in that: adding the mutant of claim 1 to alginate in NaAC-HAC buffer solution at pH 5-8, 0.1-0.3M NaCl, 0.05-5mM CaCl₂Under the condition of 50-80 ℃, the mutant is utilized to remove the divalent metal ion dependence of the catalytic algin reaction to prepare the alginate oligosaccharide.

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