CN110004134B - Alginate lyase mutant and application thereof - Google Patents

Abstract

The invention relates to an alginate lyase mutant and application, belonging to the field of enzyme engineering and genetic engineering, wherein the mutant mutates E at position 226 to K on the basis of a sequence SEQ ID NO.3, the enzyme activity of the mutant is improved by 1.11 times through expression, and the specific activity of a crude enzyme solution is improved by 1.03 times after purification compared with that of a truncated enzyme AlgL-T157N; the optimum temperature of the mutant is 55 ℃, the enzyme activity is basically unchanged after 1 h of heat preservation within the pH of 6.0-8.0, and the mutant has stronger pH stability; catalytic efficiency of mutants on sodium alginate, Poly M and Poly GK _cat /K _m Respectively 10.22 times, 8.59 times and 2.97 times higher than that of AlgL-T157N. The invention adopts a method combining structural sequence analysis and software auxiliary screening to determine mutation sites, obtains mutants with improved catalytic activity by PCR site-specific mutagenesis, and lays a foundation for further industrial application.

Description

Alginate lyase mutant and application thereof

Technical Field

The invention relates to an alginate lyase mutant and application thereof, belonging to the field of enzyme engineering and genetic engineering.

Background

Alginate lyase is a lyase for degrading alginate in brown algae, mainly catalyzes depolymerization of alginate through beta-elimination reaction of 4-O-glycosidic bond, and generates an oligouronic acid structure with unsaturated double bond between non-reducing end C-4 and C-5. Specific lyases can be classified into poly M, poly G and poly MG according to the substrate preference of alginate lyase; according to different action modes, the alginate polysaccharide can be divided into endonuclease and exonuclease, the endonuclease mainly cuts glycosidic bonds in the alginate polymer to generate unsaturated oligosaccharides, and the exonuclease further degrades the alginate oligosaccharides into monosaccharides.

At present, with the deep research on the brown alga oligosaccharide, more and more biological activities are found, such as anti-tumor, anti-allergy, anti-bacterial, blood coagulation, anti-stress, anti-oxidation and the like, and the wide application value of the biological activities promotes researchers to further explore the preparation method of the brown alga oligosaccharide. The traditional degradation method of brown algae oligosaccharide mainly comprises the following steps: acid hydrolysis, alkaline hydrolysis and oxidative degradation, and the degradation methods not only easily cause environmental pollution, but also the obtained oligosaccharide has low biological activity and poor effect. On the contrary, the preparation of the brown algae oligosaccharide by the enzyme method has the advantages of mild conditions, high yield, easy control, no environmental pollution and better oligosaccharide bioactivity, thereby gradually replacing the traditional chemical degradation method to become a main preparation method of the oligosaccharide. However, most of the currently screened algin lyase with poor substrate specificity and low enzyme activity has few and few algin lyase products applied to industrialization, so obtaining the algin lyase with high activity and wide substrate specificity becomes the current research focus, and especially the improvement of the catalytic activity of the algin lyase by means of genetic engineering and protein engineering is favored by most researchers.

The invention adopts a method of combining structural sequence analysis and Discovery Studio software auxiliary screening to determine mutation sites, obtains mutants with obviously improved catalytic activity by PCR site-specific mutagenesis, and lays a foundation for further industrial application of the mutants.

Disclosure of Invention

The invention aims to provide an alginate lyase mutant and application thereof, wherein a method of combining structural sequence analysis and Discovery Studio software auxiliary screening is adopted to determine mutation sites, and mutants with obviously improved catalytic activity are obtained through PCR (polymerase chain reaction) site-specific mutation, so that a foundation is laid for further industrial application of the mutants.

The following technical scheme is adopted for achieving the purpose:

alginate lyase gene of the present inventionAlgL(the nucleotide sequence is shown as SEQ ID NO. 1) is derived from seawater isolated from OdontobiasPseudoalteromonassp, zb7-4, 1203 bp in total length, encoding 400 amino acids, the 1 st to 31 st amino acids as signal peptide, the 32 nd to 131 rd amino acids as carbohydrate binding domain (i.e. CBM domain), and the 197 nd to 385 th amino acids as catalytic domain. Between 157-and 158-amino acidsTruncating and truncating 1-157 th amino acid to obtain the alginate lyase truncating enzyme AlgL-T157N, wherein the amino acid sequence of the truncating enzyme is shown as SEQ ID NO. 3.

Submitting an amino acid sequence of the truncated enzyme AlgL-T157N to a SWISS MODEL online server for homologous modeling, carrying out protein MODEL structure evaluation by adopting a Verify-3D, ERRAT program and a PROCHECK program in a Saves server, and carrying out molecular docking on a MODEL and mannuronic acid tetrasaccharide micromolecule Tetra by adopting AutoDock Tools to obtain a docking result (shown in figure 1). The method of combining structural sequence analysis and Discovery Studio software assisted screening is adopted to determine mutation sites, and on the basis of the amino acid sequence SEQ ID No.3 of AlgL-T157N, the 226 th glutamic acid is mutated into lysine at fixed points to obtain the alginate lyase mutant E226K with obviously improved catalytic activity, wherein the amino acid sequence is shown as SEQ ID No.4, and the nucleotide sequence is shown as SEQ ID No. 5.

Compared with AlgL-T157N, the enzyme activity of the mutant E226K is improved by 1.11 times, and the specific activity is improved by 1.03 times; the optimum temperature is 55 ℃, the temperature is increased by 5 ℃ compared with AlgL-T157N, the residual enzyme activity reaches more than 90% after heat preservation is carried out for 1 h within the pH range of 6.0-8.0, and the pH stability is stronger. Kinetic parameter measurement shows that E226K is applied to 3 substrates, i.e. sodium alginate, Poly M and Poly GK _m The values were reduced by 89.69%, 88.14%, 61.11%, respectively, indicating a significant increase in substrate affinity;K _cat 118.02%, 114.62% and 151.37% of AlgL-T157N, respectively,K _cat / K _m the values are respectively 10.22 times, 8.59 times and 2.97 times higher than those of AlgL-T157N.

Preparing brown algae oligosaccharide by respectively adopting mutant E226K for enzymolysis and acidolysis, and carrying out ESI-MS mass spectrometry on the product of the brown algae oligosaccharide, wherein the result shows that the brown algae oligosaccharide obtained by the enzymolysis method is unsaturated oligosaccharide and consists of monosaccharide, disaccharide and trisaccharide; the brown algae oligosaccharide obtained by acidolysis is saturated oligosaccharide, consists of monosaccharide and disaccharide, and is mainly monosaccharide. According to research, unsaturated oligosaccharides have higher biological activity than saturated oligosaccharides, so that the biological activity of the oligosaccharides can be better maintained by preparing the brown algae oligosaccharides by an enzymatic hydrolysis method.

The invention has the advantages that:

compared with AlgL-T157N, the enzyme activity of the mutant E226K is improved by 1.11 times, and the specific activity is improved by 1.03 times; the optimum temperature of the mutant E226K is 55 ℃, the temperature is increased by 5 ℃ compared with AlgL-T157N, the residual enzyme activity reaches more than 90% after heat preservation is carried out for 1 h within the pH range of 6.0-8.0, and the mutant has stronger pH stability; and the catalytic efficiency of the mutant on 3 substrates of sodium alginate, Poly M and Poly GK _cat / K _m Respectively 10.22 times, 8.59 times and 2.97 times higher than that of AlgL-T157N. The brown algae oligosaccharide prepared by the enzymolysis method can better retain the biological activity of the oligosaccharide.

Drawings

FIG. 1 depicts the docking scheme of the truncated enzyme AlgL-T157N with the mannuronic acid tetrasaccharide molecule Tetra. Note: glu226 is a site-directed mutation site.

FIG. 2 shows the electrophoretogram of the recombinant plasmid of the truncated enzyme AlgL-T157N and mutant E226K. M: 1 Kb DNA Ladder; lane 1: an AlgL-T157N recombinant plasmid; lane 2: E226K recombinant plasmid.

FIG. 3 SDS-PAGE analysis of induced expression and purification of the truncatase AlgL-T157N and mutant E226K. M: protein molecular weight standards (180 kDa protein Marker); lane 1: supernatant of non-induced recombinant bacteria; lane 2: supernatant of IPTG induced AlgL-T157N; lane 3: E226K supernatant after IPTG induction; lane 4: purified AlgL-T157N target protein; lane 5: purified E226K protein of interest.

FIG. 4 optimal reaction pH for the truncatase AlgL-T157N and mutant E226K.

FIG. 5 optimal reaction temperatures for the truncatase AlgL-T157N and mutant E226K.

FIG. 6 the pH stability of the truncatase AlgL-T157N and mutant E226K.

FIG. 7 temperature stability of the truncatase AlgL-T157N and mutant E226K.

FIG. 8 is an electrospray mass spectrometry analysis chart of the enzymatic product.

FIG. 9 is an electrospray mass spectrometry analysis chart of an acid hydrolysis product.

FIG. 10 shows a comparison of the hydroxyl radical scavenging effect of sodium alginate, enzymatic and acid hydrolyzed alginate-derived oligosaccharides.

FIG. 11 shows a comparison of the action of sodium alginate, enzymatically hydrolyzed alginate oligosaccharide product, and acidolyzed alginate oligosaccharide product on scavenging ABTS free radicals.

FIG. 12 shows the comparison of the reducing power of sodium alginate, enzymatic and acid hydrolyzed alginate-derived oligosaccharides.

Detailed Description

EXAMPLE 1 construction of alginate lyase mutant E226K

Alginate lyase gene of the present inventionAlgL(the nucleotide sequence is shown as SEQ ID NO. 1) is derived from seawater isolated from OdontobiasPseudoalteromonassp, zb7-4, 1203 bp in total length, encoding 400 amino acids, the 1 st to 31 st amino acids as signal peptide, the 32 nd to 131 rd amino acids as carbohydrate binding domain (i.e. CBM domain), and the 197 nd to 385 th amino acids as catalytic domain. Truncating between 157-158 amino acids and truncating 1-157 amino acids to obtain the alginate lyase truncatase AlgL-T157N with the sequence shown in SEQ ID NO. 3.

Through sequence alignment, it is found that AlgL-T157N and 4Q8K have the same amino acid sequence at the site of the catalytic domain, and therefore, based on the study of the structure and catalytic mechanism of 4Q8K protein, in combination with the docking results (fig. 1), virtual alanine scanning and saturation mutation are performed on amino acids in 8 a near the catalytic cavity of AlgL-T157N by using Discovery Studio 2016 software, an optimal mutant amino acid is determined, and finally, the mutant E226K is obtained through screening.

Design the primer containing the mutation site to recombine the plasmid pET-22b (+) -AlgL-T157NAs a template, the PCR technology is adopted to amplify the mutant recombinant plasmid pET-22b (+) -E226KIs transformed intoE. coliBL21(DE3) to obtain mutant recombinant engineering bacteria. The method comprises the following specific steps:

to contain the recombinant plasmid pET-22b (+) -AlgL-T157NLaboratory preservation of the strainsE.coliTop10 was the starting strain, and the recombinant plasmid was extracted using a plasmid kit from OMEGA. Designing a specific mutation primer, mutating 226 th glutamic acid of alginate lyase into lysine, wherein the positive primer and the negative primer are as follows:

E226K-F:5’-AGGTTAAAAAGAGTTTACGCGTTGCTATGA-3’，

E226K-R:5’-CGCGTAAACTCTTTTTAACCTTATACTCATGAC-3’。

underlined letters represent codons for the mutated amino acid lysine. By extracting pET-22b (+) -AlgL-T157NPlasmid is used as a template, the AlgL-T157N gene is subjected to site-directed mutagenesis by adopting a PCR technology, and a PCR amplification system comprises: 5 × TransStart FastPfu Fly Buffer 10 μ L, High Pure dNTPs (2.5 mmol/L) 4 μ L, PCR Stimulant (5 ×) 5 μ L, MgSO 5 ×)₄ 1 μL ，pET-22b(+)- E226K 1 μL，E226K-F (10 µmol/L) 2 μL，E226K-R (10 µmol/L) 2 μL，TransStart FastPfu Fly DNA Polymerase 1 μL，ddH₂And O is supplemented to 50 mu L.

PCR reaction parameters: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 15 s, annealing at 55 ℃ for 15 s, extension at 72 ℃ for 2 min, and 30 cycles; keeping the temperature at 72 ℃ for 5 min; storing at 4 ℃. 10 uL of the PCR amplification product was detected by electrophoresis on a 1% agarose gel (see FIG. 2). And adding 1 mu L of DMT enzyme into the residual PCR product, uniformly mixing, preserving the heat for 1 h at 37 ℃, and digesting the original template. 2 μ L of the digest was added to 100 μ L E.coli Slightly blowing, stirring and mixing Top10 competent cells, and performing ice bath for 30 min; heat-shocking at 42 deg.C for 45-90 s, and standing on ice for 2 min; adding 800 muL LB culture medium, culturing at 37 deg.C and 200 r/min for 45-60 min; centrifugation at 6000 Xg for 2 min, aspiration of 80. mu.L of supernatant, resuspension of the cells, and plating on LB (containing 100. mu.g/mL Amp)⁺) The plates were incubated at 37 ℃ for 12 h. And picking out clones for sequencing verification.

The nucleotide sequence of the mutant E226K is shown as SEQ ID NO.5, and the corresponding amino acid sequence is shown as SEQ ID NO. 4.

Example 2 inducible expression and purification of the truncating enzyme AlgL-T157N and mutant E226K

Extraction of recombinant plasmid pET-22b (+) -AlgL-T157NAnd pET-22b (+) -E226KRespectively transfer them intoE.coliIn BL21(DE3) competent cells, positive recombinants were picked up in 5mL LB (Amp)⁺) Culturing in liquid culture medium at 37 deg.C and 200 r/min for 12 h, and inoculating with 1% inoculum size to 25 mL LB (Amp)⁺) Culturing OD in culture medium at 37 deg.C and 200 r/min₆₀₀To a concentration of between 0.6 and 0.8, IPTG (final concentration of 0.2 mmol/L) is addedInducing at 20 deg.C for 24 hr at 200 r/min, and measuring alginate lyase activity. LB medium without IPTG addition after the same inoculation was used as a blank. The enzyme activity of the crude enzyme solution of the mutant E226K is detected to be 7.14 +/-0.09U/mL, and is improved by 1.11 times compared with that of AlgL-T157N (3.38 +/-0.11U/mL).

The activity of the alginate lyase is determined by a DNS method. Adding 0.1 mL enzyme solution into 0.9 mL substrate of 0.3% sodium alginate, keeping the temperature at 50 deg.C for 15 min, adding 1.5 mL DNS to stop reaction, boiling for 5 min for color development, cooling, and determining OD₅₄₀. Definition of enzyme activity unit: under certain reaction conditions, the enzyme amount required by catalyzing sodium alginate to generate 1 mu mol of reducing sugar per min is taken as an enzyme activity unit U.

The enzyme activity calculation formula is as follows:

in the formula:

u: enzyme activity, unit U/mL

A: OD after blank subtraction₅₄₀

n: dilution factor

K: slope of standard curve

V: volume of enzyme solution, mL

T: reaction time, min

194.14: relative molecular weight of glucuronic acid

1000: and (4) quality conversion multiple.

Example 3 isolation and purification of the truncatase AlgL-T157N and mutant E226K

(1) DEAE anion column purification: protein purification Using AKTA purification System, DEAE FF (Hi Trap) was first used^TM5mL) pre-column, the column was equilibrated with McIlvaine buffer at a flow rate of 5 mL/min and a concentration of 50 mmol/L, pH 7.0.0, and the crude enzyme solution was filtered through a 0.22 μm filter. Loading the obtained filtrate on anion column, balancing the column at flow rate of 2 mL/min, after balancing 4-5 column volumes, gradient eluting with 1 mol/L NaCl solution (50 mmol/L, prepared from McIlvaine buffer solution with pH of 7.0) prepared in advanceAnd (3) detecting protein through a UV detector by using a column, collecting protein peaks eluted by different gradients, detecting enzyme activity, and detecting the protein purity by using SDS-PAGE.

(2) And (3) nickel column purification: the protein preliminarily purified by DEAE was loaded on a nickel column (HisTrap)^TM5mL), NTA 0 (20 mmol/L phosphate buffer solution, 0.5 mol/L NaCl, 10% glycerol) is used as an equilibrium buffer solution, NTA 0 is used as a dissolving solution to prepare 500 mmol/L imidazole, the prepared imidazole gradient is used for eluting a nickel column, elution proteins with different concentrations are collected, the enzyme activity is determined, and SDS-PAGE is carried out to detect the protein purity.

(3) Desalting: the protein is subjected to imidazole removal by using a Desalting column (HiTrap desaling, 5mL), 1 mL of enzyme solution purified by a nickel column is loaded on the Desalting column, 50 mmol/L, pH 7.0.0 McIlvaine buffer solution is used as equilibrium solution, the flow rate is set to be 2 mL/min, protein peaks are collected, enzyme activity detection is carried out, and SDS-PAGE electrophoresis is carried out to obtain single-band protein (shown in figure 3). After purification, the specific activities of the truncated enzyme AlgL-T157N and the mutant E226K are respectively 77.81U/mL and 157.93U/mL, and the specific activities are improved by 1.03 times after mutation.

Example 4 enzymatic Properties of the truncatase AlgL-T157N and mutant E226K

And (3) determining the optimum reaction pH: respectively adding the truncatase AlgL-T157N and the mutant E226K into buffers (McIlvaine buffer with the pH of 4.0-7.0 and Na with the pH of 7.0-8.0) with different pH values₂HPO₄-NaH₂PO₄Buffer solution, Gly-NaOH buffer solution with the pH of 8.0-11.0) and sodium alginate substrate with the concentration of 3 percent, wherein the reaction temperature is 50 ℃, and the concentration of the buffer solution is 50 mmol/L. And (3) determining enzyme activity by adopting a DNS method, setting the enzyme activity with the highest light absorption value as 100%, calculating relative enzyme activity under different pH values, and determining the optimum reaction pH value. The results are shown in FIG. 4, where the optimum reaction pH was 7.0 for both enzymes.

Determination of optimum reaction temperature: under the condition of optimal reaction pH, the residual enzyme activities of AlgL-T157N and E226K at different temperatures are respectively measured, and the highest enzyme activity is set as 100%. As shown in FIG. 5, the optimum reaction temperature for AlgL-T157N was 50 ℃ and that for E226K was 55 ℃.

And (3) measuring the pH stability: mixing AlgL-T157N and E226K with the buffer solutions with different pH values according to a certain proportion, preserving the temperature for 1 h at 37 ℃, measuring the enzyme activity of the buffer solutions under the conditions of the optimal reaction temperature and the optimal reaction pH value, defining the maximum enzyme activity to be 100%, and calculating the residual enzyme activity under different pH values. As shown in FIG. 6, after incubation for 1 h at 37 ℃, the residual enzyme activities of AlgL-T157N and E226K in the pH range of 6.0-8.0 reach more than 90%, which indicates that the enzyme has stronger stability in the pH range.

And (3) measuring the temperature stability: respectively measuring the residual enzyme activities of AlgL-T157N and E226K which are preserved for 30 min at different temperatures under the conditions of the optimal reaction temperature and the optimal reaction pH, and respectively taking the enzyme activities which are not processed as a reference. The results are shown in FIG. 7, where the stability of both enzymes is better below 40 ℃ and the enzyme activity starts to decrease above 40 ℃.

Example 5 determination of catalytic kinetic parameters of alginate lyase

The specific method for determining the catalytic kinetic parameters is as follows:

sodium alginate, sodium polymannuronate (Poly M) and sodium polyguluronate (Poly G) substrates were prepared at different final concentrations (0.3, 0.45, 0.6, 1, 2, 3 mg/mL) with 50 mmol/L McIlvaine buffer, respectively. The enzyme activities of AlgL-T157N and E226K were determined at 45 ℃. Calculating by using a Lineweaver-Burk method, and plotting by taking reciprocal of reaction rate as ordinate and reciprocal of substrate concentration as abscissaK _m 、V _max 、K _cat AndK _cat /K _m 。

the results of the catalytic kinetic parameter measurements are shown in Table 1, and compared with the truncated enzyme AlgL-T157N, the mutant E226K shows the results of the measurement on 3 substrates, i.e., sodium alginate, Poly M and Poly GK _m The values were reduced by 89.69%, 88.14%, 61.11%, respectively, indicating that the mutations resulted in a significant increase in substrate affinity of the enzyme. By catalytic constantK _catCompared with the prior art, the catalytic constants after mutation are respectively 118.02%, 114.62% and 151.37% of AlgL-T157N, and the catalytic constants of 3 substratesK _cat / K _m The values are increased by 10.22, 8.59 and 2.97 times respectively.

TABLE 1 catalytic kinetic parameters of the truncatase AlgL-T157N and mutant E226K

Example 6 preparation and composition analysis of Brown algae oligosaccharides

And (3) carrying out enzymolysis preparation on the brown algae oligosaccharide. Sodium alginate is prepared into a reaction substrate with the concentration of 1.2 percent by using 50 mmol/L McIlvaine buffer solution with the pH value of 7.0. Preheating at 40 deg.C for 5 min, adding a certain amount (0.5U/mL) of enzyme solution for reaction, performing enzymolysis for 24 hr, taking out 0.9 mL of enzymolysis substrate, adding 0.1 mL of enzyme solution, and measuring OD at 40 deg.C₂₃₅And after the light absorption value is basically unchanged, completely carrying out enzymolysis on the substrate, and carrying out boiling water bath for 5 min to terminate the reaction. Repeatedly extracting by Sevage method to remove protein, centrifuging to obtain supernatant, concentrating by rotary evaporation, precipitating oligosaccharide with anhydrous ethanol, drying sample, and quantifying by phenol-sulfuric acid method.

Acidolysis preparation of oligosaccharide. Preparing a sodium alginate substrate with the concentration of 1.5%, adjusting the pH value to 4.0 by hydrochloric acid, carrying out acidolysis for 4 h at 120 ℃, cooling to room temperature after the acidolysis is finished, adjusting the pH value to 7.0, centrifuging to remove precipitates, obtaining supernatant, carrying out rotary evaporation and concentration, precipitating oligosaccharides by using absolute ethyl alcohol, drying a sample for later use, and quantifying by adopting a phenol-sulfuric acid method.

ESI-MS analysis. And respectively carrying out mass spectrum analysis on the enzymolysis product and the acidolysis product. The mass spectrometry conditions were ionization mode: ESI-, high resolution full scan mode: m/z 100-800. As shown in FIG. 8, ESI-MS detected the presence of oligosaccharides with 3 degrees of polymerization, which were respectively monosaccharide 175.0327(M-1Na +), disaccharide 351.0565(M + H + -2Na +), 373.0385(M-1Na +), trisaccharide 527.0886(M +2H + -3Na +), 549.0706(M + H + -2Na +), 571.0525(M-1Na +), and unsaturated oligosaccharides. As shown in FIG. 9, oligosaccharides with 2 degrees of polymerization were detected as monosaccharides 193.0348(M-1Na +), disaccharides 369.0678(M + H + -2Na +), and 391.0500(M-1Na +), respectively, and saturated oligosaccharides.

Example 7 comparison of biological Activity of Brown algae oligosaccharides

The results (fig. 10, 11, and 12) of the oxidation resistance (hydroxyl radical scavenging action, ABTS radical scavenging action, and reducing power) of sodium alginate, the alginate oligosaccharide product obtained by enzymolysis, and the alginate oligosaccharide product obtained by acidolysis were compared with each other, and the oxidation resistance of the alginate oligosaccharide obtained by enzymolysis was higher than that of the acidolysis product and the unhydrolyzed sodium alginate polysaccharide. Therefore, the brown algae oligosaccharide prepared by the enzymolysis method can better keep the bioactivity of the oligosaccharide, which is probably because the unsaturated oligosaccharide obtained by the enzymolysis has higher bioactivity.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

SEQUENCE LISTING

<110> Fuzhou university

<120> alginate lyase mutant and application thereof

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aacaccagcg atgtaaggtt tgataatttt gccttggttg agggcagcgg cagtaatgat 480

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agtggtagca tttttgattt agaaggtgat aacccaaatc ctctcgttga cgatagcacc 600

ttagtgtttg tgccgttaga ggcacaacat attacgccta atggtaatgg ctggcgtcat 660

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gctacggtaa aagttgagat gtctgatggc ggaaaaacaa ttatatcgca gcaccatgct 780

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aatgaagaaa aatttgcttt gggtacaatg accagcggtg agacatttaa cttgcgggta 960

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Thr Ile Pro Ser Ser Ile Thr Ser Gly Ser Ile Phe Asp Leu Glu Gly

20 25 30

Asp Asn Pro Asn Pro Leu Val Asp Asp Ser Thr Leu Val Phe Val Pro

35 40 45

Leu Glu Ala Gln His Ile Thr Pro Asn Gly Asn Gly Trp Arg His Glu

50 55 60

Tyr Lys Val Lys Lys Ser Leu Arg Val Ala Met Thr Gln Thr Tyr Glu

65 70 75 80

Val Phe Glu Ala Thr Val Lys Val Glu Met Ser Asp Gly Gly Lys Thr

85 90 95

Ile Ile Ser Gln His His Ala Ser Asp Thr Gly Thr Ile Ser Lys Val

100 105 110

Tyr Val Ser Asp Thr Asp Glu Ser Gly Phe Asn Asp Ser Val Ala Asn

115 120 125

Asn Gly Ile Phe Asp Val Tyr Val Arg Leu Arg Asn Thr Ser Gly Asn

130 135 140

Glu Glu Lys Phe Ala Leu Gly Thr Met Thr Ser Gly Glu Thr Phe Asn

145 150 155 160

Leu Arg Val Val Asn Asn Tyr Gly Asp Val Glu Val Thr Ala Phe Gly

165 170 175

Asn Ser Phe Gly Ile Pro Val Glu Asp Asp Ser Gln Ser Tyr Phe Lys

180 185 190

Phe Gly Asn Tyr Leu Gln Ser Gln Asp Pro Tyr Thr Leu Asp Lys Cys

195 200 205

Gly Glu Ala Gly Asn Ser Asn Ser Phe Lys Asn Cys Phe Glu Asp Leu

210 215 220

Gly Ile Thr Glu Ser Lys Val Thr Met Thr Asn Val Ser Tyr Thr Arg

225 230 235 240

Glu Thr Asn

<210> 5

<211> 732

<212> DNA

<213> Artificial sequence

<400> 5

agtaatgatg gtggctcaga tggcggcagc gataactcaa atggttcaac aattcctagc 60

agcataacca gtggtagcat ttttgattta gaaggtgata acccaaatcc tctcgttgac 120

gatagcacct tagtgtttgt gccgttagag gcacaacata ttacgcctaa tggtaatggc 180

tggcgtcatg agtataaggt taaaaagagt ttacgcgttg ctatgactca aacctatgaa 240

gtgttcgaag ctacggtaaa agttgagatg tctgatggcg gaaaaacaat tatatcgcag 300

caccatgcta gcgataccgg cactatatct aaagtgtatg tgtcggatac tgatgaatcg 360

ggctttaatg atagcgtagc gaacaacgga atttttgatg tgtacgtacg tttacgtaat 420

accagcggta atgaagaaaa atttgctttg ggtacaatga ccagcggtga gacatttaac 480

ttgcgggtag ttaataacta cggcgatgta gaggttacgg cattcggtaa ctcgttcggt 540

ataccagtag aggatgattc gcagtcatac tttaagtttg gtaactacct gcaatcgcaa 600

gacccataca cattagataa atgtggtgag gccggaaact ctaactcgtt taaaaactgt 660

tttgaggatt taggcattac agagtcaaaa gtgacgatga ccaatgtgag ttatacgcgt 720

gaaactaatt aa 732

<210> 6

<211> 30

<212> DNA

<213> Artificial sequence

<400> 6

aggttaaaaa gagtttacgc gttgctatga 30

<210> 7

<211> 33

<212> DNA

<213> Artificial sequence

<400> 7

cgcgtaaact ctttttaacc ttatactcat gac 33

Claims (2)

1. An alginate lyase mutant, which is characterized in that: the amino acid sequence of the mutant is shown as SEQ ID NO.4, and the nucleotide sequence is shown as SEQ ID NO. 5.

2. The use of the alginate lyase mutant of claim 1 in the preparation of alginate oligosaccharides.

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