CN113046378B

CN113046378B - Incision alginate lyase, coding gene and application thereof

Info

Publication number: CN113046378B
Application number: CN202110203345.7A
Authority: CN
Inventors: 邵宗泽; 周梅先; 陈琳
Original assignee: Third Institute of Oceanography MNR
Current assignee: Third Institute of Oceanography MNR
Priority date: 2021-02-23
Filing date: 2021-02-23
Publication date: 2022-08-05
Anticipated expiration: 2041-02-23
Also published as: CN113046378A

Abstract

The invention discloses an incision alginate lyase, and a coding gene and application thereof. The invention takes the genome DNA of Vibrio sp.MCCC 1A13243 as a template, designs and synthesizes primers, clones the gene into pET-22b expression vector by PCR amplification of incision alginate lyase gene, performs induction expression in Escherichia coli E.coli BL21, and adopts Ni-Sepharose affinity chromatography for purification to obtain recombinant incision alginate lyase protein with higher purity. The optimum temperature of the enzyme is 35 ℃, the optimum pH is 8.5, and the stability is good in the ranges of 0-65 ℃ and pH 4.5-10.5; co ²⁺ 、Cu ²⁺ 、Mn ²⁺ And Fe ³⁺ Can obviously promote the activity of the enzyme; the enzyme has the strongest degradation effect on sodium alginate, can also degrade polyguluronic acid and polymannuronic acid, and the main products for degrading the three substrates are disaccharide. The enzyme has advantages in producing brown algae oligosaccharide, especially brown algae disaccharide.

Description

Incision alginate lyase, coding gene and application thereof

Technical Field

The invention relates to an incision alginate lyase obtained by adopting a genetic engineering means and a preparation method thereof, belonging to the technical field of biological engineering.

Background

Algin is a water-soluble acidic polysaccharide mainly extracted from the cell wall of brown algae, and is a polymer formed by randomly arranging two monomers of beta-D-mannuronic acid (M) and alpha-L-guluronic acid (G), and the two monomers are connected through 1, 4-glycosidic bonds to form polysaccharide fragments with three different forms: poly beta-D-mannuronic acid (PolyM) fragment, poly alpha-L-guluronic acid (Polyguluronic acid, PolyG) fragment, and hybrid fragments of both (PolyMG). Algin has physiological activities such as anti-tumor and blood pressure lowering, and is widely used in food, medical treatment, agriculture, energy and other fields.

The existing biological enzyme method is an important method for degrading algin, and has the advantages of mild and controllable reaction conditions, strong specificity and the like compared with chemical and physical degradation methods which pollute the environment, have low efficiency and are difficult to control the reaction conditions. The algin lyase can degrade algin into a series of unsaturated uronic acid oligosaccharides and monomers through beta-elimination reaction, and the degradation products have the physiological activities of promoting plant growth, relieving plant stress, resisting inflammation, bacteria, tumors, oxidation and the like, so that the production of algin oligosaccharides is very important. According to different action modes, the algin lyase is divided into an endo type and an exo type. So far, alginate lyase belongs to 12 Polysaccharide lyase families (PLs) in the CAZy (Carbohydrate-Active Enzymes) database, including PL5, PL6, PL7, PL14, PL15, PL17, PL18, PL31, PL32, PL34, PL36 and PL 39.

The source of the alginate lyase is wide, and the alginate lyase is found in algae, bacteria, marine mollusks, fungi and viruses at present. The species of alginate lyase from microorganisms are most abundant, and these microorganisms include members of the genera Vibrio (Vibrio), Pseudomonas (Pseudomonas), Microbulbifera (Microbulbifer), Flavobacterium (Flavobacterium), Bacillus (Bacillus) and Streptomyces (Streptomyces).

With the development of genome sequencing technology, more and more alginate lyase genes are annotated and are subjected to functional research by means of genetic engineering and the like, but most of the currently discovered endo-alginate lyases have narrow action temperature range and poor tolerance to high temperature, acid, alkali and metal ions, and degradation products are a mixture of multiple components so as to interfere with the correlation analysis of the structure and function of alginate oligosaccharides, thereby being greatly limited in industrial application.

Disclosure of Invention

The main purpose of the present invention is to provide an endoalginate lyase (named as VAL3) against the shortcomings of the prior art.

The optimum temperature of the enzyme is 35 ℃, the optimum pH is 8.5, and the stability is good in the ranges of 0-65 ℃ and pH 4.5-10.5; co ²⁺ 、Cu ²⁺ 、Mn ²⁺ And Fe ³⁺ Can obviously promote the activity of the enzyme; the enzyme has the strongest degradation effect on sodium alginate, can also degrade polyguluronic acid and polymannuronic acid, and the main products for degrading the three substrates are disaccharide.

The nucleotide sequence encoding the enzyme is as follows: (SEQ ID NO:1)

Wherein, the 57bp segment at the 5' end is a coding signal peptide sequence. The amino acid sequence of the enzyme is as follows: (SEQ ID NO:2)

Wherein, 19 amino acids at the N end are signal peptide sequences.

Another objective of the present invention is to provide a method for preparing endoalginate lyase, which comprises the following steps:

(1) the endo-alginate lyase gene VAL3 was obtained based on the analysis of Vibrio sp.MCCC 1A13243 genome sequencing data.

(2) Designing and synthesizing a primer for amplifying an endoalginate lyase gene VAL3 with a signal peptide coding sequence removed, wherein the sequence is as follows:

upstream primer VAL3F: 5'-GATCACATATGTGTACCTCTACTTCTGCT-3' (SEQ ID NO:3)

Downstream primer VAL3R: 5'-GATCACTCGAGGCCTTCGTACTTGCTATG-3' (SEQ ID NO:4)

(3) PCR amplification and recombinant plasmid construction:

amplification was performed using genomic DNA of Vibrio (Vibrio sp. MCCC 1A13243) as a template with primers VAL3F and VAL 3R. The PCR amplification conditions were as follows: pre-denaturation at 95 ℃ for 3 min; 30 cycles of 95 ℃ for 20s, 52 ℃ for 20s, 72 ℃ for 100 s; extension at 72 ℃ for 10 min. The PCR product is cut by restriction endonucleases NdeI and XhoI and then is connected to an expression vector pET-22b cut by the same restriction endonucleases, the connection product is transformed into an Escherichia coli TOP10 receptor strain, recombinant plasmids containing endoalginate lyase genes are screened, and double-enzyme cutting and sequencing verification are carried out.

(4) Expression and purification of endoalgin lyase

Transforming recombinant plasmid containing incision alginate lyase gene into Escherichia coli E.coli BL21 receptor strain, inducing incision alginate lyase protein expression with IPTG, and performing Ni-Sepharose affinity chromatography (Ni Sepharose) ^TM 6Fast Flow kit) to purify the protein. The SDS-PAGE result of the purified endoalginate lyase is shown in FIG. 1.

The endoalginate lyase belongs to PL7 family, has wide temperature and pH action range, can promote the activity of the enzyme by various common metal ions or has little influence on the activity of the enzyme, has the degradation product mainly comprising disaccharide component, and has advantages in the aspect of producing single alginate oligosaccharide.

Drawings

FIG. 1 is an SDS-PAGE electrophoresis of purified enzyme proteins;

FIG. 2 shows the effect of different temperatures on enzyme activity;

FIG. 3 shows the stability test of enzyme under different temperature conditions;

FIG. 4 is a graph showing the effect of different pH on enzyme activity;

FIG. 5 shows the stability measurements of the enzyme at different pH conditions;

FIG. 6 shows the effect of different metal ions and enzyme inhibitors on enzyme activity;

FIG. 7 shows the effect of enzymes on the hydrolysis of different substrates;

FIG. 8 analysis of degradation products of enzymes

Detailed Description

EXAMPLE 1 preparation of Endoalgin lyase

The method comprises the following steps:

experimental Material

Vibrio sp.mccc 1a13243 (deposited by china marine microorganism culture collection management center, deposit number 1a13243, available commercially), escherichia coli e.coli TOP10, escherichia coli e.coil BL21(DE3) (available from ThermoFisher corporation), expression vector pET-22b (available from ThermoFisher corporation); saibang bacteria genome DNA extraction kit (purchased from Xiamen Fine gathering Co.); DNA polymerase (from all-grass of gold); restriction enzymes Nde I and Xho I (from holo-gold); t4 ligase (available from Takara); LB medium (10 g peptone, 5g yeast extract, 10g NaCl per liter); binding buffer (1 XPBS buffer: 10mM phosphate, pH7.2-7.4); rinsing buffer (500mM NaCl, 10-20 mM imidazole, 20mM phosphate, pH 7.4); an elution buffer (500mM NaCl, 50-500 mM imidazole, 20mM phosphate, pH 7.4); 3, 5-dinitrosalicylic acid (DNS) (from shanghai bio-workers); ampicillin (from Shanghai Producer); ni Sepharose ^TM 6Fast Flow kit (purchased from taijing corporation); 10kDa ultrafiltration tube (purchased from Taijing Corp.); PolyG, PolyM, algin oligosaccharides and monosaccharides (available from boshi girl); sodium alginate (purchased from Shanghai national drug group). Ampicillin-containing LB medium used in the present invention had an ampicillin concentration of 100. mu.g/mL.

Experimental procedure

(1) Based on the full-length sequence of VAL3, a primer for amplifying and removing the endoalginate lyase gene of the signal peptide coding sequence is designed and synthesized, and the sequence is as follows:

upstream primer VAL3F: 5'-GATCACATATGTGTACCTCTACTTCTGCT-3'

Downstream primer VAL3R: 5'-GATCACTCGAGGCCTTCGTACTTGCTATG-3'

(2) PCR amplification and recombinant plasmid construction

The genomic DNA of Vibrio (Vibrio sp. MCCC 1A13243) was extracted using a Sebain bacteria genomic DNA extraction kit. Amplification was performed using the Vibrio sp. MCCC 1A13243 genomic DNA as a template and primers VAL3F and VAL 3R. The amplification system (50. mu.L) was as follows: VAL3F (10. mu.M) 1. mu.L, VAL3R (10. mu.M) 1. mu.L, 5 XStart Fastpfu Fly Buffer 10. mu.L, dNTP (2.5mM) 4. mu.L, genomic DNA 2. mu.L, Transstart Fastpfu DNA Polymerase 1. mu.L, ddH ₂ O31. mu.L. The amplification conditions were as follows: pre-denaturation at 95 ℃ for 3 min; 30 cycles of 95 ℃ for 20s, 52 ℃ for 20s, 72 ℃ for 100 s; extension at 72 ℃ for 10 min. Carrying out glue recovery on the PCR product, carrying out enzyme digestion on the pET-22b vector and the corresponding target fragment by using restriction enzymes NdeI and XhoI, and then connecting by using T4 ligase; and (2) carrying out competent mixing on the connecting product and a receptor bacterium E.coli Top10, placing the mixture on ice for 30min, carrying out heat shock at 42 ℃ for 90s, adding 800 mu L of LB liquid culture medium, recovering at 37 ℃ and 100rpm for 1h, centrifuging, coating the mixture on an LB solid culture medium containing ampicillin, carrying out overnight culture at 37 ℃, screening recombinant plasmids, and carrying out double enzyme digestion and sequencing verification on the recombinant plasmids to obtain the related nucleotide sequence of the endoalginate lyase from which the N-terminal signal peptide is removed.

(3) Gene induction expression and preparation of crude enzyme solution

The recombinant plasmid was transformed into e.coli BL21(DE3) to obtain a recombinant strain containing an endoalginate lyase gene. The recombinant strain was inoculated into 5mL of LB liquid medium containing 100. mu.g/mL ampicillin overnight for culture, and 1% of the inoculum size was inoculated into 500mL of LB liquid medium containing ampicillin, and cultured at 37 ℃ and 180rpm to OD ₆₀₀ About 0.4-0.5, adding IPTG to make the final concentration 1mmol/L for induction, inducing and expressing 24h under the condition of 18 ℃ and 180rpm, centrifuging for 10min at 5000rpm and collecting thalli. Washing thallus with 1 × PBS buffer solution (pH7.2-7.4) for 2-3 times, then suspending thallus in 60mL binding buffer solution, dividing into two tubes, standing at 4 deg.C for 2-3 h, ultrasonically crushing at power of 300W for 30min at working/gap time of 3s/4s, 4 deg.C, 5000 sCentrifuging at rpm for 20min, and collecting supernatant to obtain crude enzyme solution.

(4) Purification of enzymes

Adding the prepared crude enzyme solution into a pre-balanced His purification column Ni Sepharose ^TM 6Fast Flow, mixing uniformly at 4 ℃ for 2-3 h, and then discarding the waste liquid. This was followed by two rinses with 20mL of rinse buffer containing 10mM imidazole and then two rinses with 20mL of rinse buffer containing 20mM imidazole. After the completion of the rinsing, the column was eluted twice with an elution buffer containing 50mM imidazole (first elution volume: 20mL, second elution volume: 8mL), and then eluted once with an elution buffer containing 500mM imidazole (elution volume: 8mL), and the eluates were collected in portions and subjected to SDS-PAGE to examine the purity of the eluates. Ultrafiltration was performed using a 10kDa ultrafiltration tube to remove imidazole and high concentration NaCl to obtain a purified enzyme solution.

Example 2

Endomycephalin lyase enzymatic properties

(1) Method for measuring activity of alginate lyase

And (3) determining the content of reducing sugar by using a DNS method. 50. mu.L of the diluted enzyme solution and 200. mu.L of 0.3% sodium alginate substrate were mixed and reacted at 35 ℃ for 30 min. Adding 500 μ L DNS reagent after reaction, boiling for 5min, immediately cooling in ice water, centrifuging for a short time, collecting supernatant, and determining OD ₅₄₀ Values (with inactivated enzyme solution as a control), reduced sugar amounts and enzyme activities were calculated according to the standard curve. Definition of enzyme activity unit: under the above-described measurement conditions, the amount of enzyme required to cleave sodium alginate per minute to produce 1. mu. mol of reducing sugar is defined as one enzyme activity unit (U).

(2) Temperature of action of the enzyme

And (3) performing enzyme activity determination by taking 0.3% sodium alginate prepared from 100mM Glycine-NaOH (pH 8.5) buffer solution as a substrate at the temperature of 0-90 ℃ in the diluted enzyme solution, and calculating the relative enzyme activity at different temperatures by taking the maximum enzyme activity as 100%. The result shows that when the pH value is 8.5, the endoalginate lyase has activity within the tested temperature range, the optimal reaction temperature is 35 ℃, the enzyme activity is kept above 80% within the range of 10-45 ℃, and the enzyme activity is remained above 57% at 50 ℃, and the result is shown in figure 2.

(3) Stability of enzymes under different temperature conditions

And (3) preserving the heat of the enzyme solution at different temperatures (0-90 ℃) for 1h, reacting the enzyme solution with 0.3% sodium alginate prepared from 100mM Glycine-NaOH (pH 8.5) buffer solution to perform enzyme activity determination, and calculating the relative activity of the enzymes treated differently with the activity of the untreated enzymes as 100%. The result shows that the enzyme has good stability within the range of 0-65 ℃, the residual enzyme activity is more than 90% after heat preservation is carried out for 1h within the range of 0-45 ℃, and the residual enzyme activity is more than 80% after heat preservation is carried out for 1h within the range of 50-65 ℃, and the result is shown in figure 3.

(4) Action pH of the enzyme

The diluted enzyme solution is used for enzyme activity determination at 35 ℃ by taking 0.3% sodium alginate prepared by different buffers (100mM Na-acetate buffer solution, pH 5.0-6.0; 100mM Na-phosphate buffer solution, pH 6.0-8.0; 100mM Tris-HCl buffer solution, pH 7.0-9.0; 100mM Glycine-NaOH, pH 8.0-10.9) as a substrate, and the relative activity of the enzyme under different pH values is calculated by taking the maximum enzyme activity as 100%. The result shows that the optimum pH of the enzyme is 8.5, the enzyme has a wide pH action range, and the enzyme activity is higher within the pH range of 6.0-10.0; detecting the enzyme activity by using Tris-HCl and Glycine-NaOH buffer solutions under neutral and slightly alkaline conditions (pH7.0-9.5), wherein the enzyme activity is kept above 90%; under the same pH conditions, Na-acetate, Tris-HCl and Glycine-NaOH buffers outperformed Na-phosphate buffer, and the results are shown in FIG. 4.

(5) Stability of enzymes at different pH conditions

The enzyme solution is incubated in different buffers (100mM Na-acetate buffer, pH 4.5-6.0; 100mM Na-phosphate buffer, pH 6.0-8.0; 100mM Tris-HCl buffer, pH 7.0-9.0; 100mM Glycine-NaOH, pH 8.0-10.5) at 4 ℃ for 1h, and then reacted with 0.3% sodium alginate prepared from 100mM Glycine-NaOH (pH 8.5) buffer at 35 ℃ to perform enzyme activity determination, and the activity of the untreated enzyme is 100%, and the relative activity of the enzymes treated differently is calculated. The result shows that the stability of the enzyme in the tested pH range (pH 4.5-10.5) is good, the Na-acetate, Tris-HCl and Glycine-NaOH buffer solutions are superior to the Na-phosphate buffer solution under the same pH condition, the enzyme activity in the Na-acetate, Tris-HCl and Glycine-NaOH buffer solutions is kept above 90%, the enzyme activity in the Na-phosphate buffer solutions is kept above 80%, and the result is shown in figure 5.

(6) Action of Metal ions and enzyme inhibitors on enzymes

Under the conditions of 35 ℃ and pH8.5, different metal ions with the final concentration of 1.0mM and an enzyme inhibitor are respectively added into a reaction system, then the enzyme activity is measured, and the relative enzyme activity under the influence of different metal ions and the enzyme inhibitor is calculated by taking the relative enzyme activity under the condition of not adding any metal ions or enzyme inhibitors as 100%. The results show that Co ²⁺ 、Cu ²⁺ 、Mn ²⁺ And Fe ³⁺ Has obvious promoting effect on enzyme activity, Fe ²⁺ The enzyme activity is obviously inhibited, and other metal ions have little influence on the enzyme activity; EDTA and SDS almost completely inhibited the enzyme activity, while Imidazole (Imidazole) had substantially no effect on the enzyme activity, and the results are shown in FIG. 6.

(7) Substrate specificity of the enzyme

And respectively taking 0.3% of sodium alginate, polyG and PolyM as substrates to perform enzyme activity determination at the temperature of 35 ℃ and the pH value of 8.5, and calculating the relative activities of different substrates hydrolyzed by the endoalginate lyase by taking the maximum enzyme activity as 100%. The result shows that the enzyme has degradation activity on three substrates of sodium alginate, polyG and PolyM, wherein the degradation effect on the sodium alginate is the best, and the enzyme is a bifunctional enzyme with substrate preference, and the result is shown in figure 7.

(8) Analysis of degradation products

The endoalginate lyase product was analyzed by Thin Layer Chromatography (TLC). mu.L of pure enzyme was added to 200. mu.L of 100mM Glycine-NaOH (pH 8.5) containing 0.3% substrate (sodium alginate/polyG/PolyM), mixed well and reacted overnight at 35 ℃ with a loading of 3. mu.L. The developing solvent is n-butyl alcohol: formic acid: water (4: 6: 1, v/v/v), developer is 10% ethanol sulfate. The effect of different pH (100mM Na-phosphate buffer, pH 6.5; 100mM Tris-HCl buffer, pH 7.5; 100mM Glycine-NaOH buffer, pH8.5) on sodium alginate degradation products was examined, and the reaction conditions and degradation product examination method were the same as described above except that the substrate used was different. The results showed that the major products of the enzyme degradation of sodium alginate, PolyG and PolyM are disaccharides, indicating that the enzyme is an endoalginate lyase (fig. 8A); in addition, the components of the product of the enzyme degrading sodium alginate under the conditions of pH6.5, pH7.5 and pH8.5 are not changed, and are disaccharides, which shows that the enzyme has better stability under different pH conditions (FIG. 8B).

Sequence listing

<110> third Marine institute of Natural resources

<120> incision alginate lyase, coding gene and application thereof

<160> 4

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acctctactt ctgctccaga gtttgcaaat aacaaagaaa ccggagagcc aatccttaca 120

cctgtcgcta tcacagcgag tagccatgat ggcaacggcc cagaccgtct attcgaccaa 180

gatatcacaa cacgttggtc ttctgctggt gacggtgagt gggcaatgct ggactacggt 240

tcagttcaag aattcgatgc agttcaagcg tcattcagta aaggtaatga gcgtcagagc 300

aagtttgata tccaagtgag tgtagatggt gaaaactgga ctactgttct tgaaaatcaa 360

atgagttctg gtcaaatcat tggtctagag cgtttccagt ttgaaccagc agtaaaagct 420

cgttacgtgc gctacgtagg tcacggtaac accaaaaacg gctggaacag tgtgacagaa 480

ctggctgcgg ttaactgtaa cgtaaacgcg tgtcctgcga gccatatcgt tacccctgat 540

gtcgttgaag cagaagcggt aatgatcgct gaaatgaaag cggcagagaa agcacgtaaa 600

gaagcacgta aagaccttcg taaaggtaat tggggcgagc cagcggttta cccatgcgaa 660

accacagtta agtgtgacac tcgctcagct ctacccgttc cgactggcct cccagcaacc 720

ccagttgcag gtaatgcgcc aagtgaaaac ttcgacttga cccattggta tctgtctcaa 780

ccatttgatc atgaccaaaa cggtcgtcca gatgacgttt ctgagtggaa cctagcgaac 840

ggctatcagc atccggacgt attctacacc gcagaagatg gtggcatggt gttcaagtcg 900

tacgtaaaag gcgttcgcac ttcgaaaaat actaagtatg cgcgtactga gcttcgtgaa 960

atgatgcgtc gtggtgacca atctatcagc actaaaggcg ttaacaagaa caactgggta 1020

ttcagctctg ctccagttga agatctaaag gctgcagctg cagtagacgg cgttcttgaa 1080

gcgactctta agattgacca caccacaact accggtgacg caaacgaagt aggtcgcttc 1140

atcataggtc aaatccacga ccaaaacgat gagccaattc gtctgtacta ccgtaaactt 1200

ccaaaccaag caacaggtgc ggtttacttt gctcacgaaa gccaagacgc aactaaagaa 1260

gacttctacc ctctcgttgg cgacatgacg gctgaagttg gtgaagacgg catcgcactg 1320

ggcgagaaat tcagctaccg catcgaggtt gtgggtaaca ctatgaccgt tagcctaatg 1380

cgtgaaggcc acgacgacgt agttcaagtt gtggacatga gcgacagcgg ctacgacgtt 1440

ggcggcaagt acatgtactt caaagcgggt gtttacaacc aaaacatcaa cggcgacatg 1500

gacgactatg tacaagcaac cttctatcag ctagacgttt cgcatagcaa gtacgaaggc 1560

taa 1563

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Ala Val Gly Cys Thr Ser Thr Ser Ala Pro Glu Phe Ala Asn Asn Lys

20 25 30

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

35 40 45

His Asp Gly Asn Gly Pro Asp Arg Leu Phe Asp Gln Asp Ile Thr Thr

50 55 60

Arg Trp Ser Ser Ala Gly Asp Gly Glu Trp Ala Met Leu Asp Tyr Gly

65 70 75 80

Ser Val Gln Glu Phe Asp Ala Val Gln Ala Ser Phe Ser Lys Gly Asn

85 90 95

Glu Arg Gln Ser Lys Phe Asp Ile Gln Val Ser Val Asp Gly Glu Asn

100 105 110

Trp Thr Thr Val Leu Glu Asn Gln Met Ser Ser Gly Gln Ile Ile Gly

115 120 125

Leu Glu Arg Phe Gln Phe Glu Pro Ala Val Lys Ala Arg Tyr Val Arg

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Tyr Val Gly His Gly Asn Thr Lys Asn Gly Trp Asn Ser Val Thr Glu

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Leu Ala Ala Val Asn Cys Asn Val Asn Ala Cys Pro Ala Ser His Ile

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Val Thr Pro Asp Val Val Glu Ala Glu Ala Val Met Ile Ala Glu Met

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Lys Ala Ala Glu Lys Ala Arg Lys Glu Ala Arg Lys Asp Leu Arg Lys

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Gly Asn Trp Gly Glu Pro Ala Val Tyr Pro Cys Glu Thr Thr Val Lys

210 215 220

Cys Asp Thr Arg Ser Ala Leu Pro Val Pro Thr Gly Leu Pro Ala Thr

225 230 235 240

Pro Val Ala Gly Asn Ala Pro Ser Glu Asn Phe Asp Leu Thr His Trp

245 250 255

Tyr Leu Ser Gln Pro Phe Asp His Asp Gln Asn Gly Arg Pro Asp Asp

260 265 270

Val Ser Glu Trp Asn Leu Ala Asn Gly Tyr Gln His Pro Asp Val Phe

275 280 285

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

290 295 300

Val Arg Thr Ser Lys Asn Thr Lys Tyr Ala Arg Thr Glu Leu Arg Glu

305 310 315 320

Met Met Arg Arg Gly Asp Gln Ser Ile Ser Thr Lys Gly Val Asn Lys

325 330 335

Asn Asn Trp Val Phe Ser Ser Ala Pro Val Glu Asp Leu Lys Ala Ala

340 345 350

Ala Ala Val Asp Gly Val Leu Glu Ala Thr Leu Lys Ile Asp His Thr

355 360 365

Thr Thr Thr Gly Asp Ala Asn Glu Val Gly Arg Phe Ile Ile Gly Gln

370 375 380

Ile His Asp Gln Asn Asp Glu Pro Ile Arg Leu Tyr Tyr Arg Lys Leu

385 390 395 400

Pro Asn Gln Ala Thr Gly Ala Val Tyr Phe Ala His Glu Ser Gln Asp

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Ala Thr Lys Glu Asp Phe Tyr Pro Leu Val Gly Asp Met Thr Ala Glu

420 425 430

Val Gly Glu Asp Gly Ile Ala Leu Gly Glu Lys Phe Ser Tyr Arg Ile

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Glu Val Val Gly Asn Thr Met Thr Val Ser Leu Met Arg Glu Gly His

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Asp Asp Val Val Gln Val Val Asp Met Ser Asp Ser Gly Tyr Asp Val

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Gly Gly Lys Tyr Met Tyr Phe Lys Ala Gly Val Tyr Asn Gln Asn Ile

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Asn Gly Asp Met Asp Asp Tyr Val Gln Ala Thr Phe Tyr Gln Leu Asp

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gatcacatat gtgtacctct acttctgct 29

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Claims

1. A gene for expressing incision alginate lyase is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.

2. A gene for expressing incision alginate lyase is characterized in that the gene is obtained by removing a 57bp fragment at the 5' end of a nucleotide sequence shown in SEQ ID NO. 1.

3. A recombinant vector comprising a gene for expressing an endoalginate lyase as set forth in claim 1 or 2.

4. A recombinant bacterium comprising the recombinant vector according to claim 3.

5. An endoalginate lyase, which is characterized in that: the amino acid sequence is shown in SEQ ID NO. 2.

6. An endoalginate lyase, which is characterized in that: it is obtained by removing 19 amino acids from the N end of the amino acid sequence shown in SEQ ID NO. 2.

7. The use of the endoalginate lyase of claim 5 or 6, in brown algae utilization and/or brown alginate oligosaccharide production.

8. A preparation method of incision alginate lyase comprises the following steps:

(1) obtaining VibrioVibriosp. MCCC 1A13243 genome DNA as template, the said template contains incision alginate lyase geneVAL3(ii) a The nucleotide sequence of the endoalginate lyase gene is shown as SEQ ID NO. 1;

(2) design and Synthesis of amplification EliminationEndoalgin lyase gene of signal peptide coding sequenceVAL3The primer (2) is as follows:

upstream primer VAL3F: 5'-GATCACATATGTGTACCTCTACTTCTGCT-3'

A downstream primer VAL3R: 5'-GATCACTCGAGGCCTTCGTACTTGCTATG-3';

(3) PCR amplification and recombinant plasmid construction: amplifying by adopting the primer in the step (2) and taking the genome DNA in the step (1) as a template; the PCR product is treated with restriction enzymeNdeI andXhoi is cut and then connected to an expression vector pET-22b cut by the same restriction enzyme, and the connection product is transformed into escherichia coliEscherichia coliTOP10 receptor strain, screening recombinant plasmid containing incision alginate lyase gene, and performing double enzyme digestion and sequencing verification;

(4) expressing and purifying incision alginate lyase: transforming the recombinant plasmid containing the incision alginate lyase gene into escherichia coliE. coli BL21 acceptor strain, inducing the protein expression of endoalginate lyase, and purifying to obtain purified endoalginate lyase.