CN114854778B

CN114854778B - Fucoidan gene Fcn1 and application thereof

Info

Publication number: CN114854778B
Application number: CN202210396431.9A
Authority: CN
Inventors: 陈铁军; 王莹; 王立平; 朱昱兴; 秦敏; 战旭宁
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2022-04-15
Filing date: 2022-04-15
Publication date: 2023-05-30
Anticipated expiration: 2042-04-15
Also published as: CN114854778A

Abstract

The invention discloses a fucoidan geneFcn1And applications thereof. The saidFcn1The nucleotide sequence of the gene is shown as SEQ ID No.1, and the gene is derived from a strainFlavobacteriaceaebacteriumRC2-3. The fucoidanase geneFcn1The amino acid sequence of the coded fucoidan is shown as SEQ ID No.2, and has the capacity of degrading fucoidan sulfate. The invention also provides a method for detecting the fucoidan geneFcn1Is a primer sequence of the primer of (a). The saidFcn1The gene not only enriches the fucoidan gene family, lays a foundation for further elucidating the fucoidan catalytic action mechanism, is beneficial to promoting the development and utilization of fucoidan resources, and has important theoretical value and production guidance significance.

Description

Fucoidan gene Fcn1 and application thereof

Technical Field

The invention belongs to the field of genes, and particularly relates to a fucoidan gene Fcn1 and application thereof.

Background

Fucoidan is a special polysaccharide in marine environment, is a marine sulfated polysaccharide (10000-100000 Da) which is composed of monosaccharide and has very complex chemical structure, is mainly derived from brown algae and some marine invertebrates, and has multiple biological functions of anticoagulation, anti-tumor, antithrombotic, antivirus, antioxidation and the like. However, the complexity and diversity of fucoidan molecular structure results in an ambiguous relationship between biological functions and molecular structures thereof, which severely restricts the application of fucoidan in the fields of clinical medicine, health care, food and the like. Compared with high molecular weight fucoidin, low molecular weight fucoidin has certain advantages in bioavailability and bioactivity, and has great application potential. Therefore, how to obtain low molecular weight fucoidin with various physiological functions by degrading natural high molecular weight fucoidin has attracted attention from scientific researchers at home and abroad.

Fucoidan is a key enzyme for degrading fucoidan, and since fucoidan is abundant in variety, the strain for degrading fucoidan and the fucoidan produced therefrom are also different. The reported classification of fucoidan in CAZY database is 18, and the complexity and diversity of fucoidan molecular structure determines the diversity of fucoidan species and functions. The currently reported fucoidan-producing strain has the problems of low enzyme yield and poor enzyme activity of produced enzyme, and the current solution method mainly aims at improving the enzyme yield or the enzyme activity by optimizing a culture medium, culture conditions and a physical mutagenesis mode, but the effect is not obvious, commercialization of the fucoidan is still not realized, and the research on the fucoidan is mainly focused on heterologous expression and the most basic enzymatic characteristic characterization, and does not have more intensive research on the related mechanism and substrate specificity analysis of the fucoidan.

Disclosure of Invention

The invention aims to provide a fucoidan gene Fcn1 and application thereof. The Fcn gene can code fucoidan, and has high-efficiency and specific degradation effect on fucoidan sulfate.

In order to achieve the aim of the invention, the invention is realized by adopting the following technical scheme:

the invention provides a fucoidan gene Fcn1, and the nucleotide sequence of the Fcn gene is shown as SEQ ID No. 1.

Further, the Fcn gene is derived from strain Flavobacteriaceae bacterium RC2-3.

The invention also provides a primer for detecting the fucoidan gene Fcn1, and the sequence of the primer is as follows:

Fcn1-F：5’-GGTACCATGAATAAACTAATTTCAATATTTCTAGGAGGG-3’；

Fcn1-R：5’-GGATCCTTAATCTAACCAAGTAATTTTAGCTTC-3’。

the invention also provides the fucoidan coded by the fucoidan gene Fcn, and the amino acid sequence of the fucoidan is shown as SEQ ID No. 2.

The invention also provides a recombinant vector containing the fucoidan gene Fcn1

Further, the carrier is pLB carrier.

The invention also provides a recombinant strain containing the fucoidan gene Fcn 1.

Further, the recombinant strain is escherichia coli.

The invention also provides application of the fucoidan gene Fcn or the fucoidan in preparation of preparations for degrading fucoidan sulfate.

Further, the fucoidan enzyme coded by the fucoidan gene Fcn can degrade fucoidan sulfate by degrading alpha 1-3 glycosidic bond in the fucoidan sulfate.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. according to the invention, flavobacteriaceae bacterium RC-3 with complete genome sequencing is taken as a research object, genome bioinformatics analysis is carried out on the gene, DNAMAN sequence comparison is carried out on the gene and reported fucoidan, and a fucoidan gene Fcn1 is obtained, and the homology between the coded Fcn protein (fucoidan) sequence and FunA protein reaches to be only 70.98%.

2. The invention obtains the recombinant strain of Fcn1 gene, realizes the heterologous expression of Fcn1 gene, obtains the heterologous expression enzyme fucoidan, and proves the enzyme activity of the enzyme and the capability of degrading fucoidan sulfate. The results not only enrich the fucoidan gene family, lays a foundation for further elucidating the fucoidan catalytic action mechanism, but also is helpful for promoting the development and utilization of fucoidan resources, and has important theoretical value and production guidance significance.

Drawings

FIG. 1 is a diagram of an alignment of Fcn and FunA proteins.

FIG. 2 is a graph showing the relationship between the amino acid sequence encoded by Fcn gene and the amino acid sequence of the prior fucoidan.

FIG. 3 shows the result of Q5 super fidelity enzyme PCR amplified gene agarose gel electrophoresis, wherein the left three bands are Fcn gene bands, and the right is Marker.

FIG. 4 shows the recovery result of agarose gel electrophoresis of DNA fragments, wherein the left band is Fcn gene band and the right is Marker.

FIG. 5 shows colonies grown on LB plates containing ampicillin resistance.

FIG. 6 is a diagram showing agarose gel electrophoresis verification of positive transformants, wherein the left band is a single colony PCR verification band, and the right is a Marker.

FIG. 7 shows the result of electrophoresis of plasmid enzyme digestion.

FIG. 8 is an SDS-PAGE gel electrophoresis, wherein blank bands are BL21 E.coli supernatant of empty plasmid and induced expression, 1 band to 4 bands are successfully transformed and induced expressed BL21 E.coli, and M band is Marker.

Detailed Description

The technical scheme of the invention is further described in detail by combining the following specific examples.

In the following examples, unless otherwise specified, all experimental methods used are conventional and all materials, reagents, etc. are commercially available from biological or chemical reagent companies.

Configuration of the culture medium:

LB medium (w/v): 0.5% yeast powder, 1.0% tryptone, 1.0% NaCl, if solid medium is added with 2.0% agar. Distilled water was prepared, sterilized at 121℃for 20 minutes, cooled to room temperature, and ampicillin (Amp) was added thereto at a final concentration of 100.0. Mu.g/mL.

TB medium: (1) 12g of tryptone, 24g of yeast extract, 4mL of glycerol and deionized water are added to 900mL, completely dissolving and sterilizing; (2) KH (KH) ₂ PO ₄ 2.31g，K ₂ HPO ₄ 12.54g, deionized water was added to 100mL, dissolved completely, and sterilized. After sterilization, the solutions (1) and (2) are mixed uniformly in a sterile environment.

Example 1: acquisition of Fcn1 Gene

A marine bacterium Flavobacteriaceae bacterium RC-3 strain capable of degrading fucoidan is obtained by screening from kelp, performing whole genome sequencing (the genome sequence of the RC2-3 strain is submitted to Genome Sequence Archive (GSA) database and accession number CRA003853 is obtained), analyzing the obtained result, and establishing a Flavobacterium sp.RC2-3 strain genome database and a protein database by using BioEdit software. The gene sequence and the amino acid sequence of the fucoidan reported in the present literature are searched through NCBI database, the information of the fucoidan reported at present is visible in table 1, the gene sequence and the protein sequence of the fucoidan published in NCBI database are respectively compared with the genome database and the protein database of Flavobacteriaceae bacterium RC-3 strain, 1 fucoidan gene Fcn1 is found in the strain, the amino acid sequence encoded by Fcn1 gene guidance in Flavobacteriaceae bacterium RC-3 strain is compared with the amino acid sequence corresponding to the reported fucoidan gene searched from the literature in table 1, and the amino acid sequence is compared by MEGA 7.0 software, and the construction analysis of the evolutionary tree is performed by the Neighbor-Joining method.

Table 1 reported fucoidanase

As shown in FIGS. 1-2, the homology of the fucoidan produced by encoding the Fcn1 gene and FunA (WP_ 068826898.1) fucoidan protein sequence reaches 70.98%, and the phylogenetic tree of the amino acid sequence shows that the two enzymes are related recently, and the Fcn1 gene encoded protein and the FunA gene encoded protein belong to the same glycoside hydrolase family-GH 168 by combining the amino acid sequence encoded by the Fcn1 gene and the amino acid sequence conserved domain analysis result of the FunA gene, and the family members not only can specifically hydrolyze alpha-1, 3 glycosidic bonds between 2-O-sulfated and non-sulfated fucose residues, but also have transglycosylation activity. The nucleotide sequence of Fcn gene is shown as SEQ ID No.1, and the encoded amino acid sequence is shown as SEQ ID No. 2.

Example 2: fcn1 Gene cloning

1. Flavobacteriaceae bacterium RC2-3 Strain genome extraction

Extracting Flavobacteriaceae bacterium RC2-3 strain genome, taking Flavobacteriaceae bacterium RC-3 strain liquid after culturing, extracting bacterial genome DNA by using a kit according to the specification of a bacterial genome DNA extraction kit of TIANGEN company, and measuring the extracted genome DNA by agarose gel electrophoresis.

2. Fcn1 Gene primer design

The Primer Premier is used for designing a Fcn gene PCR Primer, and the Primer sequence is as follows:

Fcn1-F：5’-GGTACCATGAATAAACTAATTTCAATATTTCTAGGAGGG-3’(SEQ ID No.3)；

Fcn1-R：5’-GGATCCTTAATCTAACCAAGTAATTTTAGCTTC-3’(SEQ ID No.4)。

3. q5 ultra-fidelity DNA polymerase PCR amplification

The amplification of Fcn gene was performed using a MJ Mini personal PCR instrument using Q5 super fidelity DNA polymerase, the reaction system was as follows:

the PCR amplification procedure was: pre-denaturation at 98 ℃ for 30s; denaturation at 94℃for 10s, annealing at 66℃for 15s, elongation at 72℃for 1min,30 cycles; extending at 72℃for 5min.

After the completion of PCR, the Fcn gene was collected by agarose gel electrophoresis.

The agarose gel electrophoresis verification result is shown in FIG. 3, and the size of the Fcn gene amplified fragment is 1221bp.

4. Agarose gel DNA recovery

The agarose gel containing the target DNA fragment was cut by ultraviolet light, and the target DNA fragment was recovered using the kit according to the specification of the agarose gel DNA recovery kit from TIANGEN, and after recovery, agarose gel electrophoresis was performed to verify and determine the recovery concentration.

The recovery results are shown in FIG. 4, and the recovery of Fcn1 gene fragment was confirmed to be correct.

5. Fcn1-pLB vector construction

Fragments pLB and Fcn were blunt-ended according to the tia ngan company pLB background rapid cloning kit instructions, the ligation system being as follows:

6. coli transformation

(1) LB solid plates containing 100. Mu.g/mL ampicillin were placed in an incubator at 37℃for 30min;

(2) Adding 10.0 mu L of the ligation product into 100.0 mu L of DH5 alpha competent cells, gently mixing, and standing in an ice bath for 30min;

(3) After ice bath standing, carrying out heat shock for 90s in a water bath at 42 ℃, rapidly transferring to ice, and standing for 1-3min;

(4) Adding 400 mu L of preheated LB liquid medium, and culturing for 45min in a shaking table at 37 ℃ and 160 rpm;

(5) 100 mu L of the bacterial liquid is coated on a preheated LB plate, and the bacterial liquid is cultured for 12-16 hours in an inverted mode at 37 ℃.

The DH5 alpha strain is transformed by using pLB zero background rapid cloning kit, after 12 hours of culture, the recombinant strain grows on an LB plate containing 100 mug/mL of ampicillin resistance, the original strain DH5 alpha can not grow on the LB plate due to lack of ampicillin resistance gene, the pLB vector itself contains the empty plasmid recombinant strain with lethal gene not connected with Fcn1 gene, and the final growth result is shown in FIG. 5.

7. Positive transformant selection

The grown colonies were picked up and inoculated into LB liquid medium containing 100. Mu.g/mL of ampicillin, and cultured at 37℃and 180rpm for 6-8 hours. Colony PCR verification is carried out by using pLB-Simple Vector universal primer, and the PCR reaction system is as follows:

PCR amplification conditions: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 58℃for 30s, extension at 72℃for 2min,30 cycles; extending at 72℃for 5min.

As a result, as shown in FIG. 6, it was revealed from the graph that the presence of a band between 1600bp and 1000bp for the most of the colonies on the left demonstrated that the Fcn gene contained therein was a positive transformant, and that no band between 1600bp and 1000bp demonstrated that the Fcn gene contained therein was a false positive.

And (3) extracting plasmids by using a plasmid miniextraction kit of TIANGEN company after seed preservation of the obtained positive transformant bacterial liquid, sending the extracted plasmids to Qingdao qinghao biotechnology limited company for sequencing, and comparing and verifying the sequencing result with Fcn1 gene sequences. The plasmid verification results were correct, which can be used in the next experiment.

Example 3: fcn1 Gene heterologous expression and catalytic substrate specificity preliminary investigation

1. Construction of Fcn1 gene heterologous expression vector

The plasmid with the completely correct sequencing in the embodiment 2 is selected, and the Fcn1 gene sequence is connected into the expression vector pET-28a (+) by an enzyme digestion and connection method, so that the construction of the RC2-3 strain Fcn1 gene expression vector Fcn-pET-28 a (+) is completed.

According to the designed enzyme cutting sites, the pET-28a (+) vector and the pLB plasmid are respectively subjected to double enzyme cutting, and the enzyme cutting system is as follows:

/>

enzyme cutting for 2 hours at 37 ℃, and after the target fragment is recovered by glue, connecting the target fragment and the target fragment by using T4 DNA ligase, wherein the connecting system is as follows:

the verification result is shown in FIG. 7, and the Fcn1 fragment is located between 1600bp and 1000bp, so that Fcn gene fragment and pET-28a (+) vector can be recovered by cutting gel.

And (3) connecting the E.coli DH5 alpha escherichia coli after 4h of water bath connection at the temperature of 22 ℃, screening positive transformants and extracting plasmids to obtain a novel plasmid named Fcn-pET-28 a (+) recombinant plasmid, and transferring the novel Fcn-pET-28 a (+) recombinant plasmid into the escherichia coli strain BL21 for protein expression.

2. Coli induced expression

(1) Inoculating the escherichia coli recombinant strain obtained through verification into 5mL of LB liquid medium containing ampicillin, and culturing at 37 ℃ for 200r/min overnight to obtain seed liquid;

(2) Transferring the seed solution into 50mL TB liquid culture medium containing ampicillin according to the inoculation amount of 1%, and culturing at 37 ℃ and 200r/min until the OD600 reaches 1.0-2.0;

(3) Adding L-arabinose with a final concentration of 0.2% and culturing at 16 ℃ for 12-16h at 200r/min to induce protein expression;

(4) Centrifuging at 4000r/min and 4 ℃ for 20min to collect thalli, ultrasonically crushing thalli, centrifuging at 12000r/min for 30min, taking supernatant after ultrasonic crushing of the thalli, performing SDS-PAGE detection, and determining whether protein is expressed in a soluble way.

3. SDS-PAGE gel electrophoresis

Sample preparation: the protein samples were mixed with 5 Xsample buffer in an Eppendorf tube and heated at 100deg.C for 10min, and the supernatants were spotted.

Preparation of separating gel and concentrated gel, and washing agent for glass plate, sample comb and SpacerWashing with ddH ₂ Washing for several times, wiping with ethanol, and air drying.

A Spacer was added between the two cleaned glass plates, and the glass plates were assembled as indicated by the Bio-Rad Mini lI/II instructions.

The gel used for SDS-PAGE electrophoresis was configured according to the following system:

firstly pouring separating gel between glass plates, immediately covering a layer of absolute ethyl alcohol after pouring, polymerizing the gel after about 20min, pouring the absolute ethyl alcohol on the upper layer after polymerizing the separating gel, pouring concentrated gel, inserting a sample comb, loading an electrophoresis system after the gel is solidified and completely solidified, adding electrode buffer solution, and loading 20 mu L of sample; stabilizing the voltage by 200V, and stopping electrophoresis when the bromophenol blue just runs out of the separating gel, wherein the electrophoresis is about 45-60 min. Removing the rubber plate, putting the stripping rubber into the dyeing liquid, and dyeing for 1-2 h at room temperature; the decolorized solution was added and placed on a 80rpm decolorization shaker, and the decolorized solution (10 mL glacial acetic acid; 45mL ethanol; 45mL distilled water) was changed every 20min until complete removal.

As shown in FIG. 8, the predicted size of the protein encoded by the Fcn1 gene is 46.8kDa, and the bands 1 to 4 above 43kDa are one more than the left band, which proves that the Fcn gene realizes heterologous expression in Escherichia coli BL21, and the Fcn1 gene directs that the synthesized protein does not form inclusion bodies during the heterologous expression, thus forming active protein.

4. Crude enzyme extraction

And subpackaging the fermented fermentation liquor into a centrifuge tube, centrifuging for 15min at 4 ℃ and 5000r/min, taking precipitate, stirring and cleaning the precipitate with a proper amount of precooled (4 ℃) distilled water to obtain relatively clean thalli, and centrifuging under the same conditions. Repeating the steps for three times, and finally taking the precipitate to obtain the thalli.

Taking distilled water with the volume being 5 times of the weight of the bacteria, carrying out ultrasonic crushing under ice bath condition for 15min at the working interval of 5s, centrifuging at the temperature of 4 ℃ after the completion of the operation, taking the supernatant by 8000r/min to obtain crude enzyme liquid, and subpackaging 10 mL/tube in a centrifuge tube for storage at the temperature of minus 20 ℃.

5. Enzymatic substrate specificity assay

Polysaccharide solutions were prepared in a system in which 0.1006g of polysaccharide was dissolved in 50ml of 1 XPBS buffer, and fucoidan solution, laminarin solution, and fucoidan sulfate polysaccharide solution were prepared, respectively.

2mL of crude enzyme solution was boiled in a boiling water bath for 10min, and the enzyme was inactivated according to the polysaccharide solution and inactivated enzyme solution/non-inactivated enzyme solution 1:1 adding a system to prepare a reaction system, wherein three parallel samples are respectively arranged in an inactivated group and an inactivated group of each polysaccharide solution, and the reaction is carried out for 2 hours in an incubator with the setting condition of 30 ℃ and 120 r/min.

Taking 0.5mL of solution after the reaction is finished, adding 0.5mL of potassium ferricyanide working solution, carrying out water bath reaction for 15min at 80 ℃, cooling at room temperature, adding 2mL of distilled water, uniformly mixing, centrifuging for 10min at 10000pm, taking supernatant, measuring absorbance at 420nm, and taking blank as absorbance measured by using distilled water instead of a sample.

Table 1: absorbance of different samples

As shown in Table 1, the difference of OD values between the inactivated group and the non-inactivated group using fucoidan as a substrate and kelp concentrate as a substrate is not obvious, which indicates that the enzyme coded by Fcn gene has not obvious degradation capability on fucoidan and kelp concentrate, and the difference of OD values between the inactivated group and the non-inactivated group is not obvious because a large amount of reducing sugar cannot be generated to react with potassium ferricyanide.

In the experimental group using fucoidan sulfate as a substrate, the OD value of the non-inactivated group is far greater than the OD value of the inactivated group, which indicates that the enzyme coded by the Fcn gene has obvious degradation capability on the fucoidan sulfate, and a large amount of reducing sugar is generated to react with potassium ferricyanide, so that the OD value of the non-inactivated group is far greater than the OD value of the inactivated group.

As shown by the analysis, the enzyme coded by the Fcn1 gene has no obvious degradation capability on fucoidan and kelp concentrate powder, and has obvious degradation capability on fucoidan sulfate.

The main chain structure of the fucoidan sulfate with a known structure is mainly 3-alpha-L-Fuc-1 or alternately 3-alpha-L-Fuc-1 and 4-alpha-L-Fuc-1, and 2-alpha-L-Fuc-1 and sometimes occurs as a branched chain, the fucoidan sulfate mainly takes alpha 1-3 glycosidic bonds as a main chain, the protein sequence alignment result of corresponding pair Fcn1, the homology of Fcn1 protein sequence and FunA (WP_ 068826898.1) protein sequence reaches 70.98%, and the amino acid sequence phylogenetic tree shows that the protein coded by Fcn1 gene has the capability of degrading the alpha 1-3 glycosidic bonds in the latest view.

The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Sequence listing

<110> Qingdao university of agriculture

<120> a fucoidan gene Fcn1 and use thereof

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<170> SIPOSequenceListing 1.0

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<213> Artificial sequence (Artificial Sequence)

<400> 1

atgaataaac taatttcaat atttctagga gcgttgtttc tatcgtgtgc aaccacaaga 60

gaggggtatc aaaaaaaggt aacatctaaa gttgcggata aaagttctag tcttttgatt 120

agtgatggta ctgtgtttat gcgaaaatct ttttatccaa aatttaattg ggaggtaaca 180

cctcagtatt ttatgtttgg tacaggtgat agattgctta cagattctga agtaaaaact 240

attgcaagta aaacagattt tatttgtata gagaaaaatc atgcatatag aaaattgaag 300

tatgcagaaa taggtgctaa acaagaaata aaagcattta aagctgttaa gccggatata 360

aagactcttt attattttaa ttctgcttat gcttggccat ttacttccta tacagaaaat 420

ttcactaaaa ataagataga aaaatatcca gctttaaaga agtttttaat taaaaataaa 480

gaaacaggag agttagagca tagaaataac atatataatt ttgatgtgtt aaatccagaa 540

tttagagttt ggtgggtaga aacgatagtt aaaggagttg aggtttcagg tgcagatgga 600

gtgtttatag atcaaatgca tgggtttgtt tggttaaggg ctgatagaaa aaaagaagta 660

gaggcagcta tgggagaaat gatgactaac ttaaagaaag ctttagggtc agataaaata 720

gtattgggta ataatgcatc aaatgtaaaa gatgtttttc ctgcaattga cgcagctatg 780

tttgaacatt acaataataa taaattaagt aaagaaaatc ttcttaagga gtgggaggat 840

atgttggtaa atgcaaaagc aggtaaaata tctattttta ggattggggt agaggcagaa 900

aaagaagagg cgagtcaaac tttaattaca gcatctaaag atgattcttt tgaagagtta 960

tctaaagaaa gattagaata ttatcaggct tgttatttaa taggagcgca accttattct 1020

tattttcagt atgggtgggg ctggcgttta gatactggtc ctttggtaga ttacccatcg 1080

ctttataaat cattaggaac tcctaaagga gcttataaaa gggttgatga aaatggttgg 1140

cagtttacaa gagattttga acaagccaaa gtttgggtag atacagaaaa aaaagaagct 1200

aaaattactt ggttagatta a 1221

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<213> Artificial sequence (Artificial Sequence)

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Met Asn Lys Leu Ile Ser Ile Phe Leu Gly Ala Leu Phe Leu Ser Cys

1 5 10 15

Ala Thr Thr Arg Glu Gly Tyr Gln Lys Lys Val Thr Ser Lys Val Ala

20 25 30

Asp Lys Ser Ser Ser Leu Leu Ile Ser Asp Gly Thr Val Phe Met Arg

35 40 45

Lys Ser Phe Tyr Pro Lys Phe Asn Trp Glu Val Thr Pro Gln Tyr Phe

50 55 60

Met Phe Gly Thr Gly Asp Arg Leu Leu Thr Asp Ser Glu Val Lys Thr

65 70 75 80

Ile Ala Ser Lys Thr Asp Phe Ile Cys Ile Glu Lys Asn His Ala Tyr

85 90 95

Arg Lys Leu Lys Tyr Ala Glu Ile Gly Ala Lys Gln Glu Ile Lys Ala

100 105 110

Phe Lys Ala Val Lys Pro Asp Ile Lys Thr Leu Tyr Tyr Phe Asn Ser

115 120 125

Ala Tyr Ala Trp Pro Phe Thr Ser Tyr Thr Glu Asn Phe Thr Lys Asn

130 135 140

Lys Ile Glu Lys Tyr Pro Ala Leu Lys Lys Phe Leu Ile Lys Asn Lys

145 150 155 160

Glu Thr Gly Glu Leu Glu His Arg Asn Asn Ile Tyr Asn Phe Asp Val

165 170 175

Leu Asn Pro Glu Phe Arg Val Trp Trp Val Glu Thr Ile Val Lys Gly

180 185 190

Val Glu Val Ser Gly Ala Asp Gly Val Phe Ile Asp Gln Met His Gly

195 200 205

Phe Val Trp Leu Arg Ala Asp Arg Lys Lys Glu Val Glu Ala Ala Met

210 215 220

Gly Glu Met Met Thr Asn Leu Lys Lys Ala Leu Gly Ser Asp Lys Ile

225 230 235 240

Val Leu Gly Asn Asn Ala Ser Asn Val Lys Asp Val Phe Pro Ala Ile

245 250 255

Asp Ala Ala Met Phe Glu His Tyr Asn Asn Asn Lys Leu Ser Lys Glu

260 265 270

Asn Leu Leu Lys Glu Trp Glu Asp Met Leu Val Asn Ala Lys Ala Gly

275 280 285

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

290 295 300

Ser Gln Thr Leu Ile Thr Ala Ser Lys Asp Asp Ser Phe Glu Glu Leu

305 310 315 320

Ser Lys Glu Arg Leu Glu Tyr Tyr Gln Ala Cys Tyr Leu Ile Gly Ala

325 330 335

Gln Pro Tyr Ser Tyr Phe Gln Tyr Gly Trp Gly Trp Arg Leu Asp Thr

340 345 350

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

355 360 365

Lys Gly Ala Tyr Lys Arg Val Asp Glu Asn Gly Trp Gln Phe Thr Arg

370 375 380

Asp Phe Glu Gln Ala Lys Val Trp Val Asp Thr Glu Lys Lys Glu Ala

385 390 395 400

Lys Ile Thr Trp Leu Asp

405

<210> 3

<211> 39

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

ggtaccatga ataaactaat ttcaatattt ctaggaggg 39

<210> 4

<211> 33

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

ggatccttaa tctaaccaag taattttagc ttc 33

Claims

1. Fucoidanase geneFcn1Characterized in that the following stepsFcn1The nucleotide sequence of the gene is shown as SEQ ID No. 1.

2. The fucoidanase gene of claim 1Fcn1The coded fucoidan is characterized in that the amino acid sequence of the fucoidan is shown as SEQ ID No. 2.

3. A method comprising the fucoidan gene of claim 1Fcn1Is a recombinant vector of (a).

4. The recombinant vector according to claim 3, wherein the vector is a pLB vector.

5. Comprising the composition of claim 1Fucoidanase geneFcn1Is a recombinant strain of (a).

6. The recombinant strain of claim 5, wherein the recombinant strain is escherichia coli.

7. The fucoidanase gene of claim 1Fcn1Or the use of a fucoidan enzyme of claim 2 for the preparation of a formulation for degrading fucoidan sulfate.