CN108531470B - Fucoidin lyase TFLFM and preparation method and application thereof - Google Patents
Fucoidin lyase TFLFM and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the field of genetic engineering, and mainly relates to a fucoidin lyase TFLFM as well as a preparation method and application thereof. The invention leads the fucoidin lyase to be expressed with high efficiency by truncating the amino acid sequence of the fucoidin lyase. The strain used by the invention is escherichia coli BL21(DE3), a gene for coding truncated fucoidin lyase is introduced, so that recombinant escherichia coli capable of efficiently expressing fucoidin lyase is obtained, and the enzyme activity is verified through in vitro experiments.
Description
Technical Field
The invention relates to the field of genetic engineering, and mainly relates to a fucoidin lyase TFLFM as well as a preparation method and application thereof.
Background
Fucoidan or fucoidan sulfate is a water-soluble heteropolysaccharide with a relatively high molecular weight, which is mainly present in brown algae (such as kelp, fucus, etc.) and some marine invertebrates (such as sea cucumber, sea urchin, etc.) and mainly consists of sulfated L-fucose (hydroxyl sulfation at position 2/3/4 of fucose), some also contains monosaccharides such as galactose, mannose, uronic acid, glucose, rhamnose, xylose, etc. and acetyl components (Ale MT, et al, Mar. drugs,2011,9,2106 2130). the structure of fucoidan derived from brown algae is very complex, including the positions and numbers of sulfation and acetylation, and branched structures, etc., and only a part of the average or partial structure of fucoidan is clear.
Although fucoidan sulfate has various activities, the structural diversity, high molecular weight, viscosity, etc. of fucoidan sulfate may limit its applications, especially in therapeutic applications. These problems can be overcome if fucoidan sulfate is degraded into low molecular weight oligosaccharides, which is also an effective means for solving the structure-activity relationship. The research proves that the low molecular weight fucoidin also shows good activities of resisting tumor, blood coagulation, thrombus and platelet, improving immunity, resisting virus, preventing tubulointerstitial fibrosis and the like. Therefore, the fucoidan oligosaccharide has more potential to be developed into medicines. The controlled degradation of fucoidan is the key to the realization of its activity application and the study of its structure-activity relationship (Chen CH, et al, sci.
Similar to the preparation of other oligosaccharides, the preparation method of the fucoidan oligosaccharide mainly comprises a physical method, a chemical method and an enzymatic method. Physical degradation methods include microwave, ultrasonic and other methods, and although the methods are simple to operate and easy to control, the degradation efficiency is low and the degradation degree is limited, and the methods are usually used in combination with other degradation methods. The chemical degradation method mainly comprises acid hydrolysis and oxidative degradation, and is usually carried out under severe conditions, so that the problems of poor controllability and repeatability, environmental pollution, special reaction equipment and the like exist. Researches prove that the content and position of the sulfate group have important influence on the activity of the fucosan sulfate and oligosaccharide. Chemical degradation easily results in the removal of sulfate groups, which seriously affects the activity of the degradation products. The enzymolysis method is to specifically degrade fucoidan sulfate by using hydrolase or lyase to obtain oligosaccharide with low molecular weight. The enzymolysis method has the advantages of high specificity, good product uniformity, mild reaction conditions, easy process control, high repeatability, less side reactions, environmental friendliness and the like, and is an ideal method for degrading the fucosan sulfate. Most importantly, the molecular weight is reduced without affecting the content and position of sulfate groups, and therefore the activity of the degradation product, fucoidan sulfate oligosaccharide (Holtkamp AD, et al, appl. microbiol. biotechnol.,2009,82, 1-11).
At present, few reports are reported on studies on fucoidan sulfate degrading enzymes, no commercial fucoidan sulfate degrading enzyme preparation is available in the market, and no fucoidan sulfate oligosaccharide product prepared by a biological method is available in the market (KusaykinMI, et al, Glycobiology,2016,26, 3-12).
Disclosure of Invention
The invention aims to provide a truncated fucoidin lyase TFLFM and a coding gene thereof.
It is another object of the present invention to provide a recombinant vector comprising a truncated fucoidan lyase TFLFM-encoding gene and a recombinant strain that efficiently expresses fucoidan lyase.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides fucoidin sulfate lyase TFLFM, the amino acid sequence of which is shown in SEQ ID NO. 4.
The invention also provides a fucoidin sulfate lyase gene which codes the fucoidin sulfate lyase TFLFM. Further preferably, the nucleotide sequence of the gene is shown as SEQ ID NO. 3.
The invention also provides a recombinant expression vector containing the fucoidin sulfate lyase gene, and preferably, the recombinant expression vector is pET28 a-TFLFM.
The invention also provides a recombinant strain containing the recombinant expression vector. Further preferably, the recombinant strain is escherichia coli BL21(DE 3).
The preparation method of the fucoidin sulfate lyase TFLFM comprises the following steps:
1) constructing a gene sequence for expressing and coding an N-terminal catalytic structural domain of the fucoidin sulfate lyase to obtain the fucoidin sulfate lyase gene, and then constructing the recombinant vector;
2) transforming host cells by using the recombinant vector obtained in the step 1) to obtain a recombinant strain;
3) culturing the recombinant strain for fermentation, and inducing the fucoidin sulfate lyase to express;
4) recovering and purifying the expressed fucoidan lyase.
The invention also provides application of the fucoidin lyase TFLFM in degrading fucoidin sulfate.
Since the gene (GenBank NoAAO00510.1) encoding the intact fucoidan sulfate Lyase FLFM (fucoidan Lyase from fucoidan marina) derived from the marine bacterium Flavobacterium marinum SA-0082 was expressed in Escherichia coli, inclusion bodies were mainly formed, and soluble proteins could not be obtained.
Conserved Domain prediction of FLFM, which includes a Domain with unknown N-terminal function, a type F5/8C Domain (also known as a spinosyn Domain), and a C-terminal Por _ Secre _ tail Domain (C-terminal alignment Domain, possibly associated with The alignment or covalent binding of proteins on The bacterial surface), was performed by The Conserved Domain Database (CCD) of NCBI (The National Center for Biotechnology information). Based on this, we speculate that the domain with unknown N-terminal function may be the catalytic domain, so the invention constructs the gene (encoding 447 amino acids at the N-terminal) only encoding the N-terminal catalytic domain, and constructs a recombinant strain for efficiently expressing the truncated fucoidin sulfate lyase. Through gene engineering technology, the expression plasmid pET28a-TFLFM (truncated FLFM) for coding truncated fucoidin lyase gene is transferred into escherichia coli BL21(DE3) to obtain recombinant escherichia coli BL21(DE 3).
The expression plasmid pET28a-TFLFM consists of a fucoidin lyase gene with a truncated N-end and an expression vector pET-28a vector. The plasmid pET28a-TFLFM for expressing the N-end truncated fucoidin lyase gene is introduced into a strain of escherichia coli BL21(DE3) for high-efficiency expression, the yield of target protein reaches 100mg/L, and the enzyme activity of the fucoidin sulfate lyase TFLFM is verified through in vitro experiments.
The invention has the advantages that: the truncated fucoidin sulfate lyase can specifically degrade fucoidin sulfate enzyme, and provides a tool enzyme for degrading fucoidin sulfate. The invention further constructs a recombinant strain for efficiently expressing the truncated fucoidin lyase by constructing the fucoidin lyase gene with the truncated N-terminal, so that the fucoidin lyase can be efficiently expressed in the strain without forming an inclusion body.
Drawings
FIG. 1 is a schematic diagram of the structure of an expression plasmid of an N-terminally truncated fucoidan lyase gene of the present invention.
FIG. 2 is an SDS-PAGE electrophoresis of the nickel column purification of the present invention expressing an N-terminally truncated fucoidan lyase (the band in frame is the purified protein of interest TFLFM).
FIG. 3 is an HPLC chart of the present invention expressing the product of the N-terminally truncated fucoidan lyase-catalyzed reaction (solid line-enzyme-catalyzed reaction; dotted line …: control, no enzyme added) (catalytic reaction product at arrow).
FIG. 4 is a first order mass spectrum of the product of the catalytic reaction of fucoidan sulfate lyase expressed as truncated N-terminal of the present invention (molecular weight of the catalytic reaction product: 563.348, predicted molecular weight 564.47 at arrow).
FIG. 5 is a secondary mass spectrum (secondary mass spectrum of 563.348) of the present invention expressing the product of the N-terminally truncated fucoidan lyase catalyzed reaction.
Detailed Description
The technical solution of the present invention will be described in detail with reference to examples. The reagents and biomaterials used below were all commercial products unless otherwise specified. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.
In the invention, the Plasmid extraction adopts OMEGA Plasmid Mini Kit I Kit (D6943-01) BL21(DE3) to sense cells from the sugar bioengineering subject group of the institute of Process engineering of Chinese academy of sciences.
Example 1 construction of intact fucoidin sulfate lyase FLFM-expressing Strain
The present invention refers to a gene (GenBank No: AAO00510.1) encoding fucoidan sulfate lyase, entrusts bioengineering (Shanghai) to a company Limited to biological engineering (Shanghai) through codon optimization to synthesize a gene encoding fucoidan sulfate lyase (excluding signal peptide), has 2022 bases in total, has a nucleotide sequence shown in SEQ ID NO.1, clones of pET28a, cloning sites NdeI and XhoI, resistance of the vector is kanamycin (Kan), and optimizes species E.coli (FIG. 1). Escherichia coli DH5 α carrying expression Plasmid pET28a-FLFM is cultured, plasmids are extracted using Plasmid Mini Kit I Kit, and then the expression plasmids are introduced into competent Escherichia coli BL21
(DE3), recombinant strains were obtained. The amino acid sequence is shown in SEQ ID NO.2, the protein contains 673 amino acids, and the predicted molecular weight of the protein is 73.0 kDa.
SEQ IDNO.1
(1) Sequence characterization
Length: 2022bp
Type (2): base sequence
Chain type: double chain
Topological structure: linearity
(2) Molecular type: DNA
(3) Suppose that: whether or not
(4) Antisense: whether or not
(5) The initial sources were: AAO00510.1
(6) Specific name: fucoidin sulfate lyase gene
SEQ ID NO.2
(1) Sequence characterization
Length: 673
Type (2): amino acid sequence
(2) Molecular type: protein
(3) Suppose that: whether or not
(4) Antisense: whether or not
(5) The initial sources were: AAO00510.1
(6) Specific name: fucoidan lyase.
Example 2 expression and detection of intact fucoidan lyase FLFM
(1) Preparing an LB culture medium containing Kan resistance: 5g/L yeast extract, 10g/L tryptone, 10g/L sodium chloride, 120 ℃ sterilization for 20min, cooling to room temperature and adding Kan to make the final concentration 50. mu.g/mL.
(2) The recombinant strain obtained in example 1 was inoculated into a solid LB medium containing Kan resistance, cultured overnight at 37 ℃, a single colony was selected and inoculated into 5mL of a liquid LB medium containing Kan resistance, shake-cultured at 37 ℃ and 200rmp for 24 hours, and then the above-mentioned bacterial liquid was inoculated into 500mL of a liquid LB medium containing Kan resistance, and bed-cultured at 37 ℃ and 200rmp to OD600nmWhen the expression level is 0.6, 0.5mM IPTG is added, the expression is induced at 16 ℃ and 200rmp for 24 hours, and the cells are collected by centrifugation at 5000 rmp.
(3) Taking a small amount of thalli to carry out SDS-PAGE analysis, and detecting no expression of the target protein in a supernatant solution; by Western blot analysis, the presence of only a small amount of the protein of interest was detected.
Example 3 construction of N-terminally truncated fucoidan lyase TFLFM expressing Strain
With F1 (5' -CGGCGC)CATATGCAGACTACTACCGTATACAG-3 ') and R1 (5' -ACGGGC)CTCGAGTTAATCGGAGTCGCAGTCAATCGC-3') as a primer pair, carrying out PCR by taking a Plasmid pET28a-FLFM as a template, carrying out enzyme digestion on a PCR product and an expression vector pET28a by NdeI and XhoI respectively, recovering a target fragment, connecting a gene fragment and a vector fragment to obtain a Plasmid pET28a-TFLFM (figure 1), culturing Escherichia coli DH5 α carrying the expression Plasmid pET28a-TFLFM, extracting the Plasmid by using a Plasmid Mini Kit I Kit, and then introducing the expression Plasmid into competent Escherichia coli BL21(DE3) to obtain a recombinant strain, wherein the nucleotide sequence is shown as SEQ ID No.3, the amino acid sequence is shown as SEQ ID No.4, the predicted protein molecular weight is 48.0 kDa.
SEQ ID NO.3
(1) Sequence characterization
Length: 1344bp
Type (2): base sequence
Chain type: double chain
Topological structure: linearity
(2) Molecular type: DNA
(3) Suppose that: whether or not
(4) Antisense: whether or not
(5) The initial sources were: AAO00510.1
(6) Specific name: truncated fucoidan sulfate lyase gene.
SEQ ID NO.4
(1) Sequence characterization
Length: 447
Type (2): amino acid sequence
(2) Molecular type: protein
(3) Suppose that: whether or not
(4) Antisense: whether or not
(5) The initial sources were: AAO00510.1
(6) Specific name: a truncated fucoidan sulfate lyase.
Example 4 expression, purification and detection of N-terminally truncated fucoidan lyase TFLFM
(1) The cells collected in example 3 were suspended in buffer A (50mM Tri-HCl, pH 7.9, 500mM NaCl) and sonicated, and the supernatant was collected by centrifugation at 12000rmp and examined by SDS-PAGE (FIG. 2) to predict a protein molecular weight of 48.0 kDa.
(2) The above proteins were purified using a nickel column:
1. buffer A (50mM Tris/HCl, pH 8.0,0.5M NaCl) equilibrated the column at a flow rate of 1 mL/min.
2. The sample was loaded at a flow rate of 1mL/min and the breakthrough was collected.
3. The buffer solution A washes the column at a flow rate of 1mL/min and 30mL,
4. buffer A +20mM imidazole eluted at a flow rate of 1mL/min, 30mL of wash was performed, and one tube was collected every 5 min.
G250 testing the collected sample to see if there is protein eluted from the last tube, and if there is no protein, then eluting with imidazole of next concentration
6. Buffer A +60mM imidazole eluted at a flow rate of 1mL/min, 30mL washed, and one tube was collected every 5 min.
G250 testing the collected sample to see if there is protein eluted from the last tube, and if there is no protein, proceeding the next concentration imidazole elution
8. Buffer A +100mM imidazole, flow 1mL/min, wash 30mL, collect one tube every 5 min.
G250 testing the collected sample to see if there is protein eluted from the last tube, and if there is no protein, then eluting with imidazole of next concentration
10. Buffer A +160mM imidazole eluted at a flow rate of 1mL/min, 30mL of wash, one tube collected every 5 min.
G250 testing the collected sample to see if any protein is eluted from the last tube, and if no protein is eluted, then eluting with the next concentration of imidazole.
12. Buffer A +200mM imidazole eluted at a flow rate of 1mL/min, 30mL washed, and one tube was collected every 5 min.
G250 testing the collected sample to see if there is protein eluted from the last tube, and if there is no protein, then eluting with imidazole of next concentration
14. The group with high content of the eluted protein of each imidazole concentration, the stock solution, the sample penetration and the elution of the buffer solution A are selected for SDS-PAGE analysis (figure 2).
The relatively pure fucoidan lyase fractions were pooled and dialyzed against a 14,000Da dialysis bag.
In the embodiment, the N-end truncated fucoidin lyase TFLFM is efficiently expressed in escherichia coli, the yield of the purified protein reaches 100mg/1L of culture medium, and a foundation is laid for the application and development of the fucoidin lyase.
Example 5 enzymatic Activity analysis of N-terminally truncated fucoidan lyase TFLFM
mu.L of fucoidan (10mg/mL) solution (dissolved in 50mM sodium phosphate buffer, pH 7.5) was added 10. mu.L of 4M NaCl, 10. mu. L N-terminally truncated fucoidan sulfate lyase TFLFM (8mg/mL) and reacted at 37 ℃ at 200rpm/min for 6 hours. Then heating in water bath at 100 deg.C for 10min, centrifuging at 12000rpm/min for 5min, collecting supernatant, and performing HPLC analysis (FIG. 3): AcchromS6000HPLC system, XAmide column (4.6mm x 250mm,5 μm), mobile phase water (a), acetonitrile (B) and ammonium formate (C), gradient elution conditions: 0-40min, 90-50% (B), 10% (C), flow rate of 1.5mL/min, column temperature of 40 ℃.
Collecting peak of HPLC about 25 min, freeze drying, dissolving in small amount of water, mixing sample with matrix DHB, and analyzing ionization-time of flight mass spectrometer MALDI-TOF/TOF Ultraflexreeme with matrix-assisted laserTM(Brucker, Germany) was analyzed in reflectance mode (FIG. 4) and the peak at 563.348 was analyzed by secondary mass spectrometry (FIG. 5).
In the embodiment, the N-terminal truncated fucoidin lyase TFLFM shows good fucoidin degradation activity, and provides a cushion for the application and development of the fucoidin lyase.
The present invention may be embodied in many different forms and modifications may be effected therein by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
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Gly Ser Val Ile Asn His Arg Lys His Ser Gly Val Leu Ile Arg Gly
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Tyr Lys Leu Pro Ala Gly Tyr Met Met Ser Leu Gln Glu Gly Gly Ile
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Arg Ala Thr Asp Asn Pro Thr Val Leu Asn Cys Thr Ile Lys Asn Ala
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Ile Arg Val Ser Asp Gln Asn Lys Asn Ile Ile Asn Lys Ile Val Lys
660 665 670
Leu
<210>3
<211>1344
<212>DNA
<213>Artificial
<400>3
atgcagacta ctaccgtata cagcctggag gacctgctgc catatctgaa gcaagataac 60
gtggatgtca agctggctcc tggtacttac aacgtcaacg gtttcgacgt cggtgaagac 120
cgtctgttct ctactactcc actgttcctg tttgaaggtt ccaacagcac ctacgacttt 180
accgacgtga agctgaacat caacaccgtc gtgctgacta agttcggtaa caacgaggtc 240
aacgaaatcc agattctggg taacaataat gtgctgaaaa acctgaaact ggaggatatc 300
ggtaccaccg cgccgtccaa ccgtgctcag agcatcgtta tcgacggtcg cgacaaccgt 360
atcgagggtt tccacctgac tatccgtggt tcttatcctt acggttacgg cgacgcattt 420
ggtaaaggtg gcggttctgt tatcaaccac cgcaaacact ccggtgtgct gattcgtggt 480
ctgcgcaatc atctgaaaga ttgcaccatc atctcccgtt cttatggtca catcgtcttc 540
atgcaggccg catcctaccc aactgtggaa ggttgctaca tcgaaggtga gatgcgttct 600
accgacgaca tgctggcaga agaaggtact ggttctccag cagataaagt ggacttcatg 660
accgtgtggg gttacaagct gcctgctggt tatatgatga gcctgcaaga gggtggtatc 720
cgtgcatata acgcaggtac tacttacatc gacggcgtgg aaattcagcg tgcaactgac 780
aacccgactg ttctgaactg cactatcaag aacgcacgta ccggtgtgac cctggctcat 840
gctaacggca ccaaatatgt tgaaggctgt acggttctgg gttgtgaaaa cggttactct 900
atcggttccg gtactgtggt gaactgcggt gctgatgcta tctacggccc ggtgtttaaa 960
aacacctacg gctctgataa aggctacaat gctgacatta ccatcctgcc gccgtctgat 1020
gcttactaca acggccatga tgcggtagcg tatatcggcg gctctaatca caacctgact 1080
ttccgttctg aaatcacgga aatcccgtct aatctgaaaa tcatggtatc tggcgacctg 1140
cagggcctgc gtgtactgca cggcagcaat ccgtctcaga ataatttcgc tggcaccaac 1200
atcgttctgc gtaacctgac caacttcccg gtagacctgc attctgattc ttctaacatc 1260
acggttacct cttgcgacac ggacaacatt acggataacg gtaccaacaa cagcatcgag 1320
gcgattgact gcgactccga ttaa 1344
<210>4
<211>447
<212>PRT
<213>Artificial
<400>4
Met Gln Thr Thr Thr Val Tyr Ser Leu Glu Asp Leu Leu Pro Tyr Leu
1 5 10 15
Lys Gln Asp Asn Val Asp Val Lys Leu Ala Pro Gly Thr Tyr Asn Val
20 25 30
Asn Gly Phe Asp Val Gly Glu Asp Arg Leu Phe Ser Thr Thr Pro Leu
35 40 45
Phe Leu Phe Glu Gly Ser Asn Ser Thr Tyr Asp Phe Thr Asp Val Lys
50 55 60
Leu Asn Ile Asn Thr Val Val Leu Thr Lys Phe Gly Asn Asn Glu Val
65 70 75 80
Asn Glu Ile Gln Ile Leu Gly Asn Asn Asn Val Leu Lys Asn Leu Lys
85 90 95
Leu Glu Asp Ile Gly Thr Thr Ala Pro Ser Asn Arg Ala Gln Ser Ile
100 105 110
Val Ile Asp Gly Arg Asp Asn Arg Ile Glu Gly Phe His Leu Thr Ile
115 120 125
Arg Gly Ser Tyr Pro Tyr Gly Tyr Gly Asp Ala Phe Gly Lys Gly Gly
130 135 140
Gly Ser Val Ile Asn His Arg Lys His Ser Gly Val Leu Ile Arg Gly
145 150 155 160
Leu Arg Asn His Leu Lys Asp Cys Thr Ile Ile Ser Arg Ser Tyr Gly
165 170 175
His Ile Val Phe Met Gln Ala Ala Ser Tyr Pro Thr Val Glu Gly Cys
180 185 190
Tyr Ile Glu Gly Glu Met Arg Ser Thr Asp Asp Met Leu Ala Glu Glu
195 200 205
Gly Thr Gly Ser Pro Ala Asp Lys Val Asp Phe Met Thr Val Trp Gly
210 215 220
Tyr Lys Leu Pro Ala Gly Tyr Met Met Ser Leu Gln Glu Gly Gly Ile
225 230 235 240
Arg Ala Tyr Asn Ala Gly Thr Thr Tyr Ile Asp Gly Val Glu Ile Gln
245 250 255
Arg Ala Thr Asp Asn Pro Thr Val Leu Asn Cys Thr Ile Lys Asn Ala
260 265 270
Arg Thr Gly Val Thr Leu Ala His Ala Asn Gly Thr Lys Tyr Val Glu
275 280 285
Gly Cys Thr Val Leu Gly Cys Glu Asn Gly Tyr Ser Ile Gly Ser Gly
290 295 300
Thr Val Val Asn Cys Gly Ala Asp Ala Ile Tyr Gly Pro Val Phe Lys
305 310 315 320
Asn Thr Tyr Gly Ser Asp Lys Gly Tyr Asn Ala Asp Ile Thr Ile Leu
325 330 335
Pro Pro Ser Asp Ala Tyr Tyr Asn Gly His Asp Ala Val Ala Tyr Ile
340 345 350
Gly Gly Ser Asn His Asn Leu Thr Phe Arg Ser Glu Ile Thr Glu Ile
355 360 365
Pro Ser Asn Leu Lys Ile Met Val Ser Gly Asp Leu Gln Gly Leu Arg
370 375 380
Val Leu His Gly Ser Asn Pro Ser Gln Asn Asn Phe Ala Gly Thr Asn
385 390 395 400
Ile Val Leu Arg Asn Leu Thr Asn Phe Pro Val Asp Leu His Ser Asp
405 410 415
Ser Ser Asn Ile Thr Val Thr Ser Cys Asp Thr Asp Asn Ile Thr Asp
420 425 430
Asn Gly Thr Asn Asn Ser Ile Glu Ala Ile Asp Cys Asp Ser Asp
435 440 445
Claims (9)
1. A fucoidan lyase TFLFM characterized by: the amino acid sequence of the fucoidin sulfate lyase is shown as SEQ ID NO. 4.
2. A fucoidin sulfate lyase gene, which is characterized in that: encoding the fucoidan lyase TFLFM according to claim 1.
3. The fucoidan lyase gene according to claim 2, wherein: the nucleotide sequence of the gene is shown in SEQ ID NO. 3.
4. A recombinant expression vector comprising the fucoidan lyase gene according to claim 2 or 3.
5. The recombinant expression vector of claim 4, wherein: the recombinant expression vector is pET28a-TFLFM, and the expression plasmid pET28a-TFLFM consists of the fucoidin lyase gene of claim 2 or 3 and an expression vector pET-28a vector.
6. A recombinant strain comprising the recombinant expression vector of claim 4 or 5.
7. The recombinant strain of claim 6, wherein the recombinant strain is Escherichia coli BL21(DE 3).
8. A method for preparing the fucoidan lyase TFLFM of claim 1, comprising the steps of:
1) constructing a gene sequence for expressing an N-terminal catalytic domain encoding fucoidan lyase to obtain the fucoidan sulfate lyase gene of claim 2 or 3, and then constructing a recombinant expression vector of claim 4 or 5;
2) transforming a host cell with the recombinant expression vector of claim 4 or 5 to obtain a recombinant strain;
3) culturing the recombinant strain for fermentation, and inducing the fucoidin sulfate lyase to express;
4) recovering and purifying the expressed fucoidan lyase.
9. Use of the fucoidan lyase TFLFM according to claim 1 for degrading fucoidan.
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