CN108531470A - A kind of sulfuric acid fucoidin lyases TFLFM and its preparation method and application - Google Patents
A kind of sulfuric acid fucoidin lyases TFLFM and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to genetic engineering field, a kind of sulfuric acid fucoidin lyases TFLFM and its preparation method and application is related generally to.The present invention makes its high efficient expression by truncating the amino acid sequence of sulfuric acid fucoidin lyases.Bacterial strain uses therefor of the present invention is e. coli bl21 (DE3), imports the gene that coding truncates sulfuric acid fucoidin lyases, to obtain capableing of the recombination bacillus coli of high efficient expression sulfuric acid fucoidin lyases, and confirms enzyme activity by experiment in vitro.
Description
Technical field
The present invention relates to genetic engineering field, a kind of sulfuric acid fucoidin lyases TFLFM and its preparation side are related generally to
Method and application.
Background technology
Sulfuric acid fucosan or fucoidan are a kind of higher water-soluble heteroglycan of molecular weight, are primarily present in
In brown alga (such as kelp, bladder-wrack) and some oceanic invertebrates (such as sea cucumber, sea urchin).It is mainly by sulphation L- rocks
Algae sugar (2/3/4 hydroxyl sulphation of fucose) composition, some also contain galactolipin, mannose, uronic acid, glucose, mouse
The monosaccharide and acetyl group etc. such as Lee's sugar, xylose components (Ale MT, et al., Mar.Drugs, 2011,9,2106-2130).It is brown
The structure of algae source sulfuric acid fucosan is extremely complex, including the position of sulphation and acetylation and quantity and branched structure
Deng only the average or part-structure of some sulfuric acid fucosan is clearer.The main chain of brown alga source sulfuric acid fucosan
It include mainly two types structural framework：Type I is only made of α -1 → 3- fucose glycosidic bonds, Type II by alternately α -1 → 3- and
α -1 → 4- fucose glycosidic bonds composition.Structure from oceanic invertebrate sulfuric acid fucosan is more relatively easy, linear main
Chain is mainly formed by α -1 → 3- or α -1 → 4- fucose glycosidic bonds, and is had certain regularity, by the sulphation rock algae repeated
The single or structural units such as three or tetrose composition.Many studies have shown that sulfuric acid fucosan have anticoagulation, it is antitumor, anti-oxidant,
Anti-inflammatory, antithrombotic, reducing blood lipid, improve immunity, antibacterial, antiviral, anticomplement isoreactivity (Holtkamp AD, et al.,
Appl.Microbiol.Biotechnol.,2009,82,1-11)。
Although there is sulfuric acid fucosan various active, the structure diversity of sulfuric acid fucosan high molecular weight, to glue
The features such as consistence, may limit their application, especially in terms for the treatment of.If sulfuric acid fucosan is degraded to low molecular weight widow
Sugar just can overcome the problems, such as these, this is also the effective means for solving its structure-activity relationship.Research has shown that, low molecular weight sulfuric acid rock algae
Oligosaccharides also show good antitumor, anticoagulation, antithrombotic, antiplatelet, improve immunity, be antiviral, to prevent kidney small
Pipe interstitial fibrosis isoreactivity.Therefore, sulfuric acid fucose is with more the potentiality that exploitation is drug.Sulfuric acid fucosan is carried out
Controlled degradation be the key that realize its activity application and study its structure-activity relation (Chen CH, et al., Sci.Rep.,
2017,40183)。
Similar with preparing for other oligosaccharides, the preparation method of sulfuric acid fucose also mainly has Physical, chemical method and enzyme
Method.Physical degradation methods include the methods of microwave, ultrasonic wave, although easy to operate, be easy to control, degradation efficiency is relatively low and drops
Solve limitation, it usually needs be used in combination with other biodegrading process.Chemical degradation method mainly include sour water solution and oxidative degradation,
It is generally necessary to be carried out under drastic conditions, there are controllabilitys to set with less reproducible, environmental pollution and special the reacting of needs
The problems such as standby.Research has shown that the content of sulfate and position have a major impact the activity of sulfuric acid fucosan and oligosaccharides.And change
Learn the activity that edman degradation Edman easilys lead to sulfate and is also removed, therefore will seriously affect catabolite.Enzymatic isolation method is to utilize water
It solves enzyme or cracks the degradation sulfuric acid fucosan of enzyme spcificity, obtain the oligosaccharides of low molecular weight.Enzymatic isolation method has specificity height, production
Object homogeneity is good, reaction condition is mild, technique is easily controllable, it is repeatable it is high, side reaction is few, advantages of environment protection, be reason
Think the method for degradation sulfuric acid fucosan.Content that most importantly will not be to sulfate while reducing molecular weight and position
It has an impact, therefore the activity of catabolite-sulfuric acid fucose will not be had an impact (Holtkamp AD, et al.,
Appl.Microbiol.Biotechnol.,2009,82,1-11)。
It is also fewer for the report of sulfuric acid fucoidin degrading enzyme research at present, it yet there are no the sulfuric acid of commercialization in the market
Fucoidin catabolic enzyme preparation, while yet there are no the sulfuric acid fucose product (Kusaykin prepared using bioanalysis in the market
MI,et al.,Glycobiology,2016,26,3-12)。
Invention content
Present invention aims at a kind of truncated sulfuric acid fucoidin lyases TFLFM of offer and its encoding genes.
It is a further object of the present invention to provide include a kind of truncated sulfuric acid fucoidin lyases TFLFM encoding genes
The recombinant bacterial strain of recombinant vector and high efficient expression sulfuric acid fucoidin lyases.
To achieve the above object, the technical solution adopted by the present invention is：
The present invention provides a kind of sulfuric acid fucoidin lyases TFLFM, amino acid sequence such as SEQ ID NO.4 institutes
Show.
The present invention also provides a kind of sulfuric acid fucoidin lyase genes, encode above-mentioned sulfuric acid fucoidin lyases
TFLFM.It is further preferred that the nucleotide sequence of the gene is as shown in SEQ ID NO.3.
The present invention also provides the recombinant expression carriers for including above-mentioned sulfuric acid fucoidin lyase gene, preferably recombinate table
It is pET28a-TFLFM up to carrier.
The present invention also provides the recombinant bacterial strains for including above-mentioned recombinant expression carrier.It is further preferred that the recombinant bacterium
Strain is e. coli bl21 (DE3).
The preparation method of the above-mentioned sulfuric acid fucoidin lyases TFLFM of the present invention, includes the following steps：
1) structure expression encodes the gene order of the ends the N- catalyst structure domain of sulfuric acid fucoidin lyases, obtains above-mentioned sulphur
Sour fucoidin lyase gene, then builds above-mentioned recombinant vector；
2) it uses the recombinant vector of step 1) to convert host cell, obtains recombinant bacterial strain；
3) culture recombinant bacterial strain makes its fermentation, induction sulfuric acid fucoidin crack expression of enzymes；
4) it recycles and purifies expressed sulfuric acid fucoidin lyases.
The present invention also provides applications of the above-mentioned sulfuric acid fucoidin lyases TFLFM in sulfuric acid fucoidin of degrading.
The sulphur of marine bacteria Flavobacterium section bacterium Fucobacter marina SA-0082 is completely derived from due to encoding
Gene (the GenBank No of sour fucoidin lyases FLFM (Fucoidan Lyase from Fucobacter marina)
AAO00510.1) in expression in escherichia coli, inclusion body is primarily formed, is unable to get soluble protein.
Pass through the conserved structure of NCBI (The National Center for Biotechnology Information)
Regional data base (CCD, The Conserved Domain Database) carries out conserved domain prediction to FLFM, and FLFM includes
The structural domain of the ends N- Unknown Function, the Por_Secre_ of F5/8 Type Cs structural domain (also referred to as cup fungi element structural domain) and the ends C-
Tail structural domains (ends C- arrangement architecture domain, may be related in the arrangement of bacterium surface or covalent bond to protein).Based on this,
It is presumed that the structural domain of the ends N- Unknown Function may be catalyst structure domain, therefore the present invention constructs the only ends coding N- catalysis knot
The gene (447 amino acid at the ends coding N-) in structure domain, constructs the truncated sulfuric acid fucoidin lyases of one plant of high efficient expression
Recombinant bacterial strain.By technique for gene engineering, it is transferred to the truncated sulfuric acid fucoidin of coding in e. coli bl21 (DE3) and splits
The expression plasmid pET28a-TFLFM (Truncated FLFM) for solving enzyme gene, obtains recombination bacillus coli BL21 (DE3).
The expression plasmid pET28a-TFLFM is carried by the truncated sulfuric acid fucoidin lyase gene in the ends N- and expression
Body pET-28a carriers form.By the plasmid pET28a- of the expression truncated sulfuric acid fucoidin lyase gene in the ends N-
TFLFM, which is imported into e. coli bl21 (DE3) bacterial strain, carries out high efficient expression, and the yield of target protein has reached~100mg/
L, and confirm by experiment in vitro the enzyme activity of the sulfuric acid fucoidin lyases TFLFM.
Advantage for present invention：The truncated sulfuric acid fucoidin lyases of the present invention being capable of single-minded degradation sulfuric acid rock
The enzyme of polysaccharides provides a kind of toolenzyme for the degradation of sulfuric acid fucoidin.The present invention is by building the truncated sulfuric acid in the ends N-
Fucoidin lyase gene further constructs the recombinant bacterial strain of the truncated sulfuric acid fucoidin lyases of one plant of high efficient expression
Make sulfuric acid fucoidin lyases can be with high efficient expression in bacterial strain, and does not form inclusion body.
Description of the drawings
Fig. 1 is the expression plasmid structural schematic diagram of the truncated sulfuric acid fucoidin lyase gene in the ends N- of the present invention.
Fig. 2 is the SDS-PAGE electrophoresis of the present invention expression truncated sulfuric acid fucoidin lyases ni-sepharose purification in the ends N-
(band in frame is the target protein TFLFM of purifying).
Fig. 3 is that the HPLC figures of the present invention expression truncated sulfuric acid fucoidin lyases catalytic reaction products in the ends N- are (real
Line-：Enzymic catalytic reaction；Dotted line ...：Control, not enzyme) (being catalytic reaction products at arrow).
Fig. 4 is the first mass spectrometric figure of the present invention expression truncated sulfuric acid fucoidin lyases catalytic reaction products in the ends N-
(it is the molecular weight of catalytic reaction products at arrow：563.348 predicted molecular weight 564.47).
Fig. 5 is the second order ms figure of the present invention expression truncated sulfuric acid fucoidin lyases catalytic reaction products in the ends N-
(563.348 second order ms figures).
Specific implementation mode
Technical scheme of the present invention is described in detail with reference to embodiment.The reagent and biomaterial used below
If not otherwise specified, it is commercially produced product.The person that is not specified actual conditions in embodiment suggests according to normal condition or manufacturer
Condition carry out.
Plasmid extraction uses OMEGA companies Plasmid Mini Kit I kits (D6943-01) BL21 in the present invention
(DE3) permissive cell comes from sugar bioengineering seminar of Chinese Academy Of Sciences Process Engineering Research Institute.
The structure of embodiment 1 complete sulfuric acid fucoidin lyases FLFM expression bacterial strains
Gene (GenBank No of the present invention with reference to coding sulfuric acid fucoidin lyases：AAO00510.1), by close
Numeral optimizes, and it (does not include letter that commission bioengineering (Shanghai) limited liability company, which has synthesized coding sulfuric acid fucoidin lyases,
Number peptide) gene, share 2022 bases, nucleotide sequence is as shown in SEQ ID NO.1, cloning vector pET28a, clone
Site NdeI and XhoI, carrier resistance are kanamycins (Kan), optimization species E.coli (Fig. 1).To carrying expression plasmid
The bacillus coli DH 5 alpha of pET28a-FLFM is cultivated, and plasmid is extracted using Plasmid Mini Kit I kits, then will
Expression plasmid imports competent E.coli BL21
(DE3) in, recombinant bacterial strain is obtained.Amino acid sequence contains 673 amino acid, prediction as shown in SEQ ID NO.2
Molecular weight of albumen is 73.0kDa.
SEQ ID NO.1
(1) sequence signature
Length：2022bp
Type：Base sequence
Chain：Double-strand
Topological structure：Linearly
(2) molecule type：DNA
(3) assume：It is no
(4) antisense：It is no
(5) initial source：AAO00510.1
(6) specific title：Sulfuric acid fucoidin lyase gene
SEQ ID NO.2
(1) sequence signature
Length：673
Type：Amino acid sequence
(2) molecule type：Protein
(3) assume：It is no
(4) antisense：It is no
(5) initial source：AAO00510.1
(6) specific title：Sulfuric acid fucoidin lyases.
The expression and detection of 2 complete sulfuric acid fucoidin lyases FLFM of embodiment
(1) the LB culture mediums containing Kan resistances are prepared：5g/L yeast extracts, 10g/L tryptones, 10g/L chlorinations
Sodium, 120 DEG C of sterilizing 20min, being cooled to room temperature addition Kan makes 50 μ g/mL of its final concentration.
(2) recombinant bacterial strain for obtaining embodiment 1 is inoculated on the solid LB media containing Kan resistances, and 37 DEG C overnight
Culture, picking single bacterium colony are inoculated into the liquid LB cultures that 5mL contains Kan resistances, 37 DEG C, 200rmp shaking table cultures 24 hours
Afterwards, above-mentioned bacterium solution is inoculated into the liquid LB cultures that 500ml contains Kan resistances, 37 DEG C, 200rmp cultures to OD600nm=
When 0.6, it is added 0.5mM IPTG, 16 DEG C, 200rmp induced expressions 24 hours, thalline is collected in 5000rmp centrifugations.
(3) it takes a small amount of thalline to carry out SDS-PAGE analyses, the expression of target protein is not detected in supernatant solution；It is logical
Western blot analyses are crossed, only detect the presence of a small amount of target protein.
3 ends N- of embodiment truncate the structure of sulfuric acid fucoidin lyases TFLFM expression bacterial strains
With F1 (5 '-CGGCGCCATATGCAGACTACTACCGTATACAG-3 ') and R1 (5 '-
ACGGGCCTCGAGTTAATCGGAGTCGCAGTCAATCGC-3 ') it is used as primer pair, using plasmid pET28a-FLFM as template
PCR is carried out, by PCR product and expression vector pET28a respectively through NdeI and XhoI digestions, target fragment is recycled, by genetic fragment
It connects to obtain plasmid pET28a-TFLFM (Fig. 1) with carrier segments.To carrying the Escherichia coli of expression plasmid pET28a-TFLFM
DH5 α are cultivated, and plasmid is extracted using Plasmid Mini Kit I kits, and it is big that expression plasmid is then imported competence
In enterobacteria BL21 (DE3), recombinant bacterial strain is obtained.Nucleotide sequence is as shown in SEQ ID NO.3, amino acid sequence such as SEQ ID
Shown in NO.4, contain 447 amino acid, prediction molecular weight of albumen is 48.0kDa.
SEQ ID NO.3
(1) sequence signature
Length：1344bp
Type：Base sequence
Chain：Double-strand
Topological structure：Linearly
(2) molecule type：DNA
(3) assume：It is no
(4) antisense：It is no
(5) initial source：AAO00510.1
(6) specific title：Truncated sulfuric acid fucoidin lyase gene.
SEQ ID NO.4
(1) sequence signature
Length：447
Type：Amino acid sequence
(2) molecule type：Protein
(3) assume：It is no
(4) antisense：It is no
(5) initial source：AAO00510.1
(6) specific title：Truncated sulfuric acid fucoidin lyases.
4 ends N- of embodiment truncate expression, purifying and the detection of sulfuric acid fucoidin lyases TFLFM
(1) embodiment 3 is collected into thalline, is suspended to buffer solution A (50mM Tri-HCl, pH 7.9,500mM
NaCl ultrasonication is carried out in), supernatant is collected by centrifugation in 12000rmp, carries out SDS-PAGE detections (Fig. 2) and predicts molecular weight of albumen
48.0kDa。
(2) above-mentioned albumen is purified using nickel column：
1. buffer solution A (50mM Tris/HCl, pH 8.0,0.5M NaCl) balances pillar, flow velocity 1mL/min.
2. loading, flow velocity 1mL/min, collection penetrate.
3. buffer solution A washs pillar, flow velocity 1mL/min washs 30mL,
4. buffer solution A+20mM imidazoles elutes, flow velocity 1mL/min, 30ml is washed, a pipe is collected per 5min.
Sample is collected in 5.G250 detections, sees whether last pipe also has albumen to be eluted, if without albumen, is carried out
Next concentration imidazoles elution
6. buffer solution A+60mM imidazoles elutes, flow velocity 1mL/min, 30mL is washed, a pipe is collected per 5min.
Sample is collected in 7.G250 detections, sees whether last pipe also has albumen to be eluted, and is carried out if without albumen
Next concentration imidazoles elution
8. buffer solution A+100mM imidazoles elutes, flow velocity 1mL/min, 30mL is washed, a pipe is collected per 5min.
Sample is collected in 9.G250 detections, sees whether last pipe also has albumen to be eluted, if without albumen, is carried out
Next concentration imidazoles elution
10. buffer solution A+160mM imidazoles elutes, flow velocity 1mL/min, 30mL is washed, a pipe is collected per 5min.
Sample is collected in 11.G250 detections, sees whether last pipe also has albumen to be eluted, if without albumen, into
The next concentration imidazoles elution of row.
12. buffer solution A+200mM imidazoles elutes, flow velocity 1mL/min, 30mL is washed, a pipe is collected per 5min.
Sample is collected in 13.G250 detections, sees whether last pipe also has albumen to be eluted, if without albumen, into
The next concentration imidazoles elution of row
14. choosing, the high group of each imidazole concentration elution protein content, stoste, loading penetrates, buffer solution A is eluted, into
Row SDS-PAGE analyses (Fig. 2).
Merge purer sulfuric acid fucoidin lyases component, is dialysed with the bag filter of 14,000Da.
In the present embodiment, the ends N- truncate sulfuric acid fucoidin lyases TFLFM and obtain high efficient expression in Escherichia coli,
The protein output of purifying has reached~100mg/1L culture mediums, and base has been established for the application and development of sulfuric acid fucoidin lyases
Plinth.
5 ends N- of embodiment truncate the enzyme activity analysis of sulfuric acid fucoidin lyases TFLFM
10 μ L 4M are added in 100 μ L fucoidin (10mg/mL) solution (being dissolved in 50mM sodium phosphate buffers, pH 7.5)
NaCl, 10 ends μ L N- truncate sulfuric acid fucoidin lyases TFLFM (8mg/mL), and at 37 DEG C, 200rpm/min reacts 6 hours.
Then 100 DEG C of heating water baths 10min, 12000rpm/min centrifuge 5min, take supernatant, carry out HPLC analyses (Fig. 3)：Acchrom
S6000HPLC systems, XAmide pillars (4.6mm × 250mm, 5 μm), mobile phase include water (A), acetonitrile (B) and ammonium formate
(C), condition of gradient elution：0-40min, 90-50% (B), 10% (C), flow velocity 1.5mL/min, 40 DEG C of column temperature.
25 minutes or so the peaks HPLC are collected, is dissolved in after freeze-drying in a small amount of water, sample is mixed with matrix DHB
Afterwards, with Matrix Assisted Laser Desorption lonization-Time of Flight instrument MALDI-TOF/TOF UltraflextremeTM
(Brucker, Germany) is analyzed (Fig. 4) in a reflective mode enabling, and second mass analysis (figure is carried out to the peak at 563.348
5)。
In the present embodiment, the ends N- truncate sulfuric acid fucoidin lyases TFLFM and show degradation fucoidin well
Activity, the application and development for sulfuric acid fucoidin lyases provides place mat.
Certainly, the present invention can also there are many embodiments, without deviating from the spirit and substance of the present invention, are familiar with
Those skilled in the art can disclosure according to the present invention make various corresponding change and deformations, but these it is corresponding change and
Deformation should all belong to the scope of the claims of the present invention.
Sequence table
<110>Chinese Academy Of Sciences Process Engineering Research Institute
<120>A kind of sulfuric acid fucoidin lyases TFLFM and its preparation method and application
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<151> 2017-11-01
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aacccgactg ttctgaactg cactatcaag aacgcacgta ccggtgtgac cctggctcat 840
gctaacggca ccaaatatgt tgaaggctgt acggttctgg gttgtgaaaa cggttactct 900
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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
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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 Asn
435 440 445
Leu Ala Leu Lys Gly Glu Ala Ser Gln Ser Ser Ser Arg Pro Ser Asp
450 455 460
Gly Phe Ala Ala Asn Ala Ile Asp Gly Asn Thr Asn Gly Ala Trp Ser
465 470 475 480
Asn Asn Ser Val Ser His Thr Gly Thr Glu Glu Asn Pro Trp Trp Gln
485 490 495
Val Asp Leu Gly Thr Asp Ala Ile Ile Gly Ser Ile Asn Ile Phe Asn
500 505 510
Arg Thr Asp Gly Cys Cys Lys Gly Arg Leu Asp Asn Phe Thr Val Tyr
515 520 525
Val Ile Asp Lys Asp Asp Lys Val Thr Phe Ser Lys Thr Tyr Val Thr
530 535 540
Val Pro Asp Pro Ser Ile Thr Val Asp Ala Gly Gly Val Asn Gly Lys
545 550 555 560
Ile Val Lys Ile Val Leu Asn Asn Ser Ser Gln Ala Leu Ala Leu Ala
565 570 575
Glu Val Glu Val Tyr Gly Thr Ser Leu Ser Asn Lys Glu Thr Ile Lys
580 585 590
Asn Pro Ile His Phe Tyr Pro Asn Pro Val Glu Asp Glu Val Thr Ile
595 600 605
Ser Leu Glu Ser Ala Asp Leu Asn Leu Asn Glu Thr Arg Val Val Ile
610 615 620
Tyr Asn Ile Lys Gly Gln Lys Ile Leu Glu Thr Thr Pro Ser Asn Ser
625 630 635 640
Thr Glu Val Asn Leu Asn Leu Ser His Leu Pro Thr Gly Val Tyr Leu
645 650 655
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 kind of sulfuric acid fucoidin lyases TFLFM, it is characterised in that：The amino acid sequence of the sulfuric acid fucoidin lyases
Row are as shown in SEQ ID NO.4.
2. a kind of sulfuric acid fucoidin lyase gene, it is characterised in that：Encode sulfuric acid fucoidin cracking described in claim 1
Enzyme TFLFM.
3. sulfuric acid fucoidin lyase gene according to claim 2, it is characterised in that：The nucleotide sequence of the gene
As shown in SEQ ID NO.3.
4. including the recombinant expression carrier of sulfuric acid fucoidin lyase gene described in Claims 2 or 3.
5. recombinant expression carrier according to claim 4, it is characterised in that：The recombinant expression carrier is pET28a-
TFLFM。
6. including the recombinant bacterial strain of the recombinant expression carrier of claim 4 or 5.
7. recombinant bacterial strain according to claim 6, which is characterized in that the recombinant bacterial strain is e. coli bl21 (DE3).
8. the preparation method of sulfuric acid fucoidin lyases TFLFM, includes the following steps described in a kind of claim 1：
1) structure expression encodes the gene order of the ends the N- catalyst structure domain of sulfuric acid fucoidin lyases, obtains claim 2
Or the sulfuric acid fucoidin lyase gene described in 3, then build the recombinant expression carrier described in claim 4 or 5；
2) it uses the recombinant expression carrier described in claim 4 or 5 to convert host cell, obtains recombinant bacterial strain；
3) culture recombinant bacterial strain makes its fermentation, induction sulfuric acid fucoidin crack expression of enzymes；
4) it recycles and purifies expressed sulfuric acid fucoidin lyases.
9. applications of the sulfuric acid fucoidin lyases TFLFM described in claim 1 in sulfuric acid fucoidin of degrading.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114107260A (en) * | 2021-12-21 | 2022-03-01 | 中国海洋大学 | Fucoidan sulfate degrading enzyme OUC-FaFcn1 and application thereof |
| CN114250209A (en) * | 2021-12-14 | 2022-03-29 | 中国海洋大学 | Fucoidan and application thereof in compound enzymolysis of sea cucumbers |
| CN114686503A (en) * | 2020-12-29 | 2022-07-01 | 潍坊康地恩生物科技有限公司 | Escherichia coli mutant strain capable of stably producing alginate lyase in high yield |
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| CN1271390A (en) * | 1997-09-03 | 2000-10-25 | 宝酒造株式会社 | Gene |
| CN105821061A (en) * | 2016-03-28 | 2016-08-03 | 山东洁晶集团股份有限公司 | Nucleotide sequence of encoded fucoidin glucoside hydrolase and application thereof |
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| CN1271390A (en) * | 1997-09-03 | 2000-10-25 | 宝酒造株式会社 | Gene |
| CN105821061A (en) * | 2016-03-28 | 2016-08-03 | 山东洁晶集团股份有限公司 | Nucleotide sequence of encoded fucoidin glucoside hydrolase and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114686503A (en) * | 2020-12-29 | 2022-07-01 | 潍坊康地恩生物科技有限公司 | Escherichia coli mutant strain capable of stably producing alginate lyase in high yield |
| CN114686503B (en) * | 2020-12-29 | 2023-05-26 | 潍坊康地恩生物科技有限公司 | Coli mutant strain for stabilizing high-yield alginic acid lyase |
| CN114250209A (en) * | 2021-12-14 | 2022-03-29 | 中国海洋大学 | Fucoidan and application thereof in compound enzymolysis of sea cucumbers |
| CN114107260A (en) * | 2021-12-21 | 2022-03-01 | 中国海洋大学 | Fucoidan sulfate degrading enzyme OUC-FaFcn1 and application thereof |
| CN114107260B (en) * | 2021-12-21 | 2022-11-11 | 中国海洋大学 | Fucoidan sulfate degrading enzyme OUC-FaFcn1 and application thereof |
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