CN103710369B - Bifunctional enzyme gene for kelp mannose 6-phosphate isomerization and GDP (Guanosine Diphosphatemannose)-mannose pyrophosphorylation - Google Patents
Bifunctional enzyme gene for kelp mannose 6-phosphate isomerization and GDP (Guanosine Diphosphatemannose)-mannose pyrophosphorylation Download PDFInfo
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Abstract
The invention relates to the technical field of genetic engineering, in particular to a bifunctional enzyme gene for kelp mannose 6-phosphate isomerization and GDP (Guanosine Diphosphatemannose)-mannose pyrophosphorylation. A gene nucleotide sequence and an amino acid sequence of encoded protein are SEQ ID NO:1 and SEQ ID NO:2 respectively; the gene cloning technology is adopted to clone a gene sequence which constructs an eukaryotic expression vector, and enzyme activity detection is carried out on recombinant protein, so that the enzyme gene is proved to have a weak function of catalyzing fructose-6-phosphoric acid and mannose-6-phosphoric acid to achieve an allosteric effect, as well as remarkable activity of GDP-mannose pyrophosphorylase, and therefore, the enzyme gene belongs to a key enzyme encoding gene in the sodium alginate biosynthetic pathway. The bifunctional enzyme gene has important application value on improving contents of sodium alginate and fucoidin of algae such as kelp.
Description
Technical field
The present invention relates to the codase gene of a main laminaria phosphomannose isomery and GDP-seminose pyrophosphorylation bifunctional enzyme.In particular to the nucleotide sequence of a main laminaria (Saccharina japonica) phosphomannose isomery and GDP-seminose pyrophosphorylation bifunctional enzyme gene, and proteins encoded and in synthesis algin and the ability of fucoidin and the application in improvement Important Economic composition proterties.
Background technology
Sea-tangle is marine plant (algae) kind of whole world Main Cultivation.As a kind of ocean vegetables, sea-tangle has very high nutritive value, and meanwhile, the Main Economic compositions such as algin, fucoidin (fucoidin), can be widely used in all trades and professions as starting material.Algin of many uses, is mainly used in the aspects such as foodstuffs industry, medical and health, textile industry, scientific research.Fucoidin not only can as the bonding agent of metal ion and resistance vapor, and also have as anti HIV-1 virus, anticoagulation, antithrombotic, physiological function and the effect such as antitumor, be a kind of important medicinal substance simultaneously.
Clone products synthesis related gene also verifies its function, discloses the relation between gene and product, auxiliaryly carries out breed improvement and has become International Agriculture breeding field and improve one of effective way of economic sector content; Meanwhile, genetic engineering technique production active substance and economic product is utilized to become the core content of modern biotechnology industry development.
The alginic acid salt that algin (Algin) is the alkaline metal salts such as water miscible sodium alginate, potassium and water-insoluble alginic acid (Alginicacid) and is combined with the above metal ion of divalence; Its basic structure is by the irregular polysaccharide be formed by connecting of β-1-4 glycosidic link by α-Isosorbide-5-Nitrae-L-guluronic acid block (α-L-guluronicacid, G) and β-Isosorbide-5-Nitrae-D-MANNOSE aldehydic acid block (β-D-mannuronic, M).
Fucoidin (fucoidin sulfuric ester, fucoidan or fucan sulfate, FS) be a kind of polysaccharide be mainly present in brown alga cell walls and some marine invertebrates, be made up of L-fucose and sulfate group, also have D-wood sugar, D-semi-lactosi or uronic acid in addition.
The biogenetic derivation of algin and fucoidin is only Phaeophyceae algae, marine invertebrate and microorganism, as true sea-tangle (Saccharina japonica), Sargassum (Sargassum), azotobacter vinelandii (Azotobacter vinelandii), marine Pseudomonas (Pseudomonas sp.), P. aeruginosa bacterium (Pseudomonas aeruginosa), Halomonasmarina etc.
Different in the algin of different biogenetic derivation and fucoidin structure, but its building-up process is consistent substantially.The biosynthetic pathway research of microorganism algin and fucoidin comparatively detailed, as pseudomonas (Pseudomonasaeruginosa), vinelandii (Azotobacter vinelandii) and γ-mycetozoan (Gammaproteo bacteria).In pseudomonas, synthesis path is made up of polystep reaction, phosphomannose isomerase (AlgA), mannose-phosphate mutase (AlgC) and GDP-mannitol dehydrogenase (AlgD) are responsible for the precursor substance GDP-mannuronic acid (Guanosine diphosphate mannuronic acid, GDP-ManA) of synthesis algin and fucoidin.And this synthesis path remains indefinite in brown alga.The scholars such as Michel carry out bioinformatic analysis to long capsule water cloud (Ectocarpus siliculosus) genomic data, the algin of long capsule water cloud of having recombinated and fucoidin synthesis path.In its genome database, searched the gene PMI(phosphomannose isomerase with AlgA, AlgC, AlgD and AlgG homology, Mannose-6-phosphate isomerase), PMM(Phosphomannomutase), GMD(GDP-D-mannose dehydratase) and MC5E(MannuronanC-5-epimerase).In the microorganisms such as bacterium, AlgA is a kind of bifunctional enzyme gene, the i.e. Type2 type of PMI, the first step of energy catalytic synthesis and three-step reaction, but scholar has only searched single functional enzyme gene PMI in long capsule water cloud, do not find the enzyme gene GMP(GDP-D-mannose pyrophosphorylase of catalysis three-step reaction, GDP-D-mannosepyrophosphorylase, MPG), see Fig. 2.
The brown algas such as sea-tangle are algin and fucoidin natural origin, and the feature of its mcroorganism amount and high-content makes it be exactly the main raw material that algin and fucoidin extract all the time.But it is very weak for the gene pathway research of its synthesis algin and fucoidin, the codase gene that catalysis Man-6-P for its key is converted into the reaction of GDP-seminose still belongs to unknown, thus the brown alga plants such as sea-tangle cannot be realized based on the breed improvement of gene clone.
Summary of the invention
For the technical problem existed in currently available technology, the invention provides a kind of phosphomannose isomery and GDP-seminose pyrophosphorylation bifunctional enzyme gene, a kind of phosphomannose isomery of its codified and GDP-seminose pyrophosphorylation bifunctional enzyme albumen, described albumen only has faint catalysis fructose-6-phosphate and Man-6-P allosteric enzyme (PMI) is active, but the enzyme (MPG) with significant catalysis Man-6-P and the conversion of GDP-seminose is active, described gene and proteins encoded thereof can be used for improving algin and fucoidin synthesis content.
An object of the present invention is to provide a kind of phosphomannose isomery and GDP-seminose pyrophosphorylation bifunctional enzyme gene, described gene is the phosphomannose isomery and GDP-seminose pyrophosphorylation bifunctional enzyme gene separated from sea-tangle (Saccharina japonica), called after SjPMI4; The nucleotide sequence of described gene is as shown in SEQ ID NO:1.
Two of object of the present invention is the albumen providing a kind of described genes encoding, nucleotide sequence coded by shown in SEQ ID NO:1 of described albumen, and its aminoacid sequence is as shown in SEQ ID NO:2; It only has faint catalysis fructose-6-phosphate and Man-6-P allosteric is active, but has the activity of significant catalysis Man-6-P and the conversion of GDP-seminose.
Three of the object of the invention is described gene and the application of proteins encoded in synthesis algin and fucoidin thereof.
Four of the object of the invention is described gene and the application of proteins encoded in improvement economic sector proterties thereof.
The present invention is by the method for gene clone, a phosphomannose isomery and GDP-seminose pyrophosphorylation bifunctional enzyme gene has been cloned into from sea-tangle, and the albumen demonstrating its coding by experiment only has faint catalysis fructose-6-phosphate and Man-6-P allosteric is active, but having the activity of significant catalysis Man-6-P and the conversion of GDP-seminose, is the key gene of synthesis algin and fucoidin.Clone and analyze sea-tangle phosphomannose isomery and GDP-seminose pyrophosphorylation bifunctional enzyme gene and contribute to deeply understanding the brown alga algins such as sea-tangle and fucoidin synthesis mechanism, also can provide genetic resources for algin and fucoidin genetically engineered and molecular breeding simultaneously.The present invention is separated to phosphomannose isomery and GDP-seminose pyrophosphorylation bifunctional enzyme gene first from sea-tangle (Saccharina japonica), and by carrier for expression of eukaryon, Enzyme assay is carried out to recombinant protein, confirm that it has faint catalysis fructose-6-phosphate and Man-6-P allosteric active function, but there is the active function of significant catalysis Man-6-P and the conversion of GDP-seminose simultaneously, there is the using value of algin and fucoidin genetically engineered and molecular breeding.
Accompanying drawing explanation
Fig. 1 is algin using GDP-seminose as precursor and fucoidin synthesis path schematic diagram.
Fig. 2 is the biological function of phosphomannose isomerase (PMI).
Fig. 3 is the pcr amplification figure of SjPMI4 gene cDNA total length of the present invention.
Fig. 4 is SjPMI4 gene of the present invention and encoding amino acid sequence (being respectively initiator codon and terminator codon in square frame) thereof.
Fig. 5 is that after SjPMI4 gene transformation yeast of the present invention, PCR detects positive colony pcr amplification figure.
Fig. 6 is SDS-PAGE detection figure after SjPMI4 gene transformation yeast expression product purification of the present invention.
Fig. 7 is the Western-Blot detection figure of SjPMI4 gene transformation yeast expression product of the present invention.
Fig. 8 is that the differing temps of SjPMI4 gene transformation yeast expression product of the present invention is to the detection figure of enzymic activity.
Fig. 9 is that the pH of SjPMI4 gene transformation yeast expression product of the present invention is to the detection figure of enzymic activity.
Figure 10 is the detection figure of the different metal ions enzyme activity of SjPMI4 gene transformation yeast expression product of the present invention.
Figure 11 is the different concentration of substrate Enzyme activities figure of SjPMI4 gene transformation yeast expression product of the present invention
Figure 12 is the different concentration of substrate double reciprocal curve figure of SjPMI4 gene transformation yeast expression product of the present invention.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these examples are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted specific experiment condition in the following example, usual conveniently condition, Molecular Cloning: A Laboratory guide (Sambrook J, et al.2008.Molecular Cloning:A Laboratory Manual, condition 3rdEd.), or according to the condition that manufacturer advises.
Embodiment 1: the cloning and analysis of full length gene coding region
Sea-tangle gathers from Rongcheng City of Shandong Province, and acquisition time is in July, 2011.Adopt Trizol method to extract Female Gametophytes of Laminaria Japonica total serum IgE, use TAKARA company PrimeScript II1
ststrand cDNA Synthesis test kit for template, utilizes cDNA end rapid amplifying technology with the first chain cDNA of kelp gametophyte total serum IgE reverse transcription, uses BDSMART
tMrACE cDNA Amplification Kit carries out RACE-PCR amplification, primer is respectively 5 '-AGGGCTACGAGCACAAGGAAAAGGGGG-3 ' and 5 '-GACCTCTACAAGGATGACAACCACAAGC-3 ', RACE-PCR amplification program is: 94 DEG C of 30sec, 72 DEG C of 3min, 5 circulations; 94 DEG C of 30s, 70 DEG C of 30s, 72 DEG C of 3min, 5 circulations; 94 DEG C of 30s, 68 DEG C of 30s, 72 DEG C of 3min, 20 circulations; 72 DEG C of 10min.Adopt TouchdownPCR technology and regular-PCR technology to carry out the amplification of the CDS full length sequence of sea-tangle PMI gene according to RACE sequencing result, amplimer comprises 3 groups of (5 '-CGACCCGACCTCTACAAGGATGACAACC-3 ' and 5 '-TCATTGGCAGCCATGAAGAACGACTCTC-3 '; 5 '-CGAGACCGAGGAGGGCAAGATGTAC-3 ' and 5 '-CTCATTGGCAGCCATGAAGAACGAC-3 '; 5 '-GCCCTTCGTCACACACACGAC-3 ' and 5 '-CGGTGCAGGTTGAACACAATCT-3 ').PCR primer is after 1% agarose gel electrophoresis detects, and the blob of viscose of cutting containing object band under ultraviolet lamp, uses sepharose to reclaim test kit and reclaim object fragment, in-20 DEG C of preservations.The object fragment reclaimed, spend the night in 16 DEG C of metal baths and be connected to cloning vector pMD19-T, and be transformed in competent escherichia coli cell E.coli Top10, coat on the LB solid medium containing 100mg/mL Amp, 37 DEG C of incubated overnight, after the blue hickie screening of IPTG/X-gal, a picking 4-10 positive colony checks order.By sequencing result by sequence alignment, be separated to a sea-tangle PMI gene, called after SjPMI4.SjPMI4CDS sequence is 1020bp, and its nucleotide sequence is as shown in SEQ ID NO:1, and 339 amino acid of encoding, take ATG as initiator codon, TAA is terminator codon, is the 3'UTR sequence of 588bp afterwards.
The preparation of embodiment 2:SjPMI4 proteins encoded and analysis
Sea-tangle SjPMI4PCR product is after the agarose gel electrophoresis detection of 1%, object band is cut under ultraviolet lamp, sepharose reclaims, recovery product S jPMI4 and pPICZ α A plasmid carry out EcoRI and NotI double digestion, after 37 DEG C of metal bath 3-4h, detect with 1% agarose gel electrophoresis, use sepharose to reclaim test kit and reclaim.Be connected with plasmid pPICZ α A by object fragment SjPMI4,16 DEG C are spent the night, the recombinant plasmid called after pPICZ α A-PMI4 built.
Use restriction endonuclease DraI to carry out linearization process recombinant plasmid pPICZ alpha A-PMI4, dissolve with the distilled water of sterilizing after purifying, with in electric shock transforming Pichia pastoris X-33 competent cell.The cell culture of having hatched is coated containing different concns Zeocin
tMyPD flat board on, every 50-200 μ L is coated with one flat plate; Flat board is placed in the incubator dark culturing 2-4 days of 28 DEG C, until single bacterium colony occurs.
Freeze the method for boiling prepare pichia pastoris phaff pcr template with boiling, utilize primer (according to the sequencing result obtained, to design the forward and reverse primer with restriction enzyme site, containing the forward primer 5 '-CAGC of EcoRI restriction enzyme site
gAATTCaTGAGACGCCTGAATTGC-3 ' and the reverse primer 5 '-TAT containing NotI restriction enzyme site
gCGGCCGCtATCAAACTCCAAATCC-3 ') carry out the screening of positive colony, PCR detects recon.PCR primer detects in 1% agarose gel electrophoresis, the imaging of automatic gel image analysis instrument, uses above-mentioned primer amplification PMI4CDS full length sequence, obtains the PCR primer that length is about 1000bp, in the same size with expection.Picking electrophoresis detection inserts the correct cloning and sequencing of band, and detect with or without sudden change, whether frameshit frame changes.The bacterium liquid getting the correct positive colony preservation of order-checking is inoculated in 50mL BMGY substratum, and 28 DEG C, 250rpm/min cultivates, and it is 0.5% that 24h adds methyl alcohol to final concentration in fermented liquid.The broken bacterium liquid of ultrasonic method, collect the supernatant liquor after fragmentation, with the membrane filtration of 0.45 μm, after Ni column purification, the protein sample collected is put into dialysis tubing and is dialysed, and to remove unwanted ion, obtains restructuring PMI4 albumen, its aminoacid sequence, as shown in SEQ ID NO:2, adopts SDS-PAGE, Western-Blot to detect the expression of recombinant protein.
The functional verification of embodiment 3:SjPMI4 proteins encoded
PMI enzyme activity determination: with reference to the two step coupling methods of (2008) such as MarutaT, and slightly transform.Reaction system is as follows: the NADP+ containing 0.5mM in the damping fluid of the Tris-HCl of 50mM pH7.5,5mM MgCl2,1U/mL PGI(Glucose-6-phosphate isomerase), 1U/mL G6PDH(Glucose-6-phosphate dehydrogenase), the M-6-P(Mannose-6-phosphate of 1mM) and appropriate embodiment 3 prepare restructuring PMI4 albumen, total reaction system is 300 μ L, adds substrate M-6-P initial action.Initial action after hatching 2min after the system of above-mentioned removing substrate being mixed under relevant temperature condition, with corresponding damping fluid for blank, in the change of 340nm place difference assaying reaction 0min, 6min and 12min light absorption value, each reaction arranges 4 Duplicate Samples.After testing, PMI4 albumen PMI enzymic activity is lower, and enzyme is lived as 0.81U/mg, and optimal reactive temperature is 15 DEG C, and optimal pH is 8.5, and this enzyme is cold-adapted enzyme, basic protein.Zn
2+, Cu
2+and Mn
2+restraining effect is had to enzymic activity; And Ca
2+and Mg
2+live substantially without impact on enzyme; Co
2+there is the effect promoting that enzyme is lived.
MPG enzyme assay: the damping fluid of 1ml50mM pH7.6Tris-HCl, 4mM glucose, 1mM ADP, 1mMNADP, 10mM MnCl
2, 1U hexokinase, 1U nucleoside-50-diphospho-kinase, 1U glucose-6-phosphate dehydrogenase (G6PD) and concentration range are the GDP-seminose of 0.05 to 5mM.Reacting by adding restructuring PMI4 albumen prepared by embodiment 3 and final concentration is that the trisodium phosphate of 2mM is initial.Under relevant temperature condition, hatch initial action by after above-mentioned system mixing, with corresponding damping fluid for blank, in the change of 340nm place difference assaying reaction 0min, 3min and 6min light absorption value, each reaction arranges 4 Duplicate Samples.Enzyme is lived as 3.28U/mg, Km value is 32.44mM, and optimal reactive temperature is 40 DEG C, and optimal pH is 7.0.This enzyme is high temperature enzyme, neutral protein; Mn
2+, Ca
2+, Cu
2+and Mg
2+can promote that enzyme is lived, Zn
2+suppress it active.
Although the invention describes concrete example, having a bit is obvious to those skilled in the art, namely can make various changes the present invention and change under the premise without departing from the spirit and scope of the present invention.Therefore, claims cover all these variations within the scope of the present invention.
Claims (4)
1. encode the gene of phosphomannose isomery and GDP-seminose pyrophosphorylation bifunctional enzyme in sea-tangle, it is characterized in that, the nucleotide sequence of described gene is as shown in SEQ ID NO:1.
2. the albumen by genes encoding described in claim 1, it is characterized in that, the aminoacid sequence of described albumen is as shown in SEQ IDNO:2, it only has faint catalysis fructose-6-phosphate and Man-6-P allosteric is active, but has the activity of significant catalysis Man-6-P and the conversion of GDP-seminose.
3. the application of bifunctional enzyme gene according to claim 1 in synthesis algin and fucoidin.
4. the application of albumen described in claim 2 in synthesis algin and fucoidin.
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| CN107354164A (en) * | 2017-07-26 | 2017-11-17 | 青岛海大蓝科生物科技有限公司 | UGD genes, its protein and the purposes of the dehydrogenase of GDP glucose 6 are encoded in long capsule water cloud |
| CN107267533A (en) * | 2017-07-26 | 2017-10-20 | 青岛海大蓝科生物科技有限公司 | The GMD genes and its protein and purposes of the dehydrogenase of GDP mannoses 6 are encoded in long capsule water cloud |
| CN107287170B (en) * | 2017-08-16 | 2020-02-21 | 青岛海大蓝科生物科技有限公司 | UGD gene in kelp, protein and application thereof |
| CN107502615B (en) * | 2017-10-16 | 2020-05-05 | 中国海洋大学 | Gene encoding GDP-mannose-4,6-dehydratase in kelp, and protein and use thereof |
| CN108034000A (en) * | 2017-11-17 | 2018-05-15 | 四川大学 | Chinese cymbidium mannose-binding protein |
| CN107828750B (en) * | 2017-11-22 | 2020-11-10 | 青岛海大蓝科生物科技有限公司 | Kelp GDP fucose synthetase, and coding gene and application thereof |
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