CN107354164A - UGD genes, its protein and the purposes of the dehydrogenase of GDP glucose 6 are encoded in long capsule water cloud - Google Patents

UGD genes, its protein and the purposes of the dehydrogenase of GDP glucose 6 are encoded in long capsule water cloud Download PDF

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CN107354164A
CN107354164A CN201710618428.6A CN201710618428A CN107354164A CN 107354164 A CN107354164 A CN 107354164A CN 201710618428 A CN201710618428 A CN 201710618428A CN 107354164 A CN107354164 A CN 107354164A
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genes
gdp
ugd
esiugd
protein
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池姗
刘涛
刘翠
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Qingdao Haida Lanke Biotechnology Co Ltd
Ocean University of China
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Qingdao Haida Lanke Biotechnology Co Ltd
Ocean University of China
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    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01132—GDP-mannose 6-dehydrogenase (1.1.1.132)

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Abstract

The invention belongs to biological technical field, particularly belongs to genetic engineering field, is more particularly to encode UGD genes, its protein and the purposes of the dehydrogenase of GDP glucose 6 in a kind of long capsule water cloud.Described encoding gene is derived from, and described UGD gene sources are named as EsiUGD genes, the nucleotides sequence of described EsiUGD genes is classified as SEQ ID NO in long capsule water cloud (Ectocarpus siliculosus)：Sequence shown in 1.Present invention also offers a kind of dehydrogenase of GDP mannoses 6, for the described dehydrogenase of GDP mannoses 6 by above-mentioned EsiUGD coded by said gene, its amino acid sequence is SEQ ID NO：Sequence shown in 2, its Rate activity are up to 8.9nanokatalsmg‑1；And there is higher optimal reactive temperature and heat endurance.The present invention prepares for the biology of algin and provides excellent genetic resources and enzyme resource.

Description

UGD genes, its protein of GDP- glucose -6- dehydrogenases are encoded in long capsule water cloud And purposes
Technical field
The invention belongs to biological technical field, particularly belongs to genetic engineering field, is more particularly to a kind of long capsule water cloud UGD Gene, its protein and purposes.
Background technology
Algin, also known as brown alga acid polysaccharide, its sodium-salt form are referred to as sodium alginate (alginate), are by β-D-MANNOSE Aldehydic acid (β-D-mannuronic acid, abbreviation M) and α-L- guluronic acids (α-L-guluronic acid, abbreviation G) pass through The straight-chain polysaccharide that Isosorbide-5-Nitrae glycosidic bond is formed by connecting.So far, it is believed that algin biological source is only Phaeophyta plant, such as sea Band, sargassum, bulk kelp etc.；And a variety of opportunists, such as P. aeruginosa bacterium (Pseudomonas aeruginosa) In edaphon azotobacter vinelandii (Azotobacter vinelandii).
Algin may be used as thickener, emulsifying agent, quality improver of food etc.；Algin with medical industry, Make dextran, styptic, scald gauze, dental impression silica agent, medicine excipient, novel barium meal contrast agent, capsule etc.；At water In reason, make water softening agent and scale remover；Used in agriculturally, making insecticide, growth accelerator, water-loss reducer etc.；Algin can be made Viability dyestuff, disperse dyes, acid dyes, basic-dyeable fibre and spirit soluble dyestuff, with printing and dyeing industry；Algin can be made Sizing agent, filler, coating agent for paper industry etc.；Algin can make rubber concentrating agents, oil-proof composition with rubber industry Deng with mechanical industry, make welding compound and cutting agent；With on daily-use chemical industry, beauty hair cosmetic agent, detergent etc. can be made.This Outside, algin industrially also has important purposes in oil, mining, building materials, ceramics etc..As can be seen here, algin is given birth in life The every field of production all has been widely used.
The mode of production of algin is chemical leaching test at present, such as the He of Chinese patent application 201510743088 Content disclosed in 201010620628.In Europe, the extraction raw material of algin is mainly used as using yellow tang and Laminaria digitata； American States bulk kelp is the primary raw material for extracting algin；Manually cultivated kelp in Asia just into the main original of extraction algin Material.
On short terms, although chemical leaching test can effectively extract algin from raw material, but it is to algae resource, energy Huge hidden danger be present in source demand and environmental protection.In the art, the chemical leaching test of algin may be summarized to be following step Suddenly：The immersions of the raw materials such as sea-tangle, bulk kelp, washing, formaldehyde, acid, alkali process, digestion extraction, filtering, condensation, bleaching, dehydration, in With, dry, crush and packaging etc..It is not difficult to find out, can be used to formaldehyde, soda acid, metal during the chemical extraction of algin The various toxic raw materials such as salt, bleaching agent, on the one hand a large amount of uses of these raw materials will cause seriously to pollute to environment, the opposing party These poisonous raw material of face are all present in a certain degree of in final products, it will it is difficult to exist to consumer or terminal user With the potential safety hazard ignored.Further, it is known that chemical extraction process can consume substantial amounts of raw material, water resource and energy Source.That is it is that it is a kind of ring that chemical extraction methods, which are not, to the question of market of the wilderness demand of algin product facing Protect, energy-conservation, sustainable extracting method.
With the continuous development of genetic engineering, production is carried out in industrial production using genetic engineering culture microorganism Every aspect successfully realize.The genetic engineering process for preparing of algin can also turn into effective supplement of its synthetic method at last, Even substitute traditional chemical leaching test.
The product is needed to use to carry out the related gene of biosynthesis in life entity using genetic engineering sintetics. Algin is not direct gene outcome, and its biosynthesis is largely divided into four parts：The generation of precursor, the activation of precursor, precursor Oxidation and sugar unit polymerization, modification and output.Research ratio of the related gene in bacterium in algin biosynthesis More thoroughly.The biosynthesis of algin is a kind of complex reaction of multi-step in bacterium, be related to algA, algC, algD and Up to tens genes such as algG.In brown alga, the homologous gene of above-mentioned bacterial gene be present, be respectively designated as PMI (Phosphomannose isomerase, phosphomannose isomerase), PMM (Phosphomannomutase, phosphomannose Mutase), GMD (GDP-D-mannose dehydratase, GDP- mannose -6- dehydrogenases) and MC5E (Alginate-C5- Mannuronan-epimerase, algin-C5- mannuronic acid difference phases isomerase) etc..The product of wherein GMD genes is GDP- mannose -6- dehydrogenases, the enzyme participate in the important step of algin biosynthesis pathway in brown alga.
Therefore, GMD genes and its coded product are undoubtedly the important foundation of algin biological synthesis method, while also will be Critical elements in algin biological synthesis method.But the clone of the GMD genes of current algin biosynthesis correlation is very Lack, it has been reported that brown alga in coding GDP- mannose -6- dehydrogenases also only a GMD gene (EsGMD1).
In summary, there is provided brown alga composes the GMD genes and its product GDP- mannose -6- dehydrogenases of correlation, to brown The biological synthesis process of phycocolloid has important practical application of significance.
The present invention is found surprisingly that the volume of the UGD genes of encoding UDP-glucose -6- dehydrogenases in coding in long capsule water cloud Code product has the activity of GDP- mannose -6- dehydrogenases, and its GDP- mannose -6- dehydrogenase activity is higher than existing skill Art, therefore provide valuable gene and protein resource for the biosynthesis of algin.
The content of the invention
The present invention is found surprisingly that the volume of the UGD genes of encoding UDP-glucose -6- dehydrogenases in coding in long capsule water cloud Code product has the activity of GDP- mannose -6- dehydrogenases, and its GDP- mannose -6- dehydrogenase activity is higher than existing skill Art, therefore provide valuable gene and protein resource for the biosynthesis of algin.
For the deficiencies in the prior art and defect, the invention provides a kind of long capsule water cloud (Ectocarpus Siliculosus the UGD genes in), the product of described UGD genes is GDP- mannose -6- dehydrogenases, and the enzyme has catalysis GDP- mannoses are converted into the activity of GDP- mannuronic acids.
On one side, the invention provides a kind of UGD genes, described encoding gene is derived from long capsule water cloud (Ectocarpus siliculosus), described UGD unnamed genes are EsiUGD genes, the nucleosides of described EsiUGD genes Acid sequence is SEQ ID NO：Sequence shown in 1.
On the other hand, the invention provides a kind of GDP- mannoses -6- dehydrogenases, described GDP- mannoses -6- to take off Hydrogen enzyme is by above-mentioned EsiUGD coded by said gene；Described GDP- mannose -6- dehydrogenases are by upper SEQ ID NO：Nucleosides shown in 1 Sequences code；The amino acid sequence of described GDP- mannose -6- dehydrogenases is SEQ ID NO：Sequence shown in 2.
Another aspect, present invention also offers the carrier or genetically engineered cell containing above-mentioned UGD genes.
Described carrier can be procaryotic cell expression carrier or eukaryotic expression vector.
Described eukaryotic expression vector is selected from：Yeast expression carrier, insect cell expression vector or mammal are thin Cellular expression carrier.
Described procaryotic cell expression carrier can be pET expression vectors.
Described pET expression vectors include but is not limited to：PET-22 carriers, pET-28 carriers, pET-30 carriers, pET-32 Carrier, pET-34 carriers, pET-40 carriers or pET-42 carriers etc..
Described genetically engineered cell includes but is not limited to：Bacillus coli cells, B. subtilis cell, lactic acid bacteria are thin Born of the same parents or Pichia pastoris etc..
Another aspect, present invention also offers purposes of the above-mentioned UGD genes in algin is prepared.Described purposes refers to By the UGD channel genes genetically engineered cells, so that genetically engineered cell can produce algin.
On the other hand, present invention also offers made by the GDP- mannose -6- dehydrogenases of above-mentioned UGD coded by said gene Purposes in standby algin.
Compared to the prior art, beneficial effects of the present invention are：The present invention is different from the conventional fabrication process of algin, It is exactly chemical leaching test.GDP- mannose -6- the dehydrogenases of UGD gene codes provided by the invention are in algin biosynthesis Important enzyme, the gene or enzyme prepare for the biology of algin and provide important living resources.UGD genes disclosed by the invention It is first disclosed brown alga UGD genes.GDP- mannose -6- the dehydrogenases of UGD coded by said gene in the present invention have higher Catalytic activity, its Rate activity is up to 8.9nanokatalsmg-1；And there is higher optimal reactive temperature and heat endurance. The present invention prepares for the biology of algin and provides excellent genetic resources and enzyme resource.
Brief description of the drawings
Fig. 1 is the polyacrylamide gel electrophoresis figure in embodiment 3.
Fig. 2 is the optimal reaction pH curve maps in embodiment 5.
Fig. 3 is the optimal reactive temperature curve map in embodiment 6.
Embodiment
The explanation of following examples is only intended to help the method and its core concept for understanding the present invention.It should be pointed out that pair For those skilled in the art, under the premise without departing from the principles of the invention, the present invention can also be carried out Some improvement and modification, these are improved and modification is also fallen into the protection domain of the claims in the present invention.To disclosed implementation The description below of example, enables professional and technical personnel in the field to realize or using the present invention.A variety of modifications to these embodiments It will be apparent for those skilled in the art, generic principles defined herein can not depart from this In the case of the spirit or scope of invention, realize in other embodiments.Therefore, the present invention is not intended to be limited to illustrated herein These embodiments in, but can apply to meet the broader model consistent with principles disclosed herein and features of novelty Enclose.Although it can be used and heretofore described similar or of equal value any method and material in the implementation or test of the present invention Material, preferable method and material are enumerated by place herein.
Unless otherwise defined, all technologies used herein and scientific terminology have and the technical field of the invention The identical meaning that those of ordinary skill is generally understood that.
In the examples where no specific technique or condition is specified, according to the technology or condition described by document in the art (such as write with reference to J. Pehanorm Brookers etc., what Huang Peitang etc. was translated《Molecular Cloning:A Laboratory guide》, the third edition, Science Press) or Person is carried out according to product description.
The acquisition of UGD genes in the long capsule water cloud of embodiment 1
According to long capsule water cloud whole genome sequence information, 1 UGD gene is obtained, is respectively designated as EsiUGD genes, it is described The nucleotides sequences of EsiUGD genes be classified as SEQ ID NO：Sequence shown in 1.Complete sequence synthesis (committee is carried out to EsiUGD genes Support Shanghai Xu Guan biotechnologies Development Co., Ltd synthesized), while rise both ends introduce EcoRI and NotI restriction enzyme sites and Corresponding protection base, obtain EsiUGD gene orders.
The clonal expression of embodiment 2EsiUGD genes and isolate and purify
Embodiment 1 is synthesized to the EcoRI that obtained EsiUGD genes are connected to pET32a carriers (being purchased from Novagen companies) Between NotI sites, pET32a-EsiUGD recombinant vectors are obtained；The pET32a-EsiUGD recombinant vectors conversion of acquisition is big In enterobacteria E.coli BL21 (DE3) competent cell (being purchased from Takara companies), and it is coated on blue or green containing 100 μ g/mL ammonia benzyls On the LB solid culture flat boards of mycin, 37 DEG C of overnight incubations；Positive colony is seeded to containing 100 μ g/mL ampicillins In LB solid mediums, 37 DEG C culture to bacterium solution OD600 be 0.4 when, add final concentration of 0.5mM IPTG, 25 DEG C, 160rpm inductive condition induces the expression of destination protein overnight；Induction takes 2mL bacterium solutions to be centrifuged in 4 DEG C, 12000rpm after terminating 5min, supernatant is abandoned, pipe is inverted on blotting paper；Precipitation adds the PBS (PH that 1/2 volume shifts to an earlier date the good 50mM of precooling =7.5) (i.e. 1mL PBS are added in precipitation after 2mL centrifugations) in, fully mixed；Ultrasonic method crushes bacterium solution, collects after crushing Supernatant, with 0.45 μm of membrane filtration, Ni posts (being purchased from GE companies) purifying (adds 200uM during purifying in solution system used NAD is active to ensure the recombinant protein of purifying) after, dialysed, obtained using PH=7.5 50mM PBS Recombinant protein, the recombinant protein be EsiUGD gene codes protein, its amino acid sequence such as SEQ ID NO：Shown in 2, life Entitled EsiUGD albumen, function are GDP- mannose -6- dehydrogenases.
The identification of the recombinant protein of embodiment 3
Sds polyacrylamide gel electrophoresis, polyacrylamide gel electricity are carried out to the EsiUGD albumen that embodiment 2 obtains respectively Swimming figure meets expected size as shown in figure 1, the molecular weight of EsiUGD albumen is about 50KD.
The functional verification of embodiment 4GDP- mannose -6- dehydrogenases and Rate activity measure
Enzyme activity determination method：The Tris-glycine of pH 8.75 comprising final concentration of 50mM in 1mL reaction system Buffer, final concentration of 1mM NAD (being purchased from Roche Holding Ag), final concentration of 0.5mM GDP- mannoses are (public purchased from Sigma Department), 30 μ g recombinant protein (the EsiUGD albumen that wherein recombinant protein obtains for embodiment 2)；Reacted at room temperature, The change of measure reaction 0min, 6min and 12min light absorption value respectively at 340nm, calculate NADH growing amount.
Measurement result：Compared with blank control (being not added with recombinant protein in identical reaction system to be reacted), NADH growing amount substantially increases, it was demonstrated that the EsiUGD albumen that embodiment 2 obtains has GDP- mannose -6- dehydrogenase activities； After identifying reaction substrate and reaction product, respectively GDP- mannoses and GDP- mannuronic acids.
Using the Rate activity of enzyme activity determination method measure EsiUGD albumen, and with unique disclosed GMD bases in current brown alga Because the GDP- mannose -6- dehydrogenases of coding are compared, as a result such as following table：Understood by measurement result provided by the invention For the vigor of the GDP- mannose -6- dehydrogenases of EsiUGD gene codes apparently higher than prior art, Rate activity is disclosed About 3 times of the GDP- mannose -6- dehydrogenases of GMD gene codes.That is EsiUGD genes and coding provided by the invention GDP- mannose -6- dehydrogenases there is higher combined coefficient in brown alga composes path.
Gene Encoding proteins function Rate activity (nanokatals mg-1)
Published EsGMD1 genes GDP- mannose -6- dehydrogenases 3.3
The EsiUGD genes of the present invention GDP- mannose -6- dehydrogenases 8.9
The measure of the optimal reaction pH value of embodiment 5
Enzyme activity determination method in embodiment 5, distinguish (pH5.0, pH 6.0, pH 7.0, pH at various ph values 8.75th, pH 9.0, pH 10.0) measure recombinant protein (the EsiUGD albumen i.e. in embodiment 2) activity, drafting optimal reaction PH curves, optimal reaction pH curves according to Fig. 2 as shown in Fig. 2 show：The optimal reaction pH of EsiUGD albumen is 9.
The measure of the optimal reactive temperature of embodiment 6
Enzyme activity determination method in embodiment 5, respectively at different temperatures (5 DEG C, 10 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 37 DEG C, 40 DEG C, 50 DEG C, 60 DEG C) measure recombinant protein (the EsiUGD albumen i.e. in embodiment 2) activity, drafting optimal reaction Temperature curve, optimal reactive temperature curve according to Fig. 3 as shown in figure 3, show：The optimal reactive temperature of EsiUGD albumen is 40- 50℃.GDP- mannose -6- the dehydrogenases of GMD genes (EsGMD1 genes) coding is most suitable anti-disclosed in unique in brown alga at present It is only 30 DEG C to answer temperature, that is to say, that and the present invention compared with prior art, has higher optimal reactive temperature, that is, more Add suitable for the synthetic reaction under higher temperature.
The thermal stability determination of embodiment 7
By recombinant protein (the EsiUGD albumen i.e. in embodiment 2) respectively 30 DEG C, 37 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 Its activity is being determined after certain time is heated at DEG C.As a result show：EsiUGD albumen is resistant to up to 60 DEG C of heat treatment, Temperature more than 60 DEG C after just occur enzyme activity lose phenomenon.And unique disclosed GMD genes (EsGMD1 genes) are compiled in brown alga at present GDP- mannose -6- the dehydrogenases of code, when more than 30 DEG C deactivation phenomenom occurs, it can be seen that it is not applied in it Actual building-up process, that is to say, that GDP- mannoses -6- dehydrogenases provided by the invention than prior art heat endurance more Height, its vigor is more durable in biosynthetic process, is more suitable for actual building-up process.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention God any modification, equivalent substitution and improvements made etc., should be included in the scope of the protection with principle.
SEQUENCE LISTING
<110>Qingdao Hai great Landteks bio tech ltd, Chinese Marine University
<120>UGD genes, its protein and the purposes of GDP- glucose -6- dehydrogenases are encoded in long capsule water cloud
<130> 20170707
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1401
<212> DNA
<213> Ectocarpus siliculosus
<400> 1
atgggcgccg ggtacgtggg cgggccgacc atggccgtga tcgcccagcg ctgccctcac 60
atccgcgtgt gcgtggtcga catctcccaa gcgcagatcg atgcctggaa caccgacgac 120
ctccccatct acgagccagg cctgctggaa gtggtcaagg agagccgggg ccgcaacctc 180
ttcttctcca ccgacatcga cgccgagatc aagcgcgcgg acatggtgtt catctccgtg 240
aacacgccga ccaagacgac cggtatcggt gcgggcaagg cggccaacat caagaactgc 300
gagctgtgcg cccgcaagat cgccgaggtc gcggacaccc ccaaggtggt ggtggagaag 360
tcgacggttc cggtccgtac cgccgaggct gttcgccgtg tgctggcgac caatgagggc 420
aaggtcaagt tccaggtgct gtccaacccg gagttcttgg cggagggtac cgccatgccc 480
gacctgcagg accccagccg ggtgctgatc ggcggtatgc agacacccga ggggctcgcg 540
gctatccagg agctggtaga cgtgtacgcc aactgggtgc cgaaggacag aatcctcgct 600
accaacactt ggtcctcaga gctctctaag ctcgtggcca acgccttcct tgcgcagcgc 660
gtgagcagca tcaactcgat ctcggcgttg tgcgaggcta cggatgctga catctctgag 720
gtgtctcgag ctctggggta cgacccccgc atcggcaaca agttcctcaa ctcctcggtc 780
gggttcgggg gttcttgctt ccagaaggac gtgctcaacc tcgtctacat ctgcgagtcc 840
accggccttc ctgaggtggc cgagtactgg caccaggtga tcgccatgaa cgactaccag 900
aagtctcgct tcacccagct catggtgcga cgtatgttca acaccgtcac cggcaagagg 960
atcgccgttc ttggcttcgc tttcaagaag gacacgggag acaccaggga gactccggcg 1020
gtgtttgtgt gcaaggccct cctggaggag caggccaagg tccaggtgta cgaccctcaa 1080
gtgaagagaa gtcagatgtt cgtggagttc gactacacgt gcggcgtcaa caaggacaac 1140
acccccaggc tcgaggagtc gattaccact tgcgaggacg cctactccgc ggccgagggg 1200
tctcacgccc tggccatctt gaccgagtgg gacgagttca agaccctcga ctaccagcgc 1260
atctacgaca gcatggccaa gccggccttc gtcttcgacg gacgcggggt ggtcgacatc 1320
gaagccctcc gcaagatcgg cttcgaggtg tactgcatcg gaaagtccaa gcccaagtac 1380
tctctcgaca gtcctaagta a 1401
<210> 2
<211> 466
<212> PRT
<213> Ectocarpus siliculosus
<400> 2
Met Gly Ala Gly Tyr Val Gly Gly Pro Thr Met Ala Val Ile Ala Gln
1 5 10 15
Arg Cys Pro His Ile Arg Val Cys Val Val Asp Ile Ser Gln Ala Gln
20 25 30
Ile Asp Ala Trp Asn Thr Asp Asp Leu Pro Ile Tyr Glu Pro Gly Leu
35 40 45
Leu Glu Val Val Lys Glu Ser Arg Gly Arg Asn Leu Phe Phe Ser Thr
50 55 60
Asp Ile Asp Ala Glu Ile Lys Arg Ala Asp Met Val Phe Ile Ser Val
65 70 75 80
Asn Thr Pro Thr Lys Thr Thr Gly Ile Gly Ala Gly Lys Ala Ala Asn
85 90 95
Ile Lys Asn Cys Glu Leu Cys Ala Arg Lys Ile Ala Glu Val Ala Asp
100 105 110
Thr Pro Lys Val Val Val Glu Lys Ser Thr Val Pro Val Arg Thr Ala
115 120 125
Glu Ala Val Arg Arg Val Leu Ala Thr Asn Glu Gly Lys Val Lys Phe
130 135 140
Gln Val Leu Ser Asn Pro Glu Phe Leu Ala Glu Gly Thr Ala Met Pro
145 150 155 160
Asp Leu Gln Asp Pro Ser Arg Val Leu Ile Gly Gly Met Gln Thr Pro
165 170 175
Glu Gly Leu Ala Ala Ile Gln Glu Leu Val Asp Val Tyr Ala Asn Trp
180 185 190
Val Pro Lys Asp Arg Ile Leu Ala Thr Asn Thr Trp Ser Ser Glu Leu
195 200 205
Ser Lys Leu Val Ala Asn Ala Phe Leu Ala Gln Arg Val Ser Ser Ile
210 215 220
Asn Ser Ile Ser Ala Leu Cys Glu Ala Thr Asp Ala Asp Ile Ser Glu
225 230 235 240
Val Ser Arg Ala Leu Gly Tyr Asp Pro Arg Ile Gly Asn Lys Phe Leu
245 250 255
Asn Ser Ser Val Gly Phe Gly Gly Ser Cys Phe Gln Lys Asp Val Leu
260 265 270
Asn Leu Val Tyr Ile Cys Glu Ser Thr Gly Leu Pro Glu Val Ala Glu
275 280 285
Tyr Trp His Gln Val Ile Ala Met Asn Asp Tyr Gln Lys Ser Arg Phe
290 295 300
Thr Gln Leu Met Val Arg Arg Met Phe Asn Thr Val Thr Gly Lys Arg
305 310 315 320
Ile Ala Val Leu Gly Phe Ala Phe Lys Lys Asp Thr Gly Asp Thr Arg
325 330 335
Glu Thr Pro Ala Val Phe Val Cys Lys Ala Leu Leu Glu Glu Gln Ala
340 345 350
Lys Val Gln Val Tyr Asp Pro Gln Val Lys Arg Ser Gln Met Phe Val
355 360 365
Glu Phe Asp Tyr Thr Cys Gly Val Asn Lys Asp Asn Thr Pro Arg Leu
370 375 380
Glu Glu Ser Ile Thr Thr Cys Glu Asp Ala Tyr Ser Ala Ala Glu Gly
385 390 395 400
Ser His Ala Leu Ala Ile Leu Thr Glu Trp Asp Glu Phe Lys Thr Leu
405 410 415
Asp Tyr Gln Arg Ile Tyr Asp Ser Met Ala Lys Pro Ala Phe Val Phe
420 425 430
Asp Gly Arg Gly Val Val Asp Ile Glu Ala Leu Arg Lys Ile Gly Phe
435 440 445
Glu Val Tyr Cys Ile Gly Lys Ser Lys Pro Lys Tyr Ser Leu Asp Ser
450 455 460
Pro Lys
465

Claims (9)

1. the UGD genes of GDP- mannose -6- dehydrogenases are encoded in long capsule water cloud, it is characterised in that：Described UGD unnamed genes For EsiUGD genes, the nucleotides sequence of described EsiUGD genes is classified as SEQ ID NO：Sequence shown in 1.
A kind of 2. protein of UGD gene codes as described in claim 1, it is characterised in that：The amino of described protein Acid sequence is SEQ ID NO：Sequence shown in 2.
3. protein as claimed in claim 2, it is characterised in that：Described protein is GDP- mannose -6- dehydrogenases, its Function is that catalysis GDP- mannoses are converted into GDP- mannuronic acids.
4. the carrier containing the UGD genes described in claim 1.
5. carrier as claimed in claim 4, it is characterised in that：Described carrier is pET expression vectors.
6. the genetically engineered cell containing the UGD genes described in claim 1.
7. genetically engineered cell as claimed in claim 6, it is characterised in that：Described genetically engineered cell is that Escherichia coli are thin Born of the same parents.
8. purposes of the UGD genes in algin is prepared described in claim 1, described purposes refer to the UGD channel genes Genetically engineered cell, so that genetically engineered cell can produce algin.
9. purposes of the protein described in claim 2 in algin is prepared.
CN201710618428.6A 2017-07-26 2017-07-26 UGD genes, its protein and the purposes of the dehydrogenase of GDP glucose 6 are encoded in long capsule water cloud Pending CN107354164A (en)

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Citations (2)

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CN103710369A (en) * 2014-01-03 2014-04-09 中国海洋大学 Bifunctional enzyme gene for kelp mannose 6-phosphate isomerization and GDP (Guanosine Diphosphatemannose)-mannose pyrophosphorylation
CN105821063A (en) * 2015-01-05 2016-08-03 中国科学院大连化学物理研究所 Incision alginate lyase Alg2B and coding gene, preparation and application thereof

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
CN103710369A (en) * 2014-01-03 2014-04-09 中国海洋大学 Bifunctional enzyme gene for kelp mannose 6-phosphate isomerization and GDP (Guanosine Diphosphatemannose)-mannose pyrophosphorylation
CN105821063A (en) * 2015-01-05 2016-08-03 中国科学院大连化学物理研究所 Incision alginate lyase Alg2B and coding gene, preparation and application thereof

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