CN107058423A - A kind of preparation method of homogeneity brown alga oligose - Google Patents

Abstract

The present invention provides a kind of preparation method of homogeneity brown alga oligose, and it with amino acid sequence is SEQ ID NO to be:1 algin enzyme and amino acid sequence is SEQ ID NO:2 algin enzyme is come algin of degrading.The method of the present invention prepares brown alga oligose based on enzymatic isolation method, and with cleaning, environmentally friendly advantage is further, since enzyme has specificity, and the accessory substance that it reacts is few；The what is more important present invention uses compound enzyme process, and product is more single, is conducive to the preparation of follow-up homogeneity oligosaccharides.

Description

A kind of preparation method of homogeneity brown alga oligose

Technical field

The invention belongs to oligosaccharides preparing technical field, and in particular to a kind of preparation method of homogeneity brown alga oligose.

Background technology

Brown alga oligose has a variety of different biological activities such as anti-oxidant, immunological regulation, and to there is molecular weight small for oligosaccharides, The advantage utilized is readily absorbed by, therefore is had in multiple fields such as pharmaceutical preparation, functional food, chemical industry before wide application Scape.Brown alga oligose is in itself without cytotoxicity, and it strengthens defence energy of the body to tumour cell by improving immunity of organisms Power.Research shows that the change that brown alga oligose can suppress on the growth of human leukemia cell, Cell Image Analyzer shows as typical case Apoptosis；Brown alga oligose has antioxidation.Research shows that algin oligosaccharide is to O^2-, the free radical such as OH and ClO There is preferable scavenging action, and scavenging capacity is influenceed by the concentration and molecular weight of brown alga oligose.Research finds brown alga Oligosaccharides shown to ABTS, the scavenging capacity of hydroxyl and superoxide radical.Free radical scavenging activity is mostly derived from enzymolysis process The presence of the conjugation olefin(e) acid structure of middle formation.Brown alga oligose adjusts blood glucose and lipid by the generation of inducing cytokine.Due to The various biological activity of brown alga oligose, therefore in the urgent need to setting up the homogeneity brown alga oligose preparation method of green high-efficient.

The content of the invention

It is an object of the invention to provide a kind of preparation method of homogeneity brown alga oligose, so that it is few to prepare homogeneity brown alga Sugar, so as to make up the deficiencies in the prior art.

The method of the present invention, it with amino acid sequence is SEQ ID NO to be:1 algin enzyme and amino acid sequence is SEQ ID NO:2 algin enzyme is come algin of degrading；

Described method, is that a kind of mode is while come algin of degrading by two kinds of algin enzymes；

Described method, it is first SEQ ID NO with amino acid sequence that its another way, which is,:1 algin is brown to degrade Phycocolloid, then with amino acid sequence be SEQ ID NO:2 algin enzyme is come algin of degrading；

Described amino acid sequence is SEQ ID NO:1 algin enzyme, the nucleotides sequence of its encoding gene is classified as SEQ ID NO:3；

Described amino acid sequence is SEQ ID NO:2 algin enzyme, the nucleotides sequence of its encoding gene is classified as SEQ ID NO:4。

The method of the present invention prepares brown alga oligose based on enzymatic isolation method, and with cleaning, environmentally friendly advantage is further, since enzyme has Specificity, the accessory substance that it reacts is few；The what is more important present invention uses compound enzyme process, and product is more single (only Two kinds of components), be conducive to the preparation of follow-up homogeneity oligosaccharides.

Brief description of the drawings

Fig. 1：Recombinase isolates and purifies figure, wherein Lane 1.AlyB-OU02；Lane2.AlyD-OU02；

Fig. 2：Complex enzyme is to the degraded figure of algin, wherein A:Complex enzyme；B:Sequentially add AlyB-OU02 and AlyD- OU02；Fig. 2-A:1：Not enzyme-added control；2：Only add AlyB-OU02；3：Only add AlyD-OU02；4：Plus AlyB-OU02 and AlyD- OU02 complex enzymes；Fig. 2-B：1：Add after AlyB-OU02 reactions 2h；2：Add AlyD-OU02 reactions 2h；

Embodiment

Applicant has found in long-term research, by being SEQ ID NO by amino acid sequence:1 algin enzyme and ammonia Base acid sequence is SEQ ID NO:2 algin enzyme is come algin of degrading；Oligosaccharides prepared by existing method can effectively be overcome not Homogeneous the problem of, so as to facilitate the present invention.

The present invention is described in detail with reference to embodiment.

Embodiment 1：The recombination expression of enzyme

1st, the extraction of genomic DNA：

Target strain OU02 is inoculated in LB fluid nutrient mediums, 7 hours left sides are cultivated in 25 DEG C, 150rpm shaken cultivation casees The right side, takes 1.5mL bacterium solutions, and in 4 DEG C, 12,000 × g centrifugation 1min, abandoning supernatant adds bacterium solution repeated centrifugation 3 times, collects Thalline.Then DNA is extracted using genomic DNA kit.(genomic DNA kit：TIANGEN BIOTECH) extraction step It is as follows：

1) 200 μ L buffer solution GA are added in thalline, vibration makes thalline suspend, and adds 4 μ LRNaseA (100mg/mL) molten Liquid removes RNA, vortex oscillation 15s, is stored at room temperature 5min.

2) 20 μ L Proteinase K solution are added into pipe, are mixed.

3) 220 μ L buffer solution GB are added, 15s is vibrated, 70 DEG C of placement 10min, solution strain is bright, and crawl centrifuges to remove The globule of inside pipe wall.

4) 220 μ L absolute ethyl alcohols are added, flocculent deposit, crawl centrifugation should now occurs in vortex oscillation 15s.

5) mixture obtained by previous step is added in adsorption column (adsorption column is put into collecting pipe), 12,000 × g centrifugations 30s, outwells waste liquid, and adsorption column is put into collecting pipe.

6) 500 μ l buffer solutions GD (having added absolute ethyl alcohol), 12,000 × g centrifugation 30s are added, waste liquid is outwelled, will adsorb Post is put into collecting pipe.

7) 700 μ l rinsing liquids PW (having added absolute ethyl alcohol), 12,000 × g centrifugation 30s are added, waste liquid is outwelled, will adsorb Post is put into collecting pipe.

8) 500 μ l rinsing liquids PW (having added absolute ethyl alcohol), 12,000 × g centrifugation 30s are added, waste liquid is outwelled, will adsorb Post is put into collecting pipe.

9) 12,000g centrifuges 2min, outwells waste liquid.It is stored at room temperature several minutes, dries rinsing liquid remaining in sorbing material.

10) adsorption column is transferred in a clean centrifuge tube, 50-200 μ l is vacantly added dropwise to the middle part of adsorbed film Elution buffer TE, room temperature places 2-5min, 12,000 × g centrifugation 2min, and the liquid received is the bacterial genomes of extraction DNA, repeats elution adsorbed film by solution in pipe, can improve the rate of recovery, by the bacterial genomes DNA of extraction in -20 DEG C of preservations.

2nd, from the genome of the microorganism of production high activity algin catenase, alginate lyase gene AlyB- is cloned OU02 and AlyD-OU02, design primer is as follows：

AlyB-OU02-BamHI-F:CGGGATCCGACTTCCCTAACAACAAAGAAACG

AlyB-OU02-XhoI-R:CCGCTCGAGCTACTTTTTGTATTGATCGTGCG

AlyD-OU02-BamHI-F:CGGGATCCAATAACGGTGTGTCTTATCCAG

AlyD-OU02-XhoI-R:CGCTCGAGCTACTTGCCGTTCAGCTTAAGTG

3rd, target gene AlyB-OU02 and AlyD-OU02 PCR amplifications are carried out：

Using the total genomic dna of bacterial strain as masterplate, PCR reaction systems are as follows：The μ L of 5 × PrimeSTAR Buffer 10, 2.5mMdNTP Mixture 4 μ L, 10 μM of forward and reverse each 1 μ L, PrimeSTAR HS DNA Polymerase (2.5U/ μ of primer L) the μ L of 0.5 μ L, DNA masterplate 1, sterilizing ultra-pure water supplies 50 μ L.

PCR conditions are as follows：94 DEG C of 3min of pre-degeneration；It is denatured 94 DEG C of 30s；Anneal 50 DEG C of 30s；Extend 72 DEG C of 2min, 5 are followed Ring；It is denatured 94 DEG C of 30s；Anneal 58 DEG C of 30s；Extend 72 DEG C of 2min, 30 circulations；Extend 72 DEG C of 10min.1% agar of product Sugared gel electrophoresis checking, there is mesh in 1500bp, 975bp or so respectively in voltage 90V, 25min, AlyB-OU02 and AlyD-OU02 Band, and with purchased from OMEGA BIO-TEK Gel Extraction Kit gel extraction PCR primers.Glue reclaim step is such as Under：

1) glue containing purpose band is cut with clean blade, unnecessary glue is cut off as far as possible.

2) glue cut is put into the 1.5mL centrifuge tubes of sterilizing, and weighed.By gum concentration 1g/mL, add in equal volume Sol solutionses (XP2), i.e. 0.3g glue add 0.3mL sol solutionses.

3) 50-60 DEG C is incubated 7min until glue is completely dissolved, and per 2-3min, vibration centrifuge tube accelerates colloidal sol.

4) HiBind DNA MiNi adsorption columns are put into 2mL collecting pipes.

5) the DNA/ agarose solutions that the 3rd step is obtained, take and add adsorption column no more than 700 μ l.

6) room temperature 10,000 × g centrifugation 1min, outwell waste liquid, adsorption column is put into collecting pipe.

7) 5-6 steps are repeated, until all sol solutionses are transferred to adsorption column.

8) 300 μ l XP2, room temperature maximum (top) speed (>=13,000 × g) centrifugation 1min are added, waste liquid is outwelled, adsorption column is put into In collecting pipe.

9) 700 μ L SPW rinsing liquids (addition absolute ethyl alcohol), room temperature maximum (top) speed (>=13,000 × g) centrifugation are added 1min, outwells waste liquid, and adsorption column is put into collecting pipe.

10) repeat step 9.

11) suction attached column, room temperature maximum (top) speed (>=13,000 × g) centrifugation 2min, dries adsorption column matrix.

12) adsorption column is put into the centrifuge tube of 1.5mL sterilizings, takes 15-30 μ L elution buffers to be vacantly added drop-wise to absorption The center of post film.

13) 2min, maximum (top) speed (>=13,000 × g) centrifugation 1min are stored at room temperature, elution is repeated and improves the DNA rate of recovery.

14) PCR primer of recovery is carried out to 1% agarose gel electrophoresis detection again, -20 DEG C are stored in.

4th, digested plasmid and template：

Expression plasmid pGEX6P-1 and PCR recovery products are subjected to double enzymes with restriction enzyme BamH I, EcoR I respectively Cut, digestion system is：The μ L, BamH I of 20 μ L, 10 × K Buffer of DNA profiling 6, each 3 μ L of EcoR I, sterilizing ultra-pure water supply 60 μ L, 30 DEG C of digestion 5h.And reclaim digestion products with purchased from OMEGA BIO-TEK Cycle-Pure Kit.Purification step is as follows：

1) the CP buffer solutions of 4-5 times of volume are added in endonuclease reaction system, vortex oscillation makes solution fully mix, briefly Centrifuge to collect the globule in lid and tube wall.

2) HiBind DNA MiNi adsorption columns are put into 2mL collecting pipes.

3) solution of the first step is added adsorption column, room temperature maximum (top) speed (>=13,000 × g) centrifugation 1min takes collecting pipe In liquid add adsorption column in, repeat adsorb, improve product recoveries, room temperature maximum (top) speed (>=13,000 × g) centrifugation 1min, outwells waste liquid, and adsorption column is put into collecting pipe.

4) 700 μ L DNA rinsing liquids (addition absolute ethyl alcohol), room temperature maximum (top) speed (>=13,000 × g) centrifugation are added 1min, outwells waste liquid, and adsorption column is put into collecting pipe.

5) repeat step 4.

6) suction attached column, room temperature maximum (top) speed (>=13,000 × g) centrifugation 2min, dries adsorption column matrix.

7) adsorption column is put into the 1.5mL centrifuge tubes of sterilizing, takes 30-50 μ L elution buffers to be vacantly added drop-wise to adsorption column The center of film.

8) 2min, room temperature maximum (top) speed (>=13,000 × g) centrifugation 1min are stored at room temperature, elution is repeated and improves DNA recovery Rate.

14) digestion products of recovery are subjected to 1% agarose gel electrophoresis detection, are stored in -20 DEG C.

5th, target gene and expression vector are connected：

Plasmid (pGEX6P-1) after digestion is connected with target gene fragment (AlyB-OU02, AlyD-OU02) in T4DNA Connected overnight under enzyme effect, linked system is：The 1 μ L of μ L, 10 × Buffer of T4DNA ligases 1, target gene fragment and plasmid In mass ratio 10:1, sterilizing ultra-pure water supplies 10 μ L, and 16 DEG C of connections are stayed overnight.

6th, convert：

Competent cell BL21 (DE3) is taken out from -80 DEG C of refrigerators, is put into ice rapidly.After cell melts completely, Aseptically connection product is slowly added into competent cell, gently mixed, ice bath 30min.It is rapid to place 42 DEG C Water-bath heat shock 75s.Quickly centrifuge tube is put into ice, ice bath 2min.500 μ L LB liquid are added under aseptic condition into mixed liquor Body culture medium, 37 DEG C of shaken cultivation 1h, makes thalline recover.Under aseptic condition, 200 μ L bacterium solutions are taken equably to be coated on anti-containing Amp+ Property LB solid mediums on, be inverted into constant incubator after liquid is fully absorbed, 37 DEG C of culture to media surfaces have Bacterium colony is generated.

7th, bacterium solution PCR identifies positive colony

Random picking monoclonal bacterium colony, sequencing analysis, and preserve bacterium solution in -80 DEG C.Wherein algin enzyme AlyB-OU02, Its amino acid sequence is SEQ ID NO:1, coding nucleotide sequence is SEE ID NO:3.Enzyme AlyD-OU02, its amino acid sequence It is classified as SEQ ID NO:2, coding nucleotide sequence is SEE ID NO:4.Particular sequence information is as follows：

Wherein AlyB-OU02 amino acid sequence is as follows：

DFPNNKETGEALLTPVDATASSHDGNGPDRLIDQDLTTRWSSAGDGEWAMLDYGSVQEFDAVQASFSKGNERQSKFD IQVSVDGETWTTVLENQLSSGKAIGLERFQFEPAVKARYVRYVGHGNTKNGWNSVTGLAAVNCSINACPASQIITSD VVAAEAVLIAEMKAAEKARKAARKDLRSGNFGVAAVYPCETSVKCDTRSALPVPTGLPATPVAGNAPSENFDMTHWY LSQPFDHDKNGKPDDVSEWNLANGYQHPEIFYTADDGGLVFKSYVKGVRTSKNTKYARTELREMMRRGDQSISTKGV NKNNWVFSSAPEADLEAAAGIDGVLEATLKIDHATTTGNANEVGRFIIGQIHDQNDEPIRLYYRKLPNQPTGAVYFA HESQDATKEDFYPLVGDMTAEVGEDGIALGEVFSYRIDVKGNTMTVTLMREGKDDVVQVVDMSNSGYDVGGKYMYFK AGVYNQNISGDLDDYSQATFYQLDVSHDQYKK；

The nucleotide sequence of its encoding gene is as follows：

GACTTCCCTAACAACAAAGAAACGGGTGAAGCACTTCTTACTCCGGTTGATGCTACCGCTAGCAGCCACGATGGAAA TGGCCCAGATCGCCTCATTGACCAAGACCTAACAACACGTTGGTCGTCAGCGGGTGACGGTGAGTGGGCAATGCTAG ATTATGGTTCAGTACAAGAGTTTGACGCTGTTCAAGCATCCTTCAGTAAAGGTAATGAGCGCCAATCTAAATTTGAT ATTCAAGTGAGTGTCGATGGTGAAACCTGGACGACGGTACTTGAGAACCAACTCAGTTCTGGTAAAGCTATCGGCCT AGAGCGTTTCCAATTTGAGCCTGCGGTGAAAGCACGTTACGTAAGATATGTTGGTCACGGCAACACCAAAAACGGTT GGAACAGTGTGACTGGGTTAGCAGCGGTTAACTGTAGCATCAACGCTTGTCCAGCGAGTCAAATCATCACTTCAGAC GTGGTGGCTGCAGAAGCGGTGCTTATTGCTGAAATGAAAGCAGCGGAAAAAGCGCGTAAAGCAGCACGCAAAGATCT ACGCTCAGGTAACTTCGGCGTAGCAGCAGTTTACCCTTGTGAGACTTCAGTTAAATGTGACACTCGCAGCGCGCTAC CTGTTCCGACAGGCCTACCAGCGACACCCGTTGCCGGTAATGCACCGAGCGAAAACTTCGACATGACTCACTGGTAC CTGTCTCAACCATTCGACCATGACAAAAACGGTAAGCCAGATGATGTATCCGAGTGGAACCTTGCAAACGGGTATCA ACACCCAGAAATCTTCTATACCGCGGACGACGGTGGTCTAGTGTTCAAGTCTTACGTGAAAGGTGTACGTACATCTA AAAACACTAAGTACGCGCGTACCGAGCTTCGTGAGATGATGCGTCGTGGTGACCAGTCTATCAGTACGAAAGGTGTT AATAAGAACAACTGGGTATTCTCAAGCGCACCTGAAGCTGATTTAGAAGCTGCGGCTGGTATCGATGGCGTTCTAGA AGCAACATTGAAAATCGATCATGCGACAACAACGGGTAATGCGAATGAAGTAGGTCGCTTTATCATTGGTCAGATTC ACGATCAAAACGATGAGCCGATTCGTTTGTACTACCGTAAACTGCCAAACCAACCAACGGGCGCAGTTTACTTTGCA CACGAAAGCCAAGACGCGACCAAAGAGGACTTCTATCCTCTAGTGGGAGACATGACGGCTGAAGTAGGTGAAGATGG TATCGCACTTGGTGAAGTGTTCAGCTACCGTATTGACGTTAAAGGCAACACGATGACGGTAACGCTAATGCGTGAAG GCAAAGACGATGTTGTACAAGTGGTTGATATGAGCAACAGCGGCTATGACGTTGGCGGCAAGTACATGTATTTCAAA GCGGGTGTTTACAACCAAAATATCAGCGGCGACCTAGACGATTACTCACAAGCTACTTTCTACCAGCTAGACGTATC GCACGATCAATACAAAAAGTAG；

The amino acid sequence of AlyD-OU02 enzymes is as follows：

PVPADKFDMRNWKITIPSDINEDGRVDEIEGVAMMSYSHSDFFHLDKDGNLVFEVQNQAITTKNSKNARSELRQMPR GADFSIDTADKGNQWALSSHPAASEYSAVGGTLEATLKVNHVSINAKYPQRYPAHSVVVGQIHAKKHDALIKAKTGY GHGNEPLKIFYKKFPDQEMGSVFWNYERNLEKKDPNRADIAYPVWGNTWENPAEPGEAGIALGEEFSYKVEVKGTMM YLTFETARHDTVKYEVDLSKGIDELDSPTGYAEDDFYYKAGAYGQCSVSDSHPVWGPGCAGTGDFAVDKKNGDYNSV TFSALKLNGK；

The nucleotide sequence of encoding gene is as follows：

CCAGTTCCCGCTGATAAGTTTGATATGCGAAATTGGAAAATAACGATCCCTTCAGATATTAATGAAGATGGACGTGT TGACGAAATCGAAGGGGTGGCAATGATGAGCTACTCACACAGCGATTTCTTCCACCTTGATAAAGACGGTAATCTCG TCTTTGAAGTACAAAACCAAGCGATTACCACGAAGAACTCGAAAAACGCACGCTCTGAGTTACGCCAGATGCCACGA GGTGCTGATTTCTCAATCGATACGGCGGACAAAGGAAACCAGTGGGCACTATCGAGCCACCCAGCAGCGAGTGAATA CAGTGCGGTTGGTGGAACGCTAGAAGCGACGCTAAAGGTGAATCACGTTTCGATTAACGCTAAGTACCCACAAAGAT ACCCAGCTCACTCGGTAGTTGTTGGCCAGATACACGCGAAGAAACACGATGCTTTGATCAAAGCAAAAACAGGTTAC GGCCACGGTAACGAACCACTAAAAATCTTCTATAAGAAATTTCCTGATCAAGAGATGGGTTCAGTCTTTTGGAACTA TGAACGCAACTTAGAGAAGAAAGATCCTAACCGTGCTGATATTGCTTACCCAGTTTGGGGCAATACTTGGGAAAACC CAGCAGAGCCTGGTGAAGCGGGAATTGCTCTAGGTGAAGAGTTCAGCTACAAAGTGGAAGTGAAGGGTACAATGATG TATCTAACATTTGAAACGGCTCGCCACGATACCGTTAAGTATGAAGTCGACTTGAGCAAGGGCATCGATGAACTTGA TTCGCCAACAGGCTATGCGGAAGATGACTTTTACTATAAAGCGGGTGCGTACGGTCAATGCAGTGTAAGCGACTCTC ACCCAGTGTGGGGCCCGGGTTGTGCGGGTACTGGCGATTTCGCTGTCGATAAAAAGAACGGCGATTACAATAGTGTG ACTTTCTCAGCACTTAAGCTGAACGGCAAGTAG。

2nd, the preparation of recombinase

1) thalline and ultrasonication (30min), 20,000g centrifugation 20min is resuspended in pGEX-6P-1 thalline, 60ml PBS.

2) GST beads (GE) after supernatant and 2ml are balanced combine outflow under 2h, Action of Gravity Field at 4 DEG C；

3) 400ml PBSs rinse uncombined albumen；

4) 2ml PBS, and in mass ratio 50 are added:1 adds PreScission enzymes, and 4 DEG C stand digestion (often 15min featheriness is mixed once).

5) flowed out after digestion 2h, outflow carries out SDS-PAGE detections (Fig. 1)：

Embodiment 2：The preparation of high homogeneity brown alga oligose based on complex enzyme

Method 1) combined-enzyme method：Algin is carried out with AlyB-OU02 the and AlyD-OU02 algin catenases of recombination expression Degraded, substrate is 0.2% algin (m/v) 200 μ L, enzyme AlyB-OU02 (1.58 μM) and AlyD-OU02 (about 13.9 μM) each 20 μL。

Three groups of reactions are set, i.e.,：AlyB-OU02, AlyD-OU02 individually degrade algin and AlyB-OU02 and The common degraded algin of AlyD-OU02 mutual cooperations.Overall reaction system is 220 μ L, and buffer solution is 50mMTris-HCl 200mMNaCl pH7.5, (about 30 DEG C) of room temperature stands reaction 90min, and reaction terminates rear 12000rpm centrifugations 3min, taken clear and coherent TLC methods are crossed to analyze catabolite.

Method 2) priority addition method：Brown alga is carried out with AlyB-OU02 the and AlyD-OU02 algin catenases of recombination expression Glue is degraded, and substrate is that 0.2% algin (m/v) 200 μ L, enzyme AlyB-OU02 (1.58 μM) and AlyD-OU02 (about 13.9 μM) are each 20μL.One group of reaction is set, i.e., first with AlyB-OU02 degrade algin, buffer solution be 50mMTris-HCl 200mMNaCl PH7.5, (about 30 DEG C) of room temperature stands reaction 90min, and AlyD-OU02 is added afterwards and continues to react 90min.After reaction terminates, 12000rpm centrifuges 3min, takes supernatant to be analyzed by TLC methods catabolite.

Take respectively 2 μ L degrade after solution and control group solution point sample on silica gel plate (Silica gel 60F 254, Merck).Silica gel plate is placed in solvent after drying and starts chromatography, solvent is configured in the following proportions：N-butanol/formic acid/water (v/v)=4:6:1.After chromatography terminates, take out silica gel plate and dry up, developer (aniline-diphenylamines colour developing is sprayed on silica gel plate Agent), drying, then silica gel plate is placed in colour developing under hot conditions, until there is obvious spot.(Fig. 2).Such as Fig. 2-A swimming lanes 2 and Shown in Fig. 2-B swimming lanes 1, only with enzyme AlyB-OU02 degraded algins, product mainly has 4 components.After AlyB-OU02 degradeds Sample carries out Mass Spectrometer Method, determines the molecular weight of sample.INSTRUMENT MODEL is Q Exactive Focus.By Mass Spectrometric Identification, AlyB-OU02 degraded after sample (Fig. 2-A, swimming lane 1) be mainly：DP1:175Da；DP2:351Da；DP3:527Da；DP4: 703Da。

In utilization complex enzyme AlyD-OU02 or after AlyB-OU02 degradeds, by subsequently adding AlyD-OU02, product As shown in Fig. 2-A swimming lanes 4 and Fig. 2-B swimming lanes 2, as a result show that complex enzyme hydrolysis can effectively degrade two of which component (follow-up matter Spectrum is accredited as DP3 and DP4), product is mainly DP1 and DP2 after degraded.As a result illustrate that AlyD-OU02 addition improves product Homogeneity.

The step of recovery of enzymolysis product, is as follows：

After reaction terminates, reaction system is placed in 100 DEG C, water-bath 10min, inactivator terminating reaction.4 DEG C, 12 000g from Heart 20min, collects supernatant, and freeze-drying obtains oligosaccharides.The rate of recovery for calculating obtained oligosaccharides weigh 85% or so.

SEQUENCE LISTING

<110>Chinese Marine University

<120>A kind of preparation method of homogeneity brown alga oligose

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<170> PatentIn version 3.5

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ggtcacggca acaccaaaaa cggttggaac agtgtgactg ggttagcagc ggttaactgt 420

agcatcaacg cttgtccagc gagtcaaatc atcacttcag acgtggtggc tgcagaagcg 480

gtgcttattg ctgaaatgaa agcagcggaa aaagcgcgta aagcagcacg caaagatcta 540

cgctcaggta acttcggcgt agcagcagtt tacccttgtg agacttcagt taaatgtgac 600

actcgcagcg cgctacctgt tccgacaggc ctaccagcga cacccgttgc cggtaatgca 660

ccgagcgaaa acttcgacat gactcactgg tacctgtctc aaccattcga ccatgacaaa 720

aacggtaagc cagatgatgt atccgagtgg aaccttgcaa acgggtatca acacccagaa 780

atcttctata ccgcggacga cggtggtcta gtgttcaagt cttacgtgaa aggtgtacgt 840

acatctaaaa acactaagta cgcgcgtacc gagcttcgtg agatgatgcg tcgtggtgac 900

cagtctatca gtacgaaagg tgttaataag aacaactggg tattctcaag cgcacctgaa 960

gctgatttag aagctgcggc tggtatcgat ggcgttctag aagcaacatt gaaaatcgat 1020

catgcgacaa caacgggtaa tgcgaatgaa gtaggtcgct ttatcattgg tcagattcac 1080

gatcaaaacg atgagccgat tcgtttgtac taccgtaaac tgccaaacca accaacgggc 1140

gcagtttact ttgcacacga aagccaagac gcgaccaaag aggacttcta tcctctagtg 1200

ggagacatga cggctgaagt aggtgaagat ggtatcgcac ttggtgaagt gttcagctac 1260

cgtattgacg ttaaaggcaa cacgatgacg gtaacgctaa tgcgtgaagg caaagacgat 1320

gttgtacaag tggttgatat gagcaacagc ggctatgacg ttggcggcaa gtacatgtat 1380

ttcaaagcgg gtgtttacaa ccaaaatatc agcggcgacc tagacgatta ctcacaagct 1440

actttctacc agctagacgt atcgcacgat caatacaaaa agtag 1485

<210> 4

<211> 957

<212> DNA

<213> 4

<400> 4

ccagttcccg ctgataagtt tgatatgcga aattggaaaa taacgatccc ttcagatatt 60

aatgaagatg gacgtgttga cgaaatcgaa ggggtggcaa tgatgagcta ctcacacagc 120

gatttcttcc accttgataa agacggtaat ctcgtctttg aagtacaaaa ccaagcgatt 180

accacgaaga actcgaaaaa cgcacgctct gagttacgcc agatgccacg aggtgctgat 240

ttctcaatcg atacggcgga caaaggaaac cagtgggcac tatcgagcca cccagcagcg 300

agtgaataca gtgcggttgg tggaacgcta gaagcgacgc taaaggtgaa tcacgtttcg 360

attaacgcta agtacccaca aagataccca gctcactcgg tagttgttgg ccagatacac 420

gcgaagaaac acgatgcttt gatcaaagca aaaacaggtt acggccacgg taacgaacca 480

ctaaaaatct tctataagaa atttcctgat caagagatgg gttcagtctt ttggaactat 540

gaacgcaact tagagaagaa agatcctaac cgtgctgata ttgcttaccc agtttggggc 600

aatacttggg aaaacccagc agagcctggt gaagcgggaa ttgctctagg tgaagagttc 660

agctacaaag tggaagtgaa gggtacaatg atgtatctaa catttgaaac ggctcgccac 720

gataccgtta agtatgaagt cgacttgagc aagggcatcg atgaacttga ttcgccaaca 780

ggctatgcgg aagatgactt ttactataaa gcgggtgcgt acggtcaatg cagtgtaagc 840

gactctcacc cagtgtgggg cccgggttgt gcgggtactg gcgatttcgc tgtcgataaa 900

aagaacggcg attacaatag tgtgactttc tcagcactta agctgaacgg caagtag 957

Claims (6)

1. a kind of preparation method of homogeneity brown alga oligose, it is characterised in that described method, it with amino acid sequence is SEQ to be ID NO:1 algin enzyme and amino acid sequence is SEQ ID NO:2 algin enzyme prepares algin to degrade algin Oligosaccharides.

2. the method as described in claim 1, it is characterised in that described method is to use amino acid sequence to be SEQ ID NO: 1 algin enzyme and amino acid sequence is SEQ ID NO:2 algin enzyme is simultaneously come algin of degrading.

3. the method as described in claim 1, it is characterised in that it is first SEQ ID NO with amino acid sequence that described method, which is,: 1 algin is come algin of degrading, then with amino acid sequence is SEQ ID NO:2 algin enzyme is come algin of degrading.

4. the method as described in claim any one of 1-3, it is characterised in that described amino acid sequence is SEQ ID NO:1 Algin enzyme, the nucleotides sequence of its encoding gene is classified as SEQ ID NO:3.

5. the method as described in claim any one of 1-3, it is characterised in that described amino acid sequence is SEQ ID NO:2 Algin enzyme, the nucleotides sequence of its encoding gene is classified as SEQ ID NO:4.

6. a kind of algin oligosaccharide, it is characterised in that described algin oligosaccharide is with the side described in claim any one of 1-3 Prepared by method.