CN110317846A - A method of preparing odd number fine jade oligosaccharides - Google Patents
A method of preparing odd number fine jade oligosaccharides Download PDFInfo
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- CN110317846A CN110317846A CN201910658526.1A CN201910658526A CN110317846A CN 110317846 A CN110317846 A CN 110317846A CN 201910658526 A CN201910658526 A CN 201910658526A CN 110317846 A CN110317846 A CN 110317846A
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- fine jade
- agarose
- odd number
- agarase
- oligosaccharides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
Abstract
The present invention provides a kind of method for preparing odd number fine jade oligosaccharides, is after being digested the pickling agent of agarose using β-agarase, then carries out purifying preparation；Agarase therein is the enzyme that can be digested agarose as six sugar of new fine jade tetrose and Xin Qiong；The present invention realizes method that is special and efficiently preparing odd number fine jade trisaccharide by combining acid hydrolyzation and enzyme process.Compared with acid hydrolyzation prepares the method for odd number fine jade oligosaccharides, yield of the present invention is obviously improved, and can be realized specific preparation；Compared with enzymatic isolation method prepares odd number fine jade oligosaccharides, it is capable of handling the substrate of high concentration, so that the method for the present invention be made to have higher efficiency compared to existing method.
Description
Technical field
The invention belongs to oligosaccharides preparation technical fields, and in particular to a method of prepare odd number fine jade oligosaccharides.
Background technique
Agaropectin oligose is derived from agar polysaccharide and (is mainly isolated from certain red algaes, such as agar, Porphyra yezoensis, wart
Shape fragrant plant mentioned in ancient texts) one kind native oligosaccharides, there are a variety of physiological activity.Agaropectin oligose by (1 → 3)-O- β-D- galactose residue and
The linear chain molecule that (1 → 4)-O-3,6- inner ether-α-L- galactose residue alternately forms.It is non-also according to agaropectin oligose
Agaropectin oligose can be divided into new fine jade oligosaccharides and non-reducing end that non-reducing end is 3,6- inner ether-L- galactolipin by the difference at former end
For the fine jade oligosaccharides of D- galactolipin.Fine jade oligosaccharides has certain physiological activity, and the researchs such as Jin discovery fine jade oligosaccharides can inhibit alcohol malicious
Property, certain protective effect is provided for liver.The result of study of Ye etc. is shown in terms of showing fine jade pentasaccharides for anti-neurotrosis
Preferable physiological activity.
The method for preparing agaropectin oligose at present mainly has chemical method and two kinds of enzyme process, mainly prepares odd number by chemical method
Fine jade oligosaccharides, enzyme process, can only be by conjunction with β-agarases and newly then usually to prepare the new fine jade oligosaccharides of even number and even number fine jade oligosaccharides
Fine jade disaccharide-hydrolysing enzymes two-step method prepares odd number fine jade oligosaccharides.Wherein preparing odd number fine jade oligosaccharides by chemical method, that is, acid hydrolyzation has reaction
Rapidly, the advantages that processing concentration of substrate is larger, but product is more miscellaneous after acidolysis, separation is more difficult.Enzymatic isolation method compared to
Acid hydrolyzation has many advantages, such as that reaction is mild, product is single, is easily isolated, but single agarase can only be to prepare even number agar-agar
Oligosaccharides.Therefore, the specifical and efficient property technology of preparing for developing odd number fine jade oligosaccharides is of great significance.
Summary of the invention
Present invention aims to overcome that prepare fine jade product oligosaccharides complicated for traditional acid hydrolyzation, it is difficult to which separation and enzymatic isolation method are logical
The problem of single agarase can not prepare odd number fine jade oligosaccharides is crossed, a kind of preparation method of novel odd number fine jade oligosaccharides is provided；It is
Agarose being handled by acidolysis, agarose acid hydrolysis solution being made, recycling primary product is β-agar-agar of six sugar of new fine jade tetrose and Xin Qiong
Enzyme hydrolysis agarose acid hydrolysis solution prepares fine jade trisaccharide and fine jade pentasaccharides.
The present invention provides a kind of method for preparing new fine jade trisaccharide and Xin Qiong pentasaccharides, is that the pickling agent of agarose is used β-
Agarase is digested, and enzymolysis product carries out purifying preparation again；Agarase therein is for preparing new fine jade four from agarose
The enzyme of sugared and new six sugar of fine jade；
The enzyme is β-agarase DagA, and amino acid sequence is SEQ ID NO:1:
MVNRRDLIKWSAVALGAGAGLAGPAPAAHAADLEWEQYPVPAAPGGNRSWQLLPSHSDDFNYTGKPQT
FRGRWLDQHKDGWSGPANSLYSARHSWVADGNLIVEGRRAPDGRVYCGYVTSRTPVEYPLYTEVLMRVSGLKLSSN
FWLLSRDDVNEIDVIECYGNESLHGKHMNTAYHIFQRNPFTELARSQKGYFADGSYGYNGETGQVFGDGAGQPLLR
NGFHRYGVHWISATEFDFYFNGRLVRRLNRSNDLRDPRSRFFDQPMHLILNTESHQWRVDRGIEPTDAELADPSIN
NIYYRWVRTYQAV。
The pickling agent of the agarose is, it is preferable to use citric acid treatment agarose prepares；
Wherein a kind of specific concentration of citric acid is the monohydrate potassium of 2.5% (w/v).
The acid processing of the agarose is to handle agarose with 2.5% monohydrate potassium, reacts in 90 DEG C, cold
But to after room temperature, the pH of reaction solution is adjusted to 7.0.
The purifying, a kind of mode are purified using column gel chromatography chromatography.
The present invention is realized special and efficiently prepares odd number fine jade trisaccharide and fine jade pentasaccharides by combining acid hydrolyzation and enzyme process
Method.Compared with acid hydrolyzation prepares the method for odd number fine jade oligosaccharides, yield of the present invention is obviously improved, and can be realized specific preparation；
It is prepared (in conjunction with β-agarase and Xin Qiong disaccharide-hydrolysing enzymes) compared with odd number fine jade oligosaccharides with enzymatic isolation method, is capable of handling the bottom of high concentration
Object, so that the method for the present invention be made to have higher efficiency compared to existing method.
Detailed description of the invention
Fig. 1: fine jade trisaccharide (a) prepared by the present invention and fine jade pentasaccharides (b) structure chart and new fine jade tetrose (c) He Xinqiong six are sugared (d)
Structure chart；
Fig. 2: β of the invention-agarase DagA hydrolyzes the TLC figure of nine sugar of seven sugar of fine jade and fine jade, A3: fine jade trisaccharide, A5: fine jade five
Sugar, A7: seven sugar of fine jade, A9: nine sugar of fine jade；
The enzyme concentration that Fig. 3: β of the invention-agarase DagA hydrolyzes acidolysis agar liquid glucose optimizes figure；
Fig. 4: β of the invention-agarase DagA (b) hydrolyze acidolysis agar liquid glucose under the conditions of most suitable enzyme concentration candy output with
The liquid phase analysis result (a) of time change；
Fig. 5: DagA combination acidolysis of the invention prepares fine jade trisaccharide and fine jade pentasaccharides liquid phase result figure；
Fig. 6: DagA combination acidolysis of the invention prepares fine jade trisaccharide (a) and fine jade pentasaccharides (b) mass spectrometry results figure.
Specific embodiment
The agarose is by D- beta galactose and 3, and 6- inner ether-L- α-galactolipin repeats disaccharide unit and constitutes, by β-
1,4 glycosidic bonds and α -1,3 glycosidic bond are alternately formed by connecting.
Fine jade trisaccharide refers to that non-reducing end is the agaropectin oligose that the D- beta galactose degree of polymerization is 3, shown in structure such as Fig. 1 (a),
Fine jade pentasaccharides refers to that non-reducing end is the agaropectin oligose that the D- beta galactose degree of polymerization is 5, shown in structure such as Fig. 1 (b).
When the present invention prepares β used in fine jade trisaccharide-agarase DagA and directly acts on agarose, primary product is new fine jade
Six sugar of tetrose and Xin Qiong is prepared new shown in new six sugared structure such as Fig. 1 (c) of fine jade using 0.3% (w/v) low melting-point agarose as substrate
Six sugar of fine jade tetrose and Xin Qiong, yield is respectively 14% and 13% or so, further passes through the effect preparation of new fine jade disaccharide-hydrolysing enzymes
Fine jade trisaccharide and fine jade pentasaccharides, purified that pure fine jade trisaccharide and fine jade pentasaccharides is prepared, total yield is respectively 12% and 11% left side
It is right.
The present invention is using fine jade trisaccharide and fine jade pentasaccharides is prepared in conjunction with acidolysis and β-agarase DagA, with nine sugar of seven sugar of fine jade and fine jade
, as shown in Fig. 2, the product for acting on seven sugar of fine jade is fine jade trisaccharide, fine jade nine is acted on after β-agarase DagA effect for substrate
The product of sugar is fine jade pentasaccharides.
Method of the invention is described further below by specific example.
The preparation of 1 agarose acid hydrolysis solution of embodiment
Using the reaction system of 200mL, substrate is the agarose of 15% (w/v), and solvent is that 2.5% (w/v) one is hydrated lemon
The pH of reaction solution after being cooled to room temperature, is adjusted to by lemon aqueous acid in 90 DEG C of reaction 50min using 20% sodium hydroxide
7.0.By gained acid hydrolysis solution, ten times are diluted, crosses 0.22 μm of water phase filter membrane, carries out liquid phase detection, as shown in Fig. 4 (a), gained agar-agar
The degree of polymerization of oligosaccharides is in 4 or more.
The optimization of embodiment 2:DagA enzyme concentration
Using the optimizing reaction system of 10ml, prepare fine jade trisaccharide and fine jade pentasaccharides be respectively adopted 0.0238,0.0475,
0.0950, the enzyme concentration of 0.1900 and 0.2850U/mL DagA, reaction are boiled ten minutes for 24 hours, and centrifuging and taking supernatant dilutes ten times,
0.22 μm of water phase filter membrane is crossed, is detected to liquid phase.Liquid phase interpretation of result show that 0.1900U/mL DagA is most suitable enzyme concentration
(Fig. 3).
The preparation and purifying of embodiment 3 fine jade trisaccharide and fine jade pentasaccharides
Using the preparation system of 100mL, substrate is acidolysis neutrality liquid glucose obtained in embodiment 1.Using excellent in embodiment 2
Change obtained most suitable enzyme concentration, i.e. 0.1900U/mL DagA reacts for 24 hours in 37 DEG C of shaking baths.With reaction time into
Centrifuging and taking supernatant after 10min is boiled in row, sampling, dilutes ten times, crosses 0.22 μm of water phase filter membrane, is detected to liquid phase, draws figure
4 time graph.After reaction for 24 hours, boil centrifuging and taking supernatant after 10min, carry out P2 column gel chromatography chromatography, obtain by
The high-purity fine jade trisaccharide and high-purity fine jade pentasaccharides of DagA preparation.After freeze-drying, weighing.High performance liquid chromatography inspection is carried out after redissolving
Survey and Mass Spectrometer Method.As a result as shown in figure 5, fine jade trisaccharide and fine jade pentasaccharides that purifying obtains are without other miscellaneous sugars, purity is higher.Mass spectrum knot
Shown in fruit such as Fig. 6 (a) and 6 (b), products therefrom is fine jade trisaccharide and fine jade pentasaccharides.The calculation shows that is using 1.5g agarose as substrate, system
Standby to obtain 0.212g fine jade trisaccharide and 0.206g fine jade pentasaccharides, conversion ratio is respectively 14.13% and 13.73%.
Sequence table
<110>Chinese Marine University
<120>a kind of method for preparing odd number fine jade oligosaccharides
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 309
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
Met Val Asn Arg Arg Asp Leu Ile Lys Trp Ser Ala Val Ala Leu Gly
1 5 10 15
Ala Gly Ala Gly Leu Ala Gly Pro Ala Pro Ala Ala His Ala Ala Asp
20 25 30
Leu Glu Trp Glu Gln Tyr Pro Val Pro Ala Ala Pro Gly Gly Asn Arg
35 40 45
Ser Trp Gln Leu Leu Pro Ser His Ser Asp Asp Phe Asn Tyr Thr Gly
50 55 60
Lys Pro Gln Thr Phe Arg Gly Arg Trp Leu Asp Gln His Lys Asp Gly
65 70 75 80
Trp Ser Gly Pro Ala Asn Ser Leu Tyr Ser Ala Arg His Ser Trp Val
85 90 95
Ala Asp Gly Asn Leu Ile Val Glu Gly Arg Arg Ala Pro Asp Gly Arg
100 105 110
Val Tyr Cys Gly Tyr Val Thr Ser Arg Thr Pro Val Glu Tyr Pro Leu
115 120 125
Tyr Thr Glu Val Leu Met Arg Val Ser Gly Leu Lys Leu Ser Ser Asn
130 135 140
Phe Trp Leu Leu Ser Arg Asp Asp Val Asn Glu Ile Asp Val Ile Glu
145 150 155 160
Cys Tyr Gly Asn Glu Ser Leu His Gly Lys His Met Asn Thr Ala Tyr
165 170 175
His Ile Phe Gln Arg Asn Pro Phe Thr Glu Leu Ala Arg Ser Gln Lys
180 185 190
Gly Tyr Phe Ala Asp Gly Ser Tyr Gly Tyr Asn Gly Glu Thr Gly Gln
195 200 205
Val Phe Gly Asp Gly Ala Gly Gln Pro Leu Leu Arg Asn Gly Phe His
210 215 220
Arg Tyr Gly Val His Trp Ile Ser Ala Thr Glu Phe Asp Phe Tyr Phe
225 230 235 240
Asn Gly Arg Leu Val Arg Arg Leu Asn Arg Ser Asn Asp Leu Arg Asp
245 250 255
Pro Arg Ser Arg Phe Phe Asp Gln Pro Met His Leu Ile Leu Asn Thr
260 265 270
Glu Ser His Gln Trp Arg Val Asp Arg Gly Ile Glu Pro Thr Asp Ala
275 280 285
Glu Leu Ala Asp Pro Ser Ile Asn Asn Ile Tyr Tyr Arg Trp Val Arg
290 295 300
Thr Tyr Gln Ala Val
305
Claims (6)
1. a kind of method for preparing new fine jade trisaccharide and Xin Qiong pentasaccharides, which is characterized in that the method is at the acid by agarose
Reason liquid is digested using β-agarase, and enzymolysis product carries out purifying preparation again；Agarase therein is for from agarose
Prepare the enzyme of six sugar of new fine jade tetrose and Xin Qiong.
2. the method as described in claim 1, which is characterized in that β-agarase amino acid sequence is SEQ ID NO:
1。
3. the method as described in claim 1, which is characterized in that the pickling agent is using citric acid treatment agarose
It prepares.
4. method as claimed in claim 3, which is characterized in that the citric acid is the citrate hydrate that concentration is 2.5%
Acid.
5. method as claimed in claim 4, which is characterized in that the pickling agent is the monohydrate potassium with 2.5%
Agarose is handled, is reacted in 90 DEG C, after being cooled to room temperature, the pH of reaction solution is adjusted to 7.0 preparations.
6. the method as described in claim 1, which is characterized in that the purifying is pure using the progress of column gel chromatography chromatography
Change.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112458126A (en) * | 2020-11-26 | 2021-03-09 | 中国海洋大学 | Preparation method and application of galactose sulfate compound with anti-inflammatory activity |
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|---|---|---|---|---|
| CN1513860A (en) * | 2003-08-04 | 2004-07-21 | 中国海洋大学 | Odd number agar oligosaccharide monomer and its preparation method |
| WO2014077575A1 (en) * | 2012-11-13 | 2014-05-22 | 다인바이오 주식회사 | Agar-derived neoagarooligosaccharide composite composition prepared by daga enzyme reaction and having anti-obesity and anti-diabetes effects |
| CN105255967A (en) * | 2015-11-12 | 2016-01-20 | 福州大学 | Enzymolysis preparation method of new agaro oligosaccharides |
| CN106834379A (en) * | 2016-09-26 | 2017-06-13 | 中国海洋大学 | A kind of preparation method and applications of new fine jade tetrose |
| CN107206018A (en) * | 2014-12-30 | 2017-09-26 | 达因比奥有限公司 | The septicemia of active ingredient or the prevention of septic shock or medicine for treatment composition are used as comprising new fine jade oligosaccharides |
| CN108715841A (en) * | 2018-06-06 | 2018-10-30 | 中国海洋大学 | A kind of immobilised enzymes of production 3,6- inner ether-L- galactolipins |
| CN109890395A (en) * | 2016-10-27 | 2019-06-14 | 达因比奥有限公司 | For preventing, alleviating or the composition containing new fine jade oligosaccharides for the treatment of of arthritis or osteoporosis |
-
2019
- 2019-07-19 CN CN201910658526.1A patent/CN110317846A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1513860A (en) * | 2003-08-04 | 2004-07-21 | 中国海洋大学 | Odd number agar oligosaccharide monomer and its preparation method |
| WO2014077575A1 (en) * | 2012-11-13 | 2014-05-22 | 다인바이오 주식회사 | Agar-derived neoagarooligosaccharide composite composition prepared by daga enzyme reaction and having anti-obesity and anti-diabetes effects |
| CN107206018A (en) * | 2014-12-30 | 2017-09-26 | 达因比奥有限公司 | The septicemia of active ingredient or the prevention of septic shock or medicine for treatment composition are used as comprising new fine jade oligosaccharides |
| CN105255967A (en) * | 2015-11-12 | 2016-01-20 | 福州大学 | Enzymolysis preparation method of new agaro oligosaccharides |
| CN106834379A (en) * | 2016-09-26 | 2017-06-13 | 中国海洋大学 | A kind of preparation method and applications of new fine jade tetrose |
| CN109890395A (en) * | 2016-10-27 | 2019-06-14 | 达因比奥有限公司 | For preventing, alleviating or the composition containing new fine jade oligosaccharides for the treatment of of arthritis or osteoporosis |
| CN108715841A (en) * | 2018-06-06 | 2018-10-30 | 中国海洋大学 | A kind of immobilised enzymes of production 3,6- inner ether-L- galactolipins |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112458126A (en) * | 2020-11-26 | 2021-03-09 | 中国海洋大学 | Preparation method and application of galactose sulfate compound with anti-inflammatory activity |
| CN112458126B (en) * | 2020-11-26 | 2022-09-23 | 中国海洋大学 | Preparation method and application of galactose sulfate compound with anti-inflammatory activity |
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