CN108384822A - A kind of poly- trisaccharide in novel Portugal and its multi-enzyme method preparation method - Google Patents
A kind of poly- trisaccharide in novel Portugal and its multi-enzyme method preparation method Download PDFInfo
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- CN108384822A CN108384822A CN201810148068.2A CN201810148068A CN108384822A CN 108384822 A CN108384822 A CN 108384822A CN 201810148068 A CN201810148068 A CN 201810148068A CN 108384822 A CN108384822 A CN 108384822A
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- 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
- C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
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- 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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Abstract
The invention discloses a kind of poly- trisaccharide in novel Portugal and its multi-enzyme method preparation methods, belong to technical field of food biotechnology.The present invention using derive from a wealth of sources, purchase conveniently, be readily transported, dextrose and saccharose that price is cheap is raw material, when by sucrose phosphorylase and alternately sucrase enzyme is successively catalyzed reaction 46h, substrate conversion efficiency is up to 62.4%, the poly- trisaccharide in novel Portugal with very big potential use is prepared, larger appreciation space and Production Gain are provided for the poly- trisaccharide in the novel Portugal of industrialized production.
Description
Technical field
The present invention relates to a kind of poly- trisaccharide in novel Portugal and its multi-enzyme method preparation methods, are especially with dextrose and saccharose
Raw material, use multi-enzyme method prepare the degree of polymerization for 3 novel glucan method, belong to technical field of food biotechnology.
Background technology
The novel poly- trisaccharide in Portugal is made of glucose unit.Glucose unit by α -1,2- glucopyranoses glycosidic bonds and
α -1,6- glucopyranosides are keyed.Currently, the novel poly- trisaccharide in Portugal is rarely reported both at home and abroad, and the novel Portugal
Poly- trisaccharide has low in calories, human body alimentary canal degradation function, can be used as potential prebiotics for industries such as food, drugs
In, it has a good application prospect.
Sucrose phosphorylase is a member in GH13 families, is the enzyme-specific of catalysis transfer glucoside bond, is catalyzed 1-
Glucosyl group in glucose 1-phosphate1- is transferred to receptor.When using sucrose as donor, when using glucose as receptor, sucrose phosphorylase
Catalysis generates kojibiose, which is glucose unit by α -1, and 2- glucopyranosides are keyed.
Alternately sucrase enzyme is a member in GH70 families, has and turns glycosides activity, is one kind of dextransucrase.When anti-
Polymerisation occurs in the presence of answering substrate there was only sucrose and generates alternan polysaccharide.When there are sucrose and another kind in reaction substrate can
With connect with enzyme monosaccharide or disaccharide when, two kinds of enzymatic sugar generates the oligosaccharide of small molecule.It is anti-that receptor occurs for Alternansucrase
At once α -1 can be formed, 3 glycosidic bonds can also form α -1,6 glycosidic bonds, when receptor is free of α -1, when 6 glycosidic bond, preferentially form
α -1,6 glycosidic bonds.Further, since acceptor molecule is different from sucrose molecule, thus acceptor molecule attack glucityl or glucan base
It terminates the formation of dextran chain afterwards and generates the lower oligosaccharides of the degree of polymerization.
Invention content
The present invention is directed to prepare a kind of poly- trisaccharide in novel Portugal, the technical problem to be solved is that provide with dextrose and saccharose
For raw material, the method that enzymic catalytic reaction prepares the poly- trisaccharide in novel Portugal is carried out using sucrose phosphorylase and alternating sucrase enzyme.
The technical scheme is that using dextrose and saccharose as more enzymatic reactions of raw material.
The condition of more enzymatic reactions is：
(1) kojibiose is prepared：Using 0.1~1M sucrose and 0.1~1M glucose as substrate, 0.1~100U/ml sucrose is added
Phosphorylase, in 30~70 DEG C of shaking baths, 100~300r/min of rotating speed reacts 20~50h, and 1~20min of heating terminates anti-
It answers, generates kojibiose；
(2) the poly- trisaccharide in novel Portugal is prepared：Sequentially added into reaction solution obtained by step (1) 1~30% sucrose and 0.1~
100U/ml replaces sucrase enzyme, and in 30~70 DEG C of shaking baths, 100~300rpm of rotating speed reacts 20~50h, subsequent boiling water
1~20min of bath heating terminates reaction.
In one embodiment of the invention, the enzyme includes sucrose phosphorylase and alternating sucrase enzyme.
In one embodiment of the invention, the sucrose phosphorylase is Bifidobacterium
The sources adolescentis.
In one embodiment of the invention, the alternately sucrase enzyme is the sources Leuconostoc citreum.
In one embodiment of the invention, step (1) prepares kojibiose：With 0.1~1M sucrose and 0.1~1M grapes
Sugar is substrate, and 0.1~0.25U/ml sucrose phosphorylases are added, in 30~50 DEG C of shaking baths, 100~300r/min of rotating speed,
20~36h is reacted, 1~20min of heating terminates reaction, generates kojibiose
In one embodiment of the invention, step (1) prepares kojibiose：Using 0.5M sucrose and 0.5M glucose the bottom of as
The sucrose phosphorylase of 0.25U/ml is added in object, under the conditions of 50 DEG C of shaking baths, rotating speed 150r/min, after reacting 36h, adds
Hot 5min terminates reaction.
In one embodiment of the invention, in step (2), 1 is sequentially added into reaction solution obtained by step (1)~
10% sucrose and 0.1~5U/ml replace sucrase enzyme, in 40~50 DEG C of shaking baths, 100~300rpm of rotating speed, reaction 20~
36h, subsequent boiling water bath heat 1~20min and terminate reaction.
In one embodiment of the invention, step (2) prepares the poly- trisaccharide in novel Portugal：To reaction solution obtained by step (1)
In sequentially add 10% sucrose and 5U/g alternating sucrase enzyme, under the conditions of 40 DEG C of shaking baths, rotating speed 150rpm, react 34h
Afterwards, heating 5min terminates reaction.
In one embodiment of the invention, step (2) prepares the poly- trisaccharide in novel Portugal after reaction, is collected by centrifugation
Clear liquid is to get to the solution containing the poly- trisaccharide in novel Portugal.
The present invention using derive from a wealth of sources, purchase conveniently, be readily transported, dextrose and saccharose that price is cheap is raw material, pass through
When sucrose phosphorylase and alternately sucrase enzyme are successively catalyzed reaction 46h, substrate conversion efficiency is prepared to have and greatly be dived up to 62.4%
In the poly- trisaccharide in novel Portugal of purposes, larger appreciation space and Production Gain are provided for industrial production.
Description of the drawings
Fig. 1 is the HPLC collection of illustrative plates for the sample after reaction that kojibiose is prepared in embodiment 1.
Fig. 2 is the HPLC collection of illustrative plates for the sample after reaction that the poly- trisaccharide in novel Portugal is prepared in embodiment 1.
Specific implementation mode
Sucrose phosphorylase enzyme activity determination method：By the 50mmol/L of the sucrose solution of 1mL 5% and 0.9mL, pH6.7's
Phosphate buffer mixes well, and 10min is preheated at 55 DEG C, and the enzyme solution of 100ul is added, and 3mL DNS are added after reacting 10min, boil
Boiling 7min is cooled down rapidly, and distilled water is added to be settled to 15ml, is surveyed absorbance under 540nm and (is equally grasped using the enzyme solution of inactivation as catalyst
It is used as blank control).
Sucrose phosphorylase enzyme activity defines：Sucrose hydrolysis sugar per minute is generated enzyme amount needed for the fructose of 1 μm of ol to be defined as
The enzyme activity (U) of one unit of sucrose phosphorylase.
Alternately sucrase enzyme enzyme activity determination method：Enzyme solution to be measured is kept the temperature into 30min at 45 DEG C, 0.4mL is taken to be added to
In 3.6mL sucrose NaAc-HAc buffer solutions (50mmol/L, pH5.4), it is 10% to make sucrose ultimate density, is preheated at 40 DEG C
The enzyme solution of 100ul is added in 10 min, and 3mL DNS are added after reacting 30min, boils 7min and cools down rapidly, distilled water is added to be settled to
Absorbance is surveyed under 15ml, 540nm.
Alternately sucrase enzyme enzyme activity definition：Sucrose hydrolysis per minute is generated into enzyme amount needed for the fructose of 1 μm of ol and is defined as sugarcane
The enzyme activity (U) of one unit of saccharophosphorylase.
The HPLC of product is detected：The amount of sucrose, glucose, fructose, kojibiose in end reaction system using HPLC come
It determines.Chromatographic condition is：Agilent 1200HPLC chromatographs, Agilent autosamplers, chromatographic column NH2-50 4E(4.6
Mm × 250mm), Composition distribution is Agilent G1362A；Mobile phase uses the mixed solution of 75% (v/v) acetonitrile and water, stream
Speed is 0.8mL/min, and column temperature is set as 35 DEG C.Using external standard method, determine that corresponding alternan is few according to retention time and peak area
The concentration of sugar.
The calculation formula for turning glycosides efficiency D of the novel poly- trisaccharide in Portugal is：
D (%)=[1- (M1/M2)] * 100%
Wherein D：Sucrose inversion is the molar yield (%) of the poly- trisaccharide in novel Portugal；
M1：The molal quantity (mol) of unreacted kojibiose in enzymatic conversion reaction；
M2：Put into the molal quantity (mol) of the sucrose in reaction solution.
1 multi-enzyme method of embodiment prepares the poly- trisaccharide in novel Portugal
The enzymatic conversion system of 10mL is carried out in the closed container of 50mL.Successively utilize sucrose phosphorylase and alternan
Invertase prepares alternan oligosaccharides, and steps are as follows：
Prepare kojibiose：Using 0.5M sucrose and 0.5M glucose as substrate, 0.25U/mol sucrose phosphorylases are added.
50 DEG C of shaking baths, pH5.4, rotating speed 150r/min react 36h.It heats 5min and terminates reaction, sampling is analyzed by HPLC and produced
Object, calculates the amount of the kojibiose of generation, and chromatographic peak is shown in Fig. 1
Prepare the poly- trisaccharide in novel Portugal：10% sucrose and 5U/g alternating sucrase enzymes are sequentially added in above-mentioned reaction solution,
40 DEG C of shaking baths, pH5.4, rotating speed 150rpm react 34h.Subsequent boiling water bath heating 5min terminates reaction, and reaction solution centrifuges,
Product is analyzed by HPLC.It is 62.4% when calculating conversion ratio after reaction, chromatographic peak is shown in Fig. 2.
Embodiment 2
The poly- trisaccharide in novel Portugal is prepared using 1 identical strategy of embodiment, difference lies in be added 100U/g and replace sucrase enzyme
Prepare the poly- trisaccharide in novel Portugal, by HPLC analyze product, conversion ratio 62.9%, be added 5U/g alternating sucrase enzyme compared with,
Conversion ratio is essentially identical.
Embodiment 3
The poly- trisaccharide in novel Portugal is prepared using 1 identical strategy of embodiment, difference lies in the reactions for the step of preparing kojibiose
Time is adjusted to for 24 hours, analyzes product, conversion ratio 48.8% by HPLC, hence it is evident that be less than the conversion ratio of reaction time 36h.
Embodiment 4
The poly- trisaccharide in novel Portugal is prepared using 1 identical strategy of embodiment, difference lies in will prepare the poly- trisaccharide step in novel Portugal
In pH conditions be adjusted to 6.0, pass through HPLC analyze product, conversion ratio 54.1%.It is catalyzed and synthesized newly in alternately sucrase enzyme
In the reaction process of the poly- trisaccharide in type Portugal, the pH of reaction system can influence the yield of the poly- trisaccharide in the novel Portugal of reaction product.As it can be seen that with
PH5.4 is compared, and when pH6.0, the novel poly- trisaccharide yield in Portugal declines.
Embodiment 5
The poly- trisaccharide in novel Portugal is prepared using 1 identical strategy of embodiment, difference lies in, will prepare the poly- trisaccharide in novel Portugal this
The reaction temperature of step is adjusted to 30 DEG C, and product, conversion ratio 51.6% are analyzed by HPLC, hence it is evident that it is 40 to be less than reaction temperature
DEG C conversion ratio.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not limited to the present invention, any to be familiar with this skill
The people of art can do various change and modification, therefore the protection model of the present invention without departing from the spirit and scope of the present invention
Enclosing be subject to what claims were defined.
Claims (10)
1. a kind of method preparing the poly- trisaccharide in novel Portugal, which is characterized in that using dextrose and saccharose as raw material, utilize sucrose phosphate
Change enzyme and alternately sucrase enzyme progress enzymic catalytic reaction prepares the poly- trisaccharide in novel Portugal.
2. a kind of method preparing the poly- trisaccharide in novel Portugal according to claim 1, which is characterized in that including step：
(1) kojibiose is prepared：Using 0.1~1M sucrose and 0.1~1M glucose as substrate, 0.1~100U/ml sucrose phosphates are added
Change enzyme, in 30~70 DEG C of shaking baths, 100~300r/min of rotating speed reacts 20~50h, and 1~20min of heating terminates reaction, raw
At kojibiose；
(2) the poly- trisaccharide in novel Portugal is prepared：1~30% sucrose and 0.1~100U/ are sequentially added into reaction solution obtained by step (1)
Ml replaces sucrase enzyme, and in 30~70 DEG C of shaking baths, 100~300rpm of rotating speed reacts 20~50h, subsequent boiling water bath heating 1
~20min terminates reaction.
3. a kind of method preparing the poly- trisaccharide in novel Portugal according to claim 1 or 2, which is characterized in that the sucrose phosphorus
Phosphorylase be Bifidobacterium adolescentis, Bacillus megaterium, Leuconostoc citreum,
The sources Leuconostoc pseudomesenteroides or Leuconostoc mesenteroides.
4. a kind of method preparing the poly- trisaccharide in novel Portugal according to claim 1 or 2, which is characterized in that the alternan
Invertase be Leuconostoc citreum, Leuconostoc mesenteroides, Leuconostoc fallax,
The sources Leuconostoc gelidum or Oenococcus oeni.
5. according to a kind of any method preparing the poly- trisaccharide in novel Portugal of claim 2~4, which is characterized in that step (1)
Prepare kojibiose：Using 0.5~1M sucrose and 0.5~1M glucose as substrate, the sucrose phosphorylase of 0.25~20U/ml is added,
Under the conditions of 50~60 DEG C of shaking baths, 150~180r/min of rotating speed, after reacting 36~48h, heating termination reaction.
6. according to a kind of any method preparing the poly- trisaccharide in novel Portugal of claim 2~4, which is characterized in that step (2)
Prepare the poly- trisaccharide in novel Portugal：10~30% sucrose are sequentially added into reaction solution obtained by step (1) and 5~20U/g replaces sugarcane
Carbohydrase, under the conditions of 40~50 DEG C of shaking baths, 150~180rpm of rotating speed, after reacting 34~42h, heating termination reaction.
7. a kind of method preparing the poly- trisaccharide in novel Portugal according to claim 5, which is characterized in that step (1) prepares bent
Disaccharides：Using 0.5M sucrose and 0.5M glucose as substrate, the sucrose phosphorylase of 0.25U/ml is added, 50 DEG C of shaking baths,
Under the conditions of rotating speed 150r/min, after reacting 36h, heating 5min terminates reaction.
8. a kind of method preparing the poly- trisaccharide in novel Portugal according to claim 6, which is characterized in that step (2) prepares new
The poly- trisaccharide in type Portugal：10% sucrose is sequentially added into reaction solution obtained by step (1) and 5U/g replaces sucrase enzyme, in 40 DEG C of water-baths
Under the conditions of shaking table, rotating speed 150rpm, after reacting 34h, heating 5min terminates reaction.
9. according to a kind of any method preparing the poly- trisaccharide in novel Portugal of claim 2~8, which is characterized in that step (2)
It prepares the poly- trisaccharide in novel Portugal after reaction, supernatant is collected by centrifugation to get to the solution containing the poly- trisaccharide in novel Portugal.
10. the poly- trisaccharide in novel Portugal being prepared according to any the method for claim 1~9.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1747659A (en) * | 2003-02-08 | 2006-03-15 | 塞里斯塔控股有限公司 | Process for preparing isomalto-oligosaccharides with elongated chain and low glycemic index |
| US7182954B1 (en) * | 2003-04-04 | 2007-02-27 | The United States Of America, As Represented By The Secretary Of Agriculture | Prebiotic oligosaccharides via alternansucrase acceptor reactions |
| CN101932255A (en) * | 2008-01-31 | 2010-12-29 | 拜尔作物科学股份公司 | The use of alternan as ingredient for certain foodstuffs |
| WO2016075219A1 (en) * | 2014-11-14 | 2016-05-19 | Universiteit Gent | A sucrose phosphorylase for the production of kojibiose |
| US20170218093A1 (en) * | 2014-05-29 | 2017-08-03 | E I Du Pont De Nemours And Company | Enzymatic synthesis of soluble glucan fiber |
| CN107058205A (en) * | 2017-06-05 | 2017-08-18 | 江南大学 | A kind of recombined bacillus subtilis for producing sucrose phosphorylase and its application |
-
2018
- 2018-02-13 CN CN201810148068.2A patent/CN108384822A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1747659A (en) * | 2003-02-08 | 2006-03-15 | 塞里斯塔控股有限公司 | Process for preparing isomalto-oligosaccharides with elongated chain and low glycemic index |
| US7182954B1 (en) * | 2003-04-04 | 2007-02-27 | The United States Of America, As Represented By The Secretary Of Agriculture | Prebiotic oligosaccharides via alternansucrase acceptor reactions |
| CN101932255A (en) * | 2008-01-31 | 2010-12-29 | 拜尔作物科学股份公司 | The use of alternan as ingredient for certain foodstuffs |
| US20170218093A1 (en) * | 2014-05-29 | 2017-08-03 | E I Du Pont De Nemours And Company | Enzymatic synthesis of soluble glucan fiber |
| WO2016075219A1 (en) * | 2014-11-14 | 2016-05-19 | Universiteit Gent | A sucrose phosphorylase for the production of kojibiose |
| CN107058205A (en) * | 2017-06-05 | 2017-08-18 | 江南大学 | A kind of recombined bacillus subtilis for producing sucrose phosphorylase and its application |
Non-Patent Citations (2)
| Title |
|---|
| SATOSHI KITAO ET AL.: "Formation of Kojibiose and Nigerose by Sucrose Phosphorylase", 《BIOSCIENCE BIOTECHONLOGY AND BIOCHEMISTRY》 * |
| 白爱娟等: "新型功能葡聚糖———交替糖的研究与应用进展", 《食品工业科技》 * |
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