CN114517216A - Application of organic solvent in prolonging polymerization degree of soluble amylose synthesized in vitro - Google Patents
Application of organic solvent in prolonging polymerization degree of soluble amylose synthesized in vitro Download PDFInfo
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- CN114517216A CN114517216A CN202011312598.XA CN202011312598A CN114517216A CN 114517216 A CN114517216 A CN 114517216A CN 202011312598 A CN202011312598 A CN 202011312598A CN 114517216 A CN114517216 A CN 114517216A
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- 229920000856 Amylose Polymers 0.000 title claims abstract description 86
- 238000000338 in vitro Methods 0.000 title claims abstract description 34
- 238000006116 polymerization reaction Methods 0.000 title claims abstract description 32
- 239000003960 organic solvent Substances 0.000 title claims abstract description 14
- 229920002472 Starch Polymers 0.000 claims abstract description 74
- 238000006243 chemical reaction Methods 0.000 claims abstract description 74
- 239000008107 starch Substances 0.000 claims abstract description 74
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 68
- HXXFSFRBOHSIMQ-VFUOTHLCSA-N alpha-D-glucose 1-phosphate Chemical compound OC[C@H]1O[C@H](OP(O)(O)=O)[C@H](O)[C@@H](O)[C@@H]1O HXXFSFRBOHSIMQ-VFUOTHLCSA-N 0.000 claims abstract description 57
- 229950010772 glucose-1-phosphate Drugs 0.000 claims abstract description 57
- 108010073135 Phosphorylases Proteins 0.000 claims abstract description 35
- 102000009097 Phosphorylases Human genes 0.000 claims abstract description 35
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims abstract description 28
- 102000004190 Enzymes Human genes 0.000 claims abstract description 25
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- 238000006555 catalytic reaction Methods 0.000 claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 18
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000000758 substrate Substances 0.000 claims abstract description 11
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- UYQJCPNSAVWAFU-UHFFFAOYSA-N malto-tetraose Natural products OC1C(O)C(OC(C(O)CO)C(O)C(O)C=O)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(O)C(CO)O2)O)C(CO)O1 UYQJCPNSAVWAFU-UHFFFAOYSA-N 0.000 claims abstract description 9
- LUEWUZLMQUOBSB-OUBHKODOSA-N maltotetraose Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@H](CO)O[C@@H](O[C@@H]2[C@@H](O[C@@H](O[C@@H]3[C@@H](O[C@@H](O)[C@H](O)[C@H]3O)CO)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O LUEWUZLMQUOBSB-OUBHKODOSA-N 0.000 claims abstract description 9
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- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 7
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- 229910001629 magnesium chloride Inorganic materials 0.000 description 7
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- 229930006000 Sucrose Natural products 0.000 description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 4
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- 229910052698 phosphorus Inorganic materials 0.000 description 3
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- -1 EC2.4.1.7 Proteins 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- BNABBHGYYMZMOA-AHIHXIOASA-N alpha-maltoheptaose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)O[C@H](O[C@@H]2[C@H](O[C@H](O[C@@H]3[C@H](O[C@H](O[C@@H]4[C@H](O[C@H](O[C@@H]5[C@H](O[C@H](O[C@@H]6[C@H](O[C@H](O)[C@H](O)[C@H]6O)CO)[C@H](O)[C@H]5O)CO)[C@H](O)[C@H]4O)CO)[C@H](O)[C@H]3O)CO)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O BNABBHGYYMZMOA-AHIHXIOASA-N 0.000 description 2
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- 239000011630 iodine Substances 0.000 description 2
- TWNIBLMWSKIRAT-VFUOTHLCSA-N levoglucosan Chemical group O[C@@H]1[C@@H](O)[C@H](O)[C@H]2CO[C@@H]1O2 TWNIBLMWSKIRAT-VFUOTHLCSA-N 0.000 description 2
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- DBTMGCOVALSLOR-UHFFFAOYSA-N 32-alpha-galactosyl-3-alpha-galactosyl-galactose Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(OC2C(C(CO)OC(O)C2O)O)OC(CO)C1O DBTMGCOVALSLOR-UHFFFAOYSA-N 0.000 description 1
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- RXVWSYJTUUKTEA-UHFFFAOYSA-N D-maltotriose Natural products OC1C(O)C(OC(C(O)CO)C(O)C(O)C=O)OC(CO)C1OC1C(O)C(O)C(O)C(CO)O1 RXVWSYJTUUKTEA-UHFFFAOYSA-N 0.000 description 1
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- FTNIPWXXIGNQQF-UHFFFAOYSA-N UNPD130147 Natural products OC1C(O)C(O)C(CO)OC1OC1C(CO)OC(OC2C(OC(OC3C(OC(OC4C(OC(O)C(O)C4O)CO)C(O)C3O)CO)C(O)C2O)CO)C(O)C1O FTNIPWXXIGNQQF-UHFFFAOYSA-N 0.000 description 1
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- C12P19/00—Preparation of compounds containing saccharide radicals
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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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Abstract
The invention discloses an application of an organic solvent in the in vitro synthesis of soluble amylose to prolong the polymerization degree of the soluble amylose. An in-vitro synthesis system for synthesizing the soluble amylose by in-vitro enzyme catalysis with glucose-1-phosphate as a substrate and maltotetraose as a primer and the application of an organic solvent in improving the polymerization degree of the soluble amylose in the system are constructed. In the system for synthesizing the soluble amylose by the in vitro enzyme catalysis, firstly, the production conditions of the soluble amylose are optimized, wherein the production conditions comprise the concentration of a substrate glucose-1-phosphate, the addition amount of an organic solvent dimethyl sulfoxide (DMSO), the dosage of alpha-starch phosphorylase and the like. In addition, the potential and application of organic solvents such as dimethyl amide (DMF) and Tetrahydrofuran (THF) except dimethyl sulfoxide in the in-vitro synthesis of the soluble amylose for prolonging the polymerization degree of the soluble amylose are also analyzed. Compared with the currently reported amylose production method, the method has the advantages of high conversion rate, low production cost and the like, and is suitable for large-scale production of soluble amylose.
Description
Technical Field
The invention belongs to the field of biological manufacturing, and particularly relates to a method for preparing soluble starch by using glucose-1-phosphate as a raw material through an enzyme method, and an application of an organic solvent in-vitro synthesis of soluble amylose for prolonging the polymerization degree of the soluble amylose.
Background
Starch is an important carbohydrate which is ingested by humans from the natural world and has the general molecular formula (C)6H1205)n，(C6H1205) Represents one glucose equivalent and n is the number of glucose monomers. Starch is classified into two types, amylose and amylose. Amylose is a linear macromolecule, is formed by connecting anhydroglucose units through alpha-1, 4 glycosidic bonds, and is in a right-handed helical structure;amylopectin is a highly branched glucose polymer, which is formed by connecting anhydroglucose via alpha-1, 6 glycosidic bonds and alpha-1, 4 glycosidic bonds, and the branching position of the amylopectin is alpha-1, 6 glycosidic bonds. Among them, amylose is widely used in the food industry, textile industry, plastic industry, etc. due to its unique physicochemical properties.
Amylose is usually coiled into a helix by the interaction of intramolecular hydrogen bonds, and each helix is composed of 6 glucose monomers. When the amylose meets iodine, iodine molecules enter the helical structure of the amylose molecules to form a starch-iodine compound, and the compound has a maximum light absorption value at 620-680 nm. The branches of the amylopectin can also form a spiral structure, but the amylopectin presents purple red when encountering iodine due to the short length and the small number of corresponding molecules of complex iodine, and presents the maximum light absorption value at 530-550 nm.
The method for obtaining amylose comprises the following steps: the amylose and the amylopectin are separated through different solubilities in a solution, the amylose and the amylopectin are separated through the molecular structure characteristics, the plant capable of producing the high amylose is obtained by improving the variety of crops, and the natural starch is subjected to debranching enzyme enzymolysis. The alpha-1, 6 glycosidic bond can be hydrolyzed specifically by debranching enzyme enzymolysis, so that the lateral branches of the whole starch chain are cut off, and the hydrolysis yield is relatively high, which is considered to be the most effective method for obtaining high amylose starch at present.
With the development of in vitro synthetic biotechnology, there have been scientists who have begun attempting to enzymatically synthesize amylose. The in vitro synthesis of artificial Starch from Potato-derived Starch phosphorylase (Potato phosphorylase, EC 2.4.1.1, PGP) was reported by Science News Letter in 1941 (Synthetic Starch by Enzyme action on Science News Letter,1941,40 (2): 26). The method for synthesizing amylose in vitro is established on the basis of the characteristic that starch phosphorylase (alpha GP) can catalyze reversible phosphorylation of alpha-1, 4 glucan. In 1986, The American scientist Whitesids team reported an in vitro synthesis route for amylose synthesis by Sucrose phosphorylase (Sucrose phosphorylase, EC2.4.1.7, SP), Potato-derived starch phosphorylase (Potato phosphorylase, EC 2.4.1.1, PGP) using Sucrose as a substrate and maltoheptaose as a primer (Waldmann, H., et al. (1986) The enzymic digestion of Sucrose in The synthesis of and derivatives of amylose 157: c4-c 7). In addition, Kazutoshi, F.et al, (2003) Bioengineering and application of novel glucose polymers and Biotransformation 21(4/5): 167. about.172; Yang, M.et al, (2005) relational effect of amino acids and derivatives 71(9): 5433. about.39; Yang. about.1632. about.11. about.23. about., ltd., osaka (jp); sanwa Kosan Kabushiki Kaisha, Nara (JP), assign (2001) Biodegradable articles isolated from enzymationally synthesized epoxy. US patent No. 7759316B 2). The enzymes for in vitro amylose synthesis include Sucrose phosphorylase (EC 2.4.1.7, SP), Cellobiose phosphorylase (Cellobiose phosphorylase, EC2.4.1.20, CBP), and starch phosphorylase (alpha-glucan phosphorylase, EC 2.4.1.1, alpha GP). The amylose synthesized by the in vitro synthesis route can realize the artificial regulation and control of the Degree of Polymerization (DP) of the starch by regulating the ratio of the substrate to the primer, and the obtained amylose has narrow molecular weight distribution.
However, Nikuni scientists, Osaka university, Japan, as early as 1983, found that amylose can be synthesized by adding endogenous primers to Potato-derived starch phosphorylase (PGP) solution without the need for adding exogenous primers (Kamogawa, A, et al (1968) starch α -glucan phosphorylase: crystallization, amino acid composition and enzymatic reaction in the absence of the added primer, the Journal of Biochemistry 63(3):361 369). In addition, in another report in 2014, it was found that in an in vitro synthesis system using Sucrose as a substrate, Sucrose Phosphorylase (SP) and potato-derived alpha-starch phosphorylase (PGP), the degree of polymerization of Amylose obtained was significantly lower than the ratio of substrate to primer (Qi, P., et al (2014) One-Point Enzyme Conversion of Sucrose to Synthetic Enzyme by using Enzyme cassettes ACS Catalysis 4 (5: 1311) -1317.). In addition, when barley-derived starch phosphorylase (Barly phosphorylase, EC 2.4.1.1, BGP) was analyzed for its enzymatic properties, it was found that no primer was involved in the in vitro starch synthesis reaction (Cuesta-Seijo, J., A., et al (2017) Functional and structural characterization of plastic stable phosphorylase and produced rod end mutation. P.O. One 12(4): e 7501488).
In addition to the fact that the Degree of Polymerization (DP) of amylose does not correspond to the ratio of substrate to primer, it was found in experiments that amylose obtained by the in vitro synthesis system of alpha-starch phosphorylase exists in a predominantly water-insoluble precipitate state.
Therefore, it is highly desired to develop a method for preparing soluble amylose.
Disclosure of Invention
The technical problem to be solved by the invention is to provide an in vitro synthesis method of soluble amylose, which takes glucose-1-phosphate as a substrate and produces the artificial amylose through the catalysis of an in vitro enzyme reaction system.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the invention provides a method for preparing soluble amylose by using an in vitro enzyme catalytic reaction, which is characterized in that glucose-1-phosphate is used as a substrate, maltotetraose is used as a primer, alpha-starch phosphorylase (alpha-glucan phosphorylase, EC 2.4.1.1, Tk alpha GP) from Thermococcus kodakarensis KOD1 is added, and the enzyme catalytic reaction is carried out in a system with dimethyl sulfoxide (DMSO).
Preferably, the concentration of glucose-1-phosphate in the enzyme-catalyzed reaction system is 50 to 500mM, more preferably 100 to 400mM, still more preferably 150 to 300mM, and most preferably 200 mM.
Preferably, buffer solution and magnesium ions should be added into the reaction system.
It will be understood by those skilled in the art that various buffers can be used in the present invention, such as HEPES buffer, Tris-HCl buffer, MOPS buffer, citrate buffer, etc., preferably, the buffer is HEPES buffer. Preferably, the pH of the buffer is 5.0-8.0, more preferably 6.0-7.5, and most preferably 7.0. Preferably, the concentration of the buffer in the reaction system is 10 to 500mM, more preferably 20 to 150mM, still more preferably 50 to 120mM, and most preferably 100 mM.
It will be appreciated by those skilled in the art that various magnesium ionic compounds may be used in the present invention, such as magnesium chloride, magnesium sulfate, and the like, preferably, the magnesium ion is magnesium chloride.
It will be appreciated by those skilled in the art that various maltopolysaccharides or maltodextrins can be used as primers for the present invention, such as maltotriose, maltotetraose, maltopentaose, maltoheptaose, and the like, with the preferred primer being maltotetraose.
In a preferred embodiment, glucose-1-phosphate is used as a substrate, maltotetraose is used as a primer, and an in vitro reaction system is constructed by adding α -starch phosphorylase (EC 2.4.1.1, Tk α GP) derived from Thermococcus kodakarensis to carry out an enzymatic reaction.
In the present invention, glucose-1-phosphate and maltotetraose may be added in any proportion to the enzyme-catalyzed reaction system.
Preferably, the ratio of the added glucose-1-phosphate to the added maltotetraose is 10,000-50,000: 1.
Preferably, the amount of T.kodakarensis α -starch phosphorylase (Tk α GP) used in the reaction system is 0.1 to 50U/mL, more preferably 0.5 to 20U/mL, still more preferably 1 to 10U/mL, and most preferably 5U/mL.
In the present invention, a variety of sources of α -starch phosphorylase can be used. For example, the α -starch phosphorylase may be derived from Thermococcus kodakarensis (Thermococcus kodakarensis), barley (Hordeum vulgare L.), Thermotoga maritima (Thermotoga maritima), Clostridium thermocellum (Clostridium thermocellum), Thermus thermophilus (Thermus thermophilus), Escherichia coli (Escherichia coli), or the like, and preferably, the α -starch phosphorylase is derived from Thermococcus kodakarensis.
Since inorganic phosphorus is gradually accumulated during the reaction, magnesium ions are added to form a phosphatase precipitate to remove the accumulation of inorganic phosphorus.
Since amylose is insoluble in water, the addition of dimethyl sulfoxide (DMSO) to the reaction system increases the degree of polymerization of amylose by increasing the solubility of amylose. Preferably, dimethyl sulfoxide (DMSO) is present in the enzyme-catalyzed reaction system in a proportion of 0.1% to 50% (v/v), more preferably 2% to 40% (v/v), even more preferably 5% to 20% (v/v), and most preferably 5% (v/v).
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
in an in vitro synthesis reaction system, glucose-1-phosphate is used as a raw material, alpha-starch phosphorylase is converted into amylose, dimethyl sulfoxide is added to improve the dissolution of the amylose, and the polymer of the amylose is improved by process optimization. The reaction system eliminates the accumulation of phosphorus by adding magnesium ions, obviously improves the conversion rate, and greatly reduces the production cost of amylose with high yield. The method has the advantages of simplicity, high utilization rate of raw materials, high yield of the amylose, environmental optimization and the like, and can realize large-scale production of the amylose.
Drawings
FIG. 1 is a schematic diagram of an in vitro enzymatic pathway for the conversion of glucose-1-phosphate to amylose; wherein: α GP is α -starch phosphorylase.
FIG. 2 shows SDS-PAGE detecting α GP; wherein: m is Marker and Tk alpha GP is purified by heat treatment.
FIG. 3 is a graph showing the in vitro enzymatic synthesis of soluble amylose from 100mM glucose-1-phosphate under initial conditions.
FIG. 4 is a curve of the optimized course of the reaction for synthesizing soluble amylose by in vitro enzyme catalysis of glucose-1-phosphate with different concentrations. Wherein FIG. 4a is a graph showing the reaction progress of synthesizing soluble amylose from 50mM glucose-1-phosphate; FIG. 4b is a graph showing the reaction progress of synthesizing soluble amylose from 100mM glucose-1-phosphate; FIG. 4c is a graph showing the reaction progress of synthesizing soluble amylose from 200mM glucose-1-phosphate; FIG. 4d is a graph showing the reaction progress of synthesizing soluble amylose from 500mM glucose-1-phosphate.
FIG. 5 is a graph showing the reaction progress for the synthesis of soluble amylose at different DMSO concentrations. Wherein FIG. 5a is a graph showing the reaction progress of glucose-1-phosphate to synthesize soluble amylose without adding DMSO; FIG. 5b is a graph showing the reaction progress of glucose-1-phosphate to synthesize soluble amylose at a DMSO concentration of 5% (v/v); FIG. 5c is a graph showing the reaction progress of glucose-1-phosphate to synthesize soluble amylose at a DMSO concentration of 10% (v/v); FIG. 5d is a graph showing the reaction progress of glucose-1-phosphate to synthesize soluble amylose at a DMSO concentration of 20% (v/v).
FIG. 6 is a graph showing the reaction progress for the synthesis of soluble amylose at different Tk α GP enzyme dosages. Wherein FIG. 6a is a reaction progress curve for synthesizing soluble amylose from glucose-1-phosphate at a dosage of 2U/mL Tk α GP enzyme; FIG. 6b is a graph showing the reaction progress of glucose-1-phosphate to soluble amylose at a dosage of 5U/mL Tk α GP enzyme; FIG. 6c is a graph showing the reaction progress of glucose-1-phosphate to soluble amylose at a dosage of 10U/mL Tk α GP enzyme.
FIG. 7 is a graph showing the progress of the reaction of synthesizing amylose from 200mM glucose-1-phosphate in vitro with an enzyme in other organic solvents. Wherein FIG. 7a is a graph showing the course of the reaction for synthesizing amylose from 200mM glucose-1-phosphate catalyzed by an in vitro enzyme without the addition of an organic solvent; FIG. 7b is a graph showing the course of the reaction of synthesizing amylose from 200mM glucose-1-phosphate catalyzed by an enzyme in vitro with the addition of Dimethylformamide (DMF) at a final concentration of 5% (v/v); FIG. 7c is a graph showing the course of the reaction of synthesizing amylose by in vitro enzymatic catalysis of 200mM glucose-1-phosphate with the addition of Tetrahydrofuran (THF) at a final concentration of 5% (v/v).
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and substitutions are intended to be within the scope of the invention.
The following materials were used in the examples of the invention:
glucose-1-phosphate, product of Sigma, product number: g9753;
dimethylsulfoxide (DMSO), product of Sigma, product number: d2650;
dimethylamide (DMF), product from Sigma, product number: 189979;
tetrahydrofuran (THF), product of Sigma, product number: 401757, respectively;
pET20b vector, Novagen, Madison, WI;
coli expression strain BL21(DE3), Invitrogen, Carlsbad, CA;
the enzyme of the present invention can be obtained by prokaryotic expression according to a genetic engineering method.
Example 1: in vitro enzymatic conversion of glucose-1-phosphate to soluble amylose
The catalytic pathway for the conversion of glucose-1-phosphate to amylose by an in vitro enzymatic catalytic system is shown in FIG. 1. Among them, alpha-starch phosphorylase is the key of the catalytic system.
In this example, the alpha-starch phosphorylase is derived from Thermococcus kodakarensis (Thermococcus kodakarensis) and the gene is numbered TK1406 on KEGG. The gene can be obtained by PCR amplification or gene synthesis, and is cloned into pET20b vector (Novagen, Madison, Wis.) by the method of Simple Cloning (You, C., et al. (2012) Simple Cloning via direct transformation of PCR product (DNA Multi) to Escherichia coli and Bacillus subtilis applied Environmental Microbiology, 78(5):1593 and 1955), so as to obtain the corresponding expression vector pET20b-Tk alpha GP. Then, the plasmid was transformed into E.coli expression strain BL21(DE3) (Invitrogen, Carlsbad, Calif.) and protein expression and purification were carried out, and the results of protein purification are shown in FIG. 2.
Then, the reaction system contained 100mM HEPES buffer (pH 7.0), 50mM glucose-1-phosphate, 5mM magnesium chloride, 5U/mL α -starch phosphorylase, and 5 μ M maltotetraose, and catalyzed at 50 ℃ for 60 hours.
The concentration of soluble amylose was measured by a total starch amount measurement kit (K-TSTA, Megazyme). The reaction sample was centrifuged at 10,000 Xg for 10min, 20. mu.L of the supernatant was added to 300. mu.L of 100mM acetate buffer (pH 5.0), 10. mu.L of alpha-amylase was added, and the mixture was subjected to boiling water bath for 6 min. Adding 10 μ L amyloglucosidase hydrolase, reacting at 50 deg.C for 30min, and determining glucose content.
As shown in FIG. 3, the amylose concentration gradually increased. The final amylose concentration was 26mM glucose equivalents with a conversion of 52% relative to starch glucose-1-phosphate (50mM, i.e., 50mM glucose equivalents, 1 molecule glucose-1-phosphate to 1 molecule glucose equivalent starch).
Example 2: by optimizing the concentration of glucose-1-phosphate, the yield and the polymerization degree of the soluble amylose are improved
The reaction system contained 100mM HEPES buffer (pH 7.0), 200mM magnesium chloride, 5U/mL of α -starch phosphorylase, 50-00mM of glucose-1-phosphate, and 10% (v/v) of dimethyl sulfoxide, and the reaction was catalyzed at 50 ℃ for 60 hours, and the preparation and purification of α -starch phosphorylase and the detection of soluble amylose were the same as in example 1.
It was determined that at 50mM glucose-1-phosphate, the final concentration of soluble starch after 60h of reaction was 48mM, and the conversion to glucose-1-phosphate was 96%, at which time the polymerization of soluble starch was 140 (FIG. 4 a); 100mM glucose-1-phosphate, the final concentration of soluble starch after 48h of reaction was 85mM, and the conversion to glucose-1-phosphate was 85%, at which time the polymerization of soluble starch was 270 (FIG. 4 b); 200mM glucose-1-phosphate, the final concentration of soluble starch after 48h of the reaction was 166mM, the conversion to glucose-1-phosphate was 83%, and the polymerization of soluble starch was 439 (FIG. 4 c); at 500mM glucose-1-phosphate, the final concentration of soluble starch after 48h of reaction was 121mM, and the conversion to glucose-1-phosphate was 24%, at which time the polymerization of soluble starch was 135% (FIG. 4 d).
Example 3: the yield and the polymerization degree of the soluble amylose are improved by adding an organic solvent, namely dimethyl sulfoxide (DMSO), for promoting the dissolution of the starch
Amylose is insoluble in water, and the degree of polymerization of amylose can be increased by adding dimethyl sulfoxide (DMSO) to the in vitro enzymatic reaction system to increase the solubility of amylose.
Preparation and purification of alpha-starch phosphorylase the same as in example 1.
The reaction system contained 100mM HEPES buffer (pH 7.0), 5mM magnesium chloride, 5U/mL of α -starch phosphorylase, 50mM glucose-1-phosphate, and 0-20% (v/v) dimethyl sulfoxide, and the reaction was catalyzed at 50 ℃ for 60 hours, and the preparation and purification of α -starch phosphorylase and the detection of soluble amylose were the same as in example 1.
When DMSO was not added to the reaction system, the final concentration of the soluble starch after 48 hours of the reaction was 148mM, and the conversion rate of glucose-1-phosphate was 74%, at which time the polymerization of the soluble starch was 237 (FIG. 5 a); 5% (v/v) DMSO, the final concentrations of soluble starch after 24h and 36h of reaction were 123mM and 106mM, respectively, the conversion rates for glucose-1-phosphate were 62% and 53%, respectively, and the polymerization rate for soluble starch after 36h was 378 (FIG. 5 b); 10% (v/v) DMSO, the final concentration of soluble starch after 48h of reaction was 171mM, the conversion to glucose-1-phosphate was 86%, and the polymerization of soluble starch was 432 (FIG. 5 c); at 20% (v/v) DMSO, the final concentration of soluble starch after 24h of reaction was 61mM, and the conversion to glucose-1-phosphate was 31%, at which time the polymerization of soluble starch was 114 (FIG. 5 d).
Example 4: by optimizing the enzyme dosage, the yield and the polymerization degree of the soluble amylose are further improved
The reaction system contained 100mM HEPES buffer (pH 7.0), 200mM magnesium chloride, 2-10U/mL of alpha-starch phosphorylase, 200mM glucose-1-phosphate, and 10% (v/v) dimethyl sulfoxide, and the reaction was catalyzed at 50 ℃ for 60 hours, and the preparation and purification of alpha-starch phosphorylase and the detection of soluble amylose were the same as in example 1.
When the dosage of the alpha-starch phosphorylase is 2U/mL, the final concentration of the soluble starch is 108mM after 48 hours of reaction, the conversion rate of the soluble starch to glucose-1-phosphate is 54 percent, and the polymerization degree of the starch is 283 (FIG. 6 a); when the amount of alpha-starch phosphorylase was 5U/mL, the final concentration of soluble starch after reaction 48 was 164mM, and the conversion rate to glucose-1-phosphate was 82%, at which point the degree of polymerization of starch was 408 (FIG. 6 b); when the amount of alpha-starch phosphorylase was 10U/mL, the final concentration of soluble starch after reaction 12 was 172mM, and the conversion rate to glucose-1-phosphate was 86%, at which point the degree of polymerization of starch was 350 (FIG. 6 c).
Example 5: application of other organic solvents in prolonging polymerization degree of soluble amylose
The reaction system contained 100mM HEPES buffer (pH 7.0), 200mM magnesium chloride, 5U/mL of α -starch phosphorylase, 200mM glucose-1-phosphate, 5% (v/v) Dimethylformamide (DMF) or 5% (v/v) Tetrahydrofuran (THF), and the reaction was catalyzed at 50 ℃ for 60 hours, and the preparation and purification of α -starch phosphorylase and the detection of soluble amylose were the same as in example 1.
It was found that when no organic solvent was added to the reaction system, the final concentration of soluble starch was 153mM and the conversion of glucose-1-phosphate was 77% after the reaction for 60 hours, and the degree of polymerization of starch was 236 (FIG. 7 a).
When 5% (v/v) of Dimethylformamide (DMF) was added to the reaction system, the concentrations of soluble starch were 123mM and 140mM, respectively, and the polymerization degrees were 259 and 173, respectively, after 24 hours and 48 hours of the reaction (FIG. 7b), and the polymerization degree of soluble starch was increased by 10% as compared with that of the control group (FIG. 7 a).
At 5% (v/v) Tetrahydrofuran (THF), the final concentration of soluble starch was 160mM and the degree of polymerization was 354 after 48 hours of reaction (FIG. 7c), and the degree of polymerization of soluble starch was increased by 50% as compared with the control (FIG. 7 a).
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. The application of an organic solvent in prolonging polymerization of soluble amylose prepared by in vitro enzyme catalysis is characterized in that glucose-1-phosphate is used as a substrate, maltotetraose is used as a primer, dimethyl sulfoxide (DMSO) is added into the system, and an in vitro enzyme catalysis reaction system is established by alpha-starch phosphorylase to carry out enzyme catalysis reaction.
2. The method of claim 1, wherein a buffer and magnesium ions are further added to the enzyme-catalyzed reaction system.
3. The method according to any of claims 1 or 2, wherein the concentration of glucose-1-phosphate in the enzyme-catalyzed reaction system is 10 to 1000mM, more preferably 50 to 500mM, even more preferably 50 to 300mM, and most preferably 200 mM.
4. The method according to any of claims 1 to 3, wherein the enzyme-catalyzed reaction system is added in a ratio of dimethyl sulfoxide (DMSO) of 1% to 50% (v/v), more preferably 5% to 20% (v/v), even more preferably 1% to 50% (v/v), and most preferably 10% (v/v).
5. The method according to any one of claims 1 to 4, wherein the amount of the α -starch phosphorylase used in the enzyme-catalyzed reaction system is 1 to 20U/mL, more preferably 2 to 20U/mL, still more preferably 5 to 10U/mL, and most preferably 10U/mL.
6. The process according to claims 1 to 5, wherein the reaction temperature of the enzymatic reaction system is 10 to 95 ℃, more preferably 20 to 80 ℃, more preferably 30 to 60 ℃, and most preferably 50 ℃.
7. The method according to any of claims 1 to 6, wherein the reaction time of the enzyme-catalyzed reaction system is 0.5 to 150 hours, more preferably 1 to 90 hours, even more preferably 12 to 60 hours, and most preferably 48 hours.
8. A method according to any one of claims 1 to 7, wherein the alpha-starch phosphorylase is derived from Thermococcus kodakarensis (Thermococcus kodakarensis), barley (Barly), Thermotoga maritima (Thermotoga maritima), Clostridium thermocellum (Clostridium thermocellum), Thermus thermophilus (Thermus thermophilus), Escherichia coli (Escherichia coli) or the like, or has an amino acid sequence which is at least 60%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to the starch phosphorylase from the above source; preferably, the alpha-starch phosphorylase is derived from, or has an amino acid sequence at least 60%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to, an alpha-starch phosphorylase from, a species of Pyrococcus kodakarensis.
9. The method according to any one of claims 1 to 8, wherein the organic solvent used in the enzyme-catalyzed reaction system is selected from the group consisting of dimethyl sulfoxide (DMSO), dimethyl amide (DMF), and Tetrahydrofuran (THF). And various concentrations of Dimethylamide (DMF), Tetrahydrofuran (THF) can be used in the present invention.
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| CN1535317A (en) * | 2001-05-28 | 2004-10-06 | 江崎格力高株式会社 | Production method and preparation method of glucans |
| CN1894418A (en) * | 2003-12-12 | 2007-01-10 | 江崎格力高株式会社 | Method of converting beta-1,4-glucan to alpha-glucan |
| JP2008280466A (en) * | 2007-05-11 | 2008-11-20 | Osaka Prefecture Univ | Non-digestible amylose particle, its preparation, food and drink, drug, and quasi drug containing it |
| JP2009270012A (en) * | 2008-05-07 | 2009-11-19 | Kagoshima Univ | Amylose synthesis in dimethyl sulfoxide |
| CN102533905A (en) * | 2012-02-23 | 2012-07-04 | 江南大学 | Enzymatic synthesis method of straight-chain dextrin |
| US20190322990A1 (en) * | 2018-04-23 | 2019-10-24 | Danisco Us Inc | Synthesis of glucan comprising beta-1,3 glycosidic linkages with phosphorylase enzymes |
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1535317A (en) * | 2001-05-28 | 2004-10-06 | 江崎格力高株式会社 | Production method and preparation method of glucans |
| CN1894418A (en) * | 2003-12-12 | 2007-01-10 | 江崎格力高株式会社 | Method of converting beta-1,4-glucan to alpha-glucan |
| JP2008280466A (en) * | 2007-05-11 | 2008-11-20 | Osaka Prefecture Univ | Non-digestible amylose particle, its preparation, food and drink, drug, and quasi drug containing it |
| JP2009270012A (en) * | 2008-05-07 | 2009-11-19 | Kagoshima Univ | Amylose synthesis in dimethyl sulfoxide |
| CN102533905A (en) * | 2012-02-23 | 2012-07-04 | 江南大学 | Enzymatic synthesis method of straight-chain dextrin |
| US20190322990A1 (en) * | 2018-04-23 | 2019-10-24 | Danisco Us Inc | Synthesis of glucan comprising beta-1,3 glycosidic linkages with phosphorylase enzymes |
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