CN114686543B - Method for precisely regulating and controlling enzymolysis reaction of alpha-glucan and directionally extracting specific molecular weight product and application thereof - Google Patents
Method for precisely regulating and controlling enzymolysis reaction of alpha-glucan and directionally extracting specific molecular weight product and application thereof Download PDFInfo
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- 229920000310 Alpha glucan Polymers 0.000 title claims abstract description 80
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 36
- 230000001276 controlling effect Effects 0.000 title claims abstract description 14
- 230000001105 regulatory effect Effects 0.000 title claims abstract description 13
- 239000012266 salt solution Substances 0.000 claims abstract description 24
- 108090000790 Enzymes Proteins 0.000 claims abstract description 19
- 102000004190 Enzymes Human genes 0.000 claims abstract description 19
- 238000011084 recovery Methods 0.000 claims abstract description 13
- 239000000203 mixture Substances 0.000 claims abstract description 6
- 239000002202 Polyethylene glycol Substances 0.000 claims description 75
- 229920001223 polyethylene glycol Polymers 0.000 claims description 75
- 239000000243 solution Substances 0.000 claims description 73
- 238000000855 fermentation Methods 0.000 claims description 38
- 230000004151 fermentation Effects 0.000 claims description 38
- 239000011259 mixed solution Substances 0.000 claims description 36
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 claims description 27
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 26
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 20
- 239000007788 liquid Substances 0.000 claims description 16
- 239000000047 product Substances 0.000 claims description 16
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 13
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 13
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 13
- 238000000605 extraction Methods 0.000 claims description 13
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 13
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 13
- 239000012086 standard solution Substances 0.000 claims description 12
- 241000894006 Bacteria Species 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 8
- 241000235058 Komagataella pastoris Species 0.000 claims description 7
- 238000010612 desalination reaction Methods 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 5
- 238000000108 ultra-filtration Methods 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 230000000694 effects Effects 0.000 claims description 3
- 230000007071 enzymatic hydrolysis Effects 0.000 claims description 3
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims description 3
- 235000013305 food Nutrition 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 239000000758 substrate Substances 0.000 claims description 3
- 239000004695 Polyether sulfone Substances 0.000 claims description 2
- 229920006393 polyether sulfone Polymers 0.000 claims description 2
- 239000011347 resin Substances 0.000 claims description 2
- 229920005989 resin Polymers 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims 1
- 239000000825 pharmaceutical preparation Substances 0.000 claims 1
- 229940127557 pharmaceutical product Drugs 0.000 claims 1
- 229920000642 polymer Polymers 0.000 abstract description 13
- 238000000926 separation method Methods 0.000 abstract description 7
- 229920001503 Glucan Polymers 0.000 abstract description 6
- 102000004169 proteins and genes Human genes 0.000 abstract description 4
- 108090000623 proteins and genes Proteins 0.000 abstract description 4
- 238000005192 partition Methods 0.000 abstract description 3
- 238000005191 phase separation Methods 0.000 abstract description 3
- 230000015572 biosynthetic process Effects 0.000 abstract description 2
- 239000012071 phase Substances 0.000 description 54
- 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 12
- 229930006000 Sucrose Natural products 0.000 description 12
- 239000005720 sucrose Substances 0.000 description 12
- 238000009826 distribution Methods 0.000 description 10
- 230000007062 hydrolysis Effects 0.000 description 8
- 238000006460 hydrolysis reaction Methods 0.000 description 8
- 238000004321 preservation Methods 0.000 description 8
- -1 salt ions Chemical class 0.000 description 7
- 239000008346 aqueous phase Substances 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 238000000746 purification Methods 0.000 description 5
- 241000192130 Leuconostoc mesenteroides Species 0.000 description 4
- 230000001133 acceleration Effects 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000012467 final product Substances 0.000 description 3
- 238000004108 freeze drying Methods 0.000 description 3
- 150000002772 monosaccharides Chemical class 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 229920002153 Hydroxypropyl cellulose Polymers 0.000 description 2
- 108010044467 Isoenzymes Proteins 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 150000001450 anions Chemical class 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 150000001768 cations Chemical class 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000010924 continuous production Methods 0.000 description 2
- 238000011033 desalting Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000012869 ethanol precipitation Methods 0.000 description 2
- 238000010812 external standard method Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000003301 hydrolyzing effect Effects 0.000 description 2
- 239000001863 hydroxypropyl cellulose Substances 0.000 description 2
- 235000010977 hydroxypropyl cellulose Nutrition 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
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- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000007738 vacuum evaporation Methods 0.000 description 2
- 229920001221 xylan Polymers 0.000 description 2
- 150000004823 xylans Chemical class 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- 241000589220 Acetobacter Species 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 241001507810 Talaromyces aculeatus Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 239000000679 carrageenan Substances 0.000 description 1
- 229920001525 carrageenan Polymers 0.000 description 1
- 235000010418 carrageenan Nutrition 0.000 description 1
- 229940113118 carrageenan Drugs 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000004042 decolorization Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000010587 phase diagram Methods 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 150000004804 polysaccharides Chemical class 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 description 1
Classifications
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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
- C12P19/08—Dextran
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
- C07H3/02—Monosaccharides
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- Life Sciences & Earth Sciences (AREA)
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- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
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- Chemical Kinetics & Catalysis (AREA)
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- General Engineering & Computer Science (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for precisely regulating and controlling an alpha-glucan enzymolysis reaction and directionally extracting a specific molecular weight product and application thereof, and belongs to the technical field of biochemical separation. The invention uses glucan as a polymer capable of forming a phase body, the molecular weight of the glucan is a key factor influencing the phase formation condition, and the final target molecular weight of the enzymolysis reaction is dropped on a phase separation point by adjusting the composition of other high polymer phase bodies. Meanwhile, the high partition coefficient of the alpha-glucanase in part of the polymer phase is utilized to realize the rapid recovery of the enzyme protein from the reaction phase, the enzymolysis reaction is stopped, and the enzyme is recovered again through a specific concentrated salt solution which is compatible with the alpha-glucanase protein after the separation of the low molecular weight alpha-glucan.
Description
Technical Field
The invention belongs to the technical field of biochemical separation, relates to an expansion application of a water phase extraction technology, and particularly relates to a method for precisely regulating and controlling an alpha-glucan enzymolysis reaction and directionally extracting a specific molecular weight product and an application thereof.
Background
The low molecular weight alpha-glucan production flow currently required by the food or pharmaceutical industry is: fermentation-ethanol precipitation-hydrolysis-separation and purification-freeze-drying. The hydrolysis method mainly comprises enzymolysis and acidolysis, and also comprises fermentation and hydrolysis steps, for example, CN101205257 is obtained by fermenting to obtain high molecular weight alpha-glucan, acidolysis to obtain target molecular weight product, and multistage membrane separation, ion exchange resin desalination, active carbon decolorization and other steps to obtain the final product. Acidolysis gives better control over the molecular weight distribution range of the final product, but strong acids cause more side reactions, especially the production of large amounts of monosaccharides, which can increase the color of the downstream product. CN103993052 is prepared in one step by directly controlling the molecular weight during fermentation using hydrolytic enzymes. The enzymolysis has higher specificity, but the hydrolysis characteristics of isozymes from different sources are different, the possible reaction strengths of the isozymes to substrates with different molecular weights are different, enzyme proteins are possibly difficult to inactivate, the molecular weight distribution of the product is easy to widen, and meanwhile, when the enzyme is used as the protein to inactivate at high temperature, maillard reaction is carried out on the enzyme and reducing sugar, and the color value is also increased. CN105671104 is prepared by respectively fixing Penicillium aculeatum and Acetobacter which secrete hydrolytic enzymes on carrageenan matrix to form microspheres, so that the enzymes can be conveniently separated from reaction substrates, and the purpose of controlling enzymatic reaction is achieved. However, immobilized fermentation generally has the problems of reduced fermentation efficiency and reduced utilization rate of carbon sources and nitrogen sources due to the blockage of solute exchange. CN200810122697.4 proposes a method for recovering xylanases remaining after hydrolysis of xylan by a two-aqueous system, which relates to the design of a continuous production plant, wherein the composition of the aqueous system is not related to xylan but only to achieve recycling of the enzyme.
The aqueous phase extraction technology is different from the traditional purification technology, and has mild reaction conditions and short time; and all reagents are non-volatile nontoxic medicines, and are particularly suitable for the purification application of biological materials. The principle of phase separation of a polymer from a salt solution is to use the fact that the interaction between cations and polyatomic anions of small high density charges is inhibited, so that salt ions are blocked when approaching the surface area of the polymer, and a salt-free and directional hydration layer area is formed, and when the concentration of the cations and polyatomic anions reaches a certain value, two phases are promoted to be separated. According to factors such as polymer molecular weight, density, interfacial tension, potential difference and the like, proteins, nucleic acids, viruses, cell particles and the like have corresponding distribution behaviors in different aqueous phase systems, so that a theoretical basis of aqueous phase extraction and purification is formed. In the prior art, the water phase extraction purification technology is mainly applied to enzyme engineering or other polymers are added for polysaccharide purification, and no report has been made that the raw materials directly participate in the composition of a water phase system to achieve the purpose of purifying the product.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the prior art and provides a method for precisely regulating and controlling the enzymolysis reaction of alpha-glucan and directionally extracting a specific molecular weight product; the invention uses dextran as the polymer of the constituent phase, the molecular weight is also the key factor influencing the phase forming condition, and the target molecular weight of the final product of the enzymolysis reaction is dropped on the phase splitting point by adjusting the composition of other high polymer phases. Meanwhile, the high partition coefficient of the alpha-glucanase in part of the polymer phase is utilized to realize the rapid recovery of the enzyme protein from the reaction phase, the enzymolysis reaction is stopped, and the enzyme is recovered through a specific concentrated salt solution which is compatible with the alpha-glucanase protein after the separation of the low molecular weight alpha-glucan.
The invention also aims to provide application of the method for precisely regulating and controlling the enzymolysis reaction of the alpha-glucan and directionally extracting the specific molecular weight product.
The aim of the invention is achieved by the following technical scheme:
A method for precisely regulating and controlling an alpha-glucan enzymolysis reaction and directionally extracting a specific molecular weight product comprises the following steps:
(1) Preparation of a Standard Curve
A) Preparing an alpha-glucan standard solution with the mass fraction of 0-25% and a polyethylene glycol solution with the mass fraction of 50-60%, dropwise adding the alpha-glucan standard solution into the polyethylene glycol solution until the mixed solution is turbid, recording the mass fraction of the polyethylene glycol solution corresponding to the mixed solution when the alpha-glucan solution with the mass fraction of 0-25% becomes turbid, and preparing an alpha-glucan-polyethylene glycol standard curve by taking the mass fraction of the alpha-glucan standard solution in the mixed solution as an abscissa and the mass fraction of the polyethylene glycol solution as an ordinate;
b) Preparing a salt solution with the mass fraction of 0% -25% and a polyethylene glycol solution with the mass fraction of 50% -60%, dropwise adding the salt solution into the polyethylene glycol solution until the mixed solution is turbid, recording the mass fraction of the polyethylene glycol solution corresponding to the mixed solution when the salt solution with the mass fraction of 2% -14% becomes turbid, and preparing a salt-polyethylene glycol standard curve by taking the mass fraction of the salt solution in the mixed solution as an abscissa and the mass fraction of the polyethylene glycol solution as an ordinate;
c) Determining the dosage of polyethylene glycol solution and salt solution in the actual extraction process according to the mass fraction of the alpha-glucan-polyethylene glycol standard curve of the step a) and the salt-polyethylene glycol standard curve of the step b) combined with the alpha-glucan in the fermentation broth;
(2) According to the standard curve indication manufactured in the step (1), taking a mixed solution of polyethylene glycol (PEG 8000) and alpha-glucan to be tested, vibrating and mixing, standing and layering, dividing into two layers from top to bottom, wherein an upper phase is a polyethylene glycol phase, a lower phase is an alpha-glucan phase, adding an alpha-glucanase solution to the vicinity of a lower phase two-phase interface, slightly vibrating, preserving heat, observing that after two phases are mixed, injecting a salt solution into the mixed solution until the final mass fraction is 2% -14%, standing and layering again, centrifuging, dividing the liquid into two phases at the moment, taking the lower phase, adding alcohol, centrifuging, and taking the precipitate to obtain the target low molecular weight alpha-glucan; adding magnesium sulfate solution into the upper phase, oscillating, standing for layering, taking the upper phase as polyethylene glycol phase, taking the lower phase as salt solution phase, and carrying out ultrafiltration desalination and concentration to obtain the alpha-glucanase recovery liquid.
The polyethylene glycol (PEG 8000) and the alpha-glucan in the mixed solution of the polyethylene glycol (PEG 8000) and the alpha-glucan are preferably calculated according to the mass ratio of 4-4.8:6.2-10.
The solvent of the mixed solution of polyethylene glycol (PEG 8000) and alpha-glucan is preferably water.
The adding amount of the alpha-glucanase is increased by 0.002-0.005U/mL according to the adding amount of the alpha-glucan added by 1% of the total reaction system.
The alpha-glucanase is derived from at least one of a bacterium and a commercial purchase; when the alpha-glucanase is derived from bacteria, the alpha-glucanase is preferably derived from a strain with a preservation number of CCTCC No: m2013096 Pichia pastoris engineering bacteria. The preservation number is CCTCC NO:2013096 Pichia pastoris engineering bacteria and a preparation method of alpha-glucanase are disclosed in patent 2013102877278.
The solvent of the alpha-glucanase solution is preferably water.
The enzyme activity of the alpha-glucanase is preferably 0.02-0.5U/mL; more preferably 0.1 to 0.4U/mL.
The temperature of the heat preservation is preferably 40-60 ℃; more preferably 45 ℃.
The salt solution is preferably at least one of an ammonium sulfate solution and a dipotassium hydrogen phosphate solution.
When the salt solution is an ammonium sulfate solution, the mass fraction of ammonium sulfate in the ammonium sulfate solution is preferably 11% -16% (w/w); more preferably from 14% to 16% (w/w).
The centrifugation conditions are preferably as follows: centrifuging at 4000-6000 rpm for 4-6 min; more preferably at 5000rpm for 5min.
The alcohol is preferably 80% -95% (v/v) alcohol; more preferably 95% (v/v) alcohol.
The final concentration of the alcohol after adding the alcohol downwards is preferably 75% -95% (v/v); more preferably 80% (v/v).
The final concentration of alcohol after adding alcohol downward is preferably 75% -95% v/v.
The conditions of centrifugation after adding alcohol are preferably as follows: centrifuging at 1000-5000 rpm for 4-6 min.
The mass fraction of magnesium sulfate in the magnesium sulfate solution is preferably 25% -36% (w/w).
The addition amount of magnesium sulfate after adding the magnesium sulfate solution to the upper layer is preferably 10% -14% (w/w) of the system.
The ultrafiltration desalination is preferably performed by a polyethersulfone resin membrane, the elution volume of the ultrafiltration desalination is preferably 4 to 6 times, and the final solution is concentrated by about 4 times.
The method for precisely regulating and controlling the enzymolysis reaction of the alpha-glucan and directionally extracting the products with specific molecular weight is applied to foods and/or medicines.
Compared with the prior art, the invention has the following advantages and effects:
(1) The finally obtained low molecular weight alpha-glucan has concentrated distribution, the reaction is easy to regulate and control, and meanwhile, the condition is mild, and the waste strong acid solution does not need to be treated additionally; the enzyme preparation used in the reaction can be simply recovered, and the production cost is saved.
(2) The invention uses glucan as a polymer capable of forming a phase body, the molecular weight of the glucan is a key factor influencing the phase formation condition, and the final target molecular weight of the enzymolysis reaction is dropped on a phase separation point by adjusting the composition of other high polymer phase bodies. Meanwhile, the high partition coefficient of the alpha-glucanase in part of the polymer phase is utilized to realize the rapid recovery of the enzyme protein from the reaction phase, the enzymolysis reaction is stopped, and the enzyme protein is recovered through concentrated salt solution after the separation of the low molecular weight alpha-glucanase.
(3) The method of the application takes the salt solution as one of the extraction phases, which is beneficial to reducing the cost of continuous production, and part of metal ions have a protective effect on alpha-glucanase.
Drawings
FIG. 1 is a graph of the standard curve of the α -glucan-polyethylene glycol prepared in example 1.
FIG. 2 is a graph of the ammonium sulfate-polyethylene glycol standard prepared in example 1.
FIG. 3 is a graph showing the results of a conventional enzymatic hydrolysis of α -glucanase compared to a liquid phase diagram of α -glucan having a weight average molecular weight of about 200kDa produced by the method described in example 1; wherein, the peak between 12.5 and 20min is alpha-glucan peak; peaks between 20 and 25min represent monosaccharide peaks.
FIG. 4 is a graph of the standard curve of the α -glucan-polyethylene glycol prepared in example 2.
FIG. 5 is a standard graph of dipotassium hydrogen phosphate-polyethylene glycol prepared in example 2.
FIG. 6 is a graph showing the results of a comparison of conventional enzymatic hydrolysis of α -glucanase with a weight average molecular weight of about 70kDa produced by the method described in example 2; wherein, the peak between 12.5 and 20min is alpha-glucan peak; peaks between 20 and 25min represent monosaccharide peaks.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
Example 1: alpha-glucan with weight average molecular weight of about 200kDa is prepared by precisely regulating and controlling enzymolysis reaction of alpha-glucan and directionally extracting specific molecular weight product
Reagents such as polyethylene glycol, potassium dihydrogen phosphate, ammonium sulfate, sodium sulfate, magnesium sulfate and the like are all analytically pure.
The alpha-glucanase is obtained by fermenting and culturing Pichia pastoris engineering bacteria with the preservation number of CCTCC No.2013096, and the preparation method of the Pichia pastoris engineering bacteria with the preservation number of CCTCC No.2013096 and the alpha-glucanase are disclosed in patent 2013102877278. The alpha-glucan is prepared from leuconostoc mesenteroides (Leuconostoc mesenteroides, purchased from the microorganism strain collection of Guangdong province) with a preservation number of GDMCC 1.473.473 through shake flask single batch fermentation, wherein the specific fermentation process is that the liquid amount of a 5L fermentation tank is 2.7L, the inoculation amount is 10% (v/v), the initial pH is 6.5, the culture temperature is 25 ℃, the fermentation is carried out under the condition of 100r/min, the pH is regulated to be 5.5 after 10 hours, the alpha-glucanase with the final concentration of 0.005U/mL is added after 12 hours of fermentation, meanwhile, the sucrose stream is supplemented with 7 g/(h.L) of flow acceleration, 280g of sucrose is added altogether, and the fermentation is stopped when the residual sugar of the fermentation liquid is reduced to 1% -2% by HPLC detection. The fermentation broth is concentrated by vacuum evaporation and then heated for sterilization, and the molecular weight of the alpha-glucan is larger than 1000kDa.
(1) Preparation of a Standard Curve
A) Preparing an alpha-glucan standard solution (the weight average molecular weight of an alpha-glucan standard substance is 200kDa and is purchased from French and America) with the mass fraction of 25% and a polyethylene glycol (PEG 8000) solution with the mass fraction of 50% -60%, dropwise adding the alpha-glucan standard solution into the polyethylene glycol (PEG 8000) solution until the mixed solution is turbid, and recording that the mass fraction of the mixed solution is 0% -25% (the specific mass fractions are respectively: 0.5%, 2.5%, 5%, 10%, 15%, 25%) of the polyethylene glycol (PEG 8000) solution corresponding to the turbidity of the alpha-glucan solution is 2.5% -15% (the specific mass fractions are respectively: 9.8%, 7.8%, 6.2%, 4.4%, 3.6%, 2.2%), and preparing an alpha-glucan-polyethylene glycol (PEG 8000) standard curve by taking the mass fraction of the alpha-glucan standard solution in the mixed solution as an abscissa and the mass fraction of the polyethylene glycol (PEG 8000) solution as an ordinate; the curve is shown in figure 1.
B) Respectively preparing an ammonium sulfate solution with the mass fraction of 0-25% and a polyethylene glycol solution with the mass fraction of 50-60%, dropwise adding the ammonium sulfate solution into the polyethylene glycol solution until the mixed solution is turbid, and recording that the mass fraction of the mixed solution is 2-14% (the specific mass fractions are respectively: 2.7%, 4.5%, 5.2%, 6.3%, 10%, 12.5%) of ammonium sulfate solution, and the mass fraction of the corresponding polyethylene glycol solution when the ammonium sulfate solution becomes turbid is 2% -40% (the specific mass fractions are respectively: 39.5%, 27.1%, 23.4%, 18.1%, 6.2%, 1.7%) of an ammonium sulfate solution in the mixed solution is taken as an abscissa, and the mass fraction of a polyethylene glycol solution is taken as an ordinate to manufacture an ammonium sulfate-polyethylene glycol standard curve; the curve is shown in fig. 2.
C) Determining the dosage of polyethylene glycol and ammonium sulfate in the actual extraction process according to the alpha-glucan-polyethylene glycol standard curve of the step (a) and the ammonium sulfate-polyethylene glycol standard curve of the step (b) combined with the mass fraction of the alpha-glucan in the fermentation broth;
(2) According to curve indication, preparing a mixed solution consisting of polyethylene glycol (PEG 8000) and alpha-glucan-containing fermentation liquor by using water as a solvent, wherein the mass fractions of polyethylene glycol (PEG 8000) and alpha-glucan in the mixed solution are respectively 4% and 6.2%, standing for layering after shaking and mixing, dividing into two layers from top to bottom, adding an alpha-glucanase aqueous solution with the final concentration of 0.1U/mL and about 0.1mL into a position close to a two-phase interface of the lower phase, slightly shaking, preserving heat at 45 ℃, observing that the middle phase and the lower phase are mixed, injecting an ammonium sulfate solution until the mass fraction is 14%, standing for re-layering, centrifuging at 5000rpm for 5min, dividing the liquid into two phases at the moment, taking the lower phase, adding alcohol until the final concentration is 80% (v/v), centrifuging at 5000rpm for 5min, taking the precipitate, detecting by using a hydroxypropyl cellulose chromatographic column Skodex SB-806M HQ, calibrating by an external standard method, and determining the product as the target molecular weight.
Adding concentrated magnesium sulfate solution into the upper phase liquid until the mass fraction reaches 14%, oscillating, standing for layering, distributing polyethylene glycol phase and salt solution phase from top to bottom, taking out the lower phase, ultrafiltering and desalting by Vivaflow 200PES 10000 for about four times, concentrating the final solution for about 4 times, and obtaining the alpha-glucanase recovery liquid.
The conversion rate of the whole process sucrose reaches 85%, the alpha-glucanohol precipitation is about 90%, and the recovery rate of the alpha-glucanase is about 70%.
In addition, preparing a process sample for producing alpha-glucan by direct enzymolysis, namely preparing the alpha-glucan by a common enzymolysis method, wherein the alpha-glucan process sample is obtained by fermenting and culturing by the following method: the liquid amount of the 5L fermentation tank is 2.7L, the inoculation amount is 10% (v/v), the initial pH is 6.5, and the fermentation is carried out under the conditions of the culture temperature of 25 ℃ and 100 r/min. After 16h of fermentation, adding alpha-glucanase to a final concentration of 0.05U/mL, and adding sucrose to the fermentation broth at a constant flow acceleration of 8 g/(h.L), and stopping fermentation when the residual sugar concentration of the fermentation broth is 1% -2% after 26h according to HPLC monitoring. After detecting that the hydrolysis degree of the alpha-glucan is close to the target molecular weight by HPLC, heating at 85 ℃ to stop the reaction, adding alcohol until the final concentration is 80% (v/v), centrifuging at 5000rpm for 5min, and taking the precipitate to obtain the alpha-glucan produced by a direct enzymolysis method. The sample Mn was 31kDa, mw was 167kDa and the distribution coefficient was 5.4. The Mn of the sample obtained by the continuous directional water phase extraction process flow is 102kDa, the Mw of the sample is 235kDa, the distribution coefficient is 2.3, and the molecular weight distribution condition is obviously narrower than that of the common enzymolysis method. The results are shown in FIG. 3.
Wherein the enzyme is defined as the hydrolysis of 1. Mu. Mol glucose by a hydrolase at 45℃for 1 min. Sucrose conversion (%) = [ α -glucan end product weight/(sucrose weight/2) ]×100%; ethanol precipitation recovery (%) = [ α -glucan weight after freeze-drying/total glucan content in solution after aqueous phase extraction reaction ] ×100%; the recovery rate (%) of α -glucanase was = [ original concentration x added volume/(concentration after concentration x volume after concentration) ]x100%.
Example 2: method for preparing alpha-glucan with weight average molecular weight of about 70kDa by precisely regulating and controlling alpha-glucan enzymolysis reaction and directionally extracting specific molecular weight product
The alpha-glucanase is obtained by fermenting and culturing Pichia pastoris engineering bacteria with the preservation number of CCTCC No.2013096, and the preparation method of the Pichia pastoris engineering bacteria with the preservation number of CCTCC No.2013096 and the alpha-glucanase are disclosed in patent 2013102877278. The alpha-glucan was prepared from Leuconostoc mesenteroides (Leuconostoc mesenteroides, available from the collection of microbial strains in Guangdong province) with accession number GDMCC 1.473.473 by shake flask single batch fermentation. The specific fermentation process is that the liquid amount of a 5L fermentation tank is 2.7L, the inoculation amount is 10% (v/v), the initial pH is 6.5, the fermentation is carried out at the culture temperature of 25 ℃ under the condition of 100r/min, the pH is regulated and controlled to be 5.5 after 10 hours, the alpha-glucanase with the final concentration of 0.005U/mL is added after 12 hours of fermentation, meanwhile, the sucrose flowing liquid is supplemented with 8 g/(h.L) of flow acceleration, 280g of sucrose is added in total, and the fermentation is stopped when the residual sugar of the fermentation liquid is reduced to 1% -2% by HPLC detection. The fermentation broth is concentrated by vacuum evaporation and then heated for sterilization, and the molecular weight of the alpha-glucan is larger than 1000kDa.
(1) Preparation of a Standard Curve
A) Preparing an alpha-glucan standard solution (the weight average molecular weight of an alpha-glucan standard substance is 70kDa and is purchased from French company of America) with the mass fraction of 0-25% and a polyethylene glycol (PEG 8000) solution with the mass fraction of 50-60%, dropwise adding the alpha-glucan standard solution into the polyethylene glycol (PEG 8000) solution until a mixed solution is turbid, and recording that the mass fraction of the mixed solution is 0-25% (the specific mass fractions are respectively: 0.5%, 2.5%, 5%, 10%, 15%, 25%) of the polyethylene glycol solution corresponding to the turbidity of the alpha-glucan solution is 2.5% -15% (the specific mass fractions are respectively: 12.8%, 8.2%, 6.4%, 4.9%, 3.8%, 3.4%), and preparing an alpha-glucan-polyethylene glycol (PEG 8000) standard curve by taking the mass fraction of the alpha-glucan standard solution in the mixed solution as an abscissa and the mass fraction of the polyethylene glycol (PEG 8000) solution as an ordinate; the curve is shown in fig. 4.
B) Preparing 0-25% of dipotassium hydrogen phosphate solution and 50-60% of polyethylene glycol (PEG 8000) solution by mass fraction, dropwise adding the dipotassium hydrogen phosphate solution into the polyethylene glycol (PEG 8000) solution until the mixed solution is turbid, and recording the dipotassium hydrogen phosphate solution with the mass fraction of 2-14% in the mixed solution (the specific mass fraction is respectively: 4.9%, 6.4%, 7.8%, 9.6%, 12.1%, 13.5%) of polyethylene glycol solution corresponding to turbidity of 5% -40% (the specific mass fractions are respectively: 44.6%, 32.7%, 26%, 19.5%, 10.7%, 7.9%), and making a standard curve of dipotassium hydrogen phosphate-polyethylene glycol (PEG 8000) by taking the mass fraction of the dipotassium hydrogen phosphate solution in the mixed solution as an abscissa and the mass fraction of the polyethylene glycol (PEG 8000) solution as an ordinate; the curve is shown in fig. 5.
C) Determining the dosage of polyethylene glycol (PEG 8000) and dipotassium hydrogen phosphate in the actual extraction process according to the alpha-glucan-polyethylene glycol (PEG 8000) standard curve of the step (a) and the dipotassium hydrogen phosphate-polyethylene glycol (PEG 8000) standard curve of the step (b) combined with the mass fraction of the alpha-glucan in the fermentation broth;
(2) According to curve indication, preparing a mixed solution consisting of polyethylene glycol (PEG 8000) and alpha-glucan-containing fermentation liquor by using water as a solvent, wherein the mass fractions of the polyethylene glycol (PEG 8000) and the alpha-glucan in the mixed solution are respectively 4.8% and 10%, standing for layering after shaking and mixing, dividing into two layers from top to bottom, wherein the upper phase is polyethylene glycol phase, the lower phase is alpha-glucan phase, adding an alpha-glucanase aqueous solution with the final concentration of 0.1U/mL and about 0.1mL into the interface of the lower phase, slightly shaking, preserving heat at 45 ℃, observing that the middle phase and the lower phase are mixed, injecting a dipotassium phosphate solution until the mass fraction is 15%, standing for re-layering, centrifuging for 5min at 5000rpm, dividing the liquid into two phases at the moment, taking the lower phase, adding alcohol until the final concentration is 80% (v/v), centrifuging for 5min at 5000rpm, taking the precipitate, detecting by using a hydroxypropyl cellulose chromatographic column Skodex SB-806M HQ, and determining that the product is the target molecular weight after calibration by an external standard method.
Adding concentrated magnesium sulfate solution into the upper phase liquid until the mass fraction reaches 14%, oscillating, standing for layering, distributing polyethylene glycol phase and salt solution phase from top to bottom, taking out the lower phase, ultrafiltering and desalting by Vivaflow 200PES 10000 for about four times, concentrating the final solution for about 4 times, and obtaining the alpha-glucanase recovery liquid.
The conversion rate of the whole process sucrose reaches 85%, the alpha-glucanohol precipitation is about 90%, and the recovery rate of the alpha-glucanase is about 70%.
In addition, preparing a process sample for producing alpha-glucan by direct enzymolysis, namely preparing the alpha-glucan by a common enzymolysis method, wherein the alpha-glucan process sample is obtained by fermenting and culturing by the following method: the liquid amount of the 5L fermentation tank is 2.7L, the inoculation amount is 10% (v/v), the initial pH is 6.5, and the fermentation is carried out under the conditions of the culture temperature of 25 ℃ and 100 r/min. After 16h of fermentation, adding alpha-glucanase to a final concentration of 0.1U/mL, and adding sucrose to the fermentation broth at a constant flow acceleration of 10 g/(h.L), and stopping fermentation when the residual sugar concentration of the fermentation broth is 1% -2% after 26h according to HPLC monitoring. After detecting that the hydrolysis degree of the alpha-glucan is close to the target molecular weight by HPLC, heating at 85 ℃ to stop the reaction, adding alcohol until the final concentration is 80% (v/v), centrifuging at 5000rpm for 5min, and taking the precipitate to obtain the alpha-glucan produced by a direct enzymolysis method. The number average molecular weight (Mn) of the sample was 16kDa, the weight average molecular weight (Mw) was 51kDa, and the distribution coefficient was 3.2. The number average molecular weight (Mn) of the sample obtained by the continuous directional aqueous phase extraction process flow is 22kDa, the weight average molecular weight (Mw) is 68kDa, the distribution coefficient is 3.1, and the molecular weight distribution condition is obviously narrower than that of the common enzymolysis method. The results are shown in FIG. 6.
Wherein the enzyme is defined as the hydrolysis of 1. Mu. Mol glucose by a hydrolase at 45℃for 1 min. Sucrose conversion (%) = [ α -glucan end product weight/(sucrose weight/2) ]×100%; ethanol precipitation recovery (%) = [ α -glucan weight after freeze-drying/total glucan content in solution after aqueous phase extraction reaction ] ×100%; the recovery rate (%) of α -glucanase was = [ original concentration x added volume/(concentration after concentration x volume after concentration) ]x100%.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (8)
1. The method for precisely regulating and controlling the enzymolysis reaction of the alpha-glucan and directionally extracting the product with specific molecular weight is characterized by comprising the following steps:
(1) Preparation of a Standard Curve
A) Preparing an alpha-glucan standard solution with the mass fraction of 0-25% and a polyethylene glycol solution with the mass fraction of 50-60%, dropwise adding the alpha-glucan standard solution into the polyethylene glycol solution until the mixed solution is turbid, recording the mass fraction of the polyethylene glycol solution corresponding to the mixed solution when the alpha-glucan solution with the mass fraction of 0-25% becomes turbid, and preparing an alpha-glucan-polyethylene glycol standard curve by taking the mass fraction of the alpha-glucan standard solution in the mixed solution as an abscissa and the mass fraction of the polyethylene glycol solution as an ordinate;
b) Preparing a salt solution with the mass fraction of 0% -25% and a polyethylene glycol solution with the mass fraction of 50% -60%, dropwise adding the salt solution into the polyethylene glycol solution until the mixed solution is turbid, recording the mass fraction of the polyethylene glycol solution corresponding to the mixed solution when the salt solution with the mass fraction of 2% -14% becomes turbid, and preparing a salt-polyethylene glycol standard curve by taking the mass fraction of the salt solution in the mixed solution as an abscissa and the mass fraction of the polyethylene glycol solution as an ordinate;
c) Determining the dosage of polyethylene glycol solution and salt solution in the actual extraction process according to the mass fraction of the alpha-glucan-polyethylene glycol standard curve of the step a) and the salt-polyethylene glycol standard curve of the step b) combined with the alpha-glucan in the fermentation broth;
(2) According to the standard curve indication produced in the step (1), taking a mixed solution of polyethylene glycol PEG8000 and alpha-glucan to be detected, vibrating and mixing, standing and layering, dividing into two layers from top to bottom, wherein an upper phase is a polyethylene glycol phase, a lower phase is an alpha-glucan phase, adding an alpha-glucanase solution to the vicinity of an interface of the lower phase, slightly vibrating, preserving heat, observing that after the two phases are mixed, injecting a salt solution into the mixed solution until the final mass fraction is 2% -14%, standing and layering again, centrifuging, dividing the liquid into two phases at the moment, taking the lower phase, adding alcohol, centrifuging, and taking the precipitate to obtain the target low molecular weight alpha-glucan; adding an upper phase into a magnesium sulfate solution, oscillating, standing for layering, taking the upper phase as a polyethylene glycol phase, taking the lower phase as a salt solution phase, and carrying out ultrafiltration desalination and concentration to obtain an alpha-glucanase recovery solution;
the solvent of the mixed solution of polyethylene glycol PEG8000 and alpha-glucan is water;
the salt solution is at least one of an ammonium sulfate solution and a dipotassium hydrogen phosphate solution;
The mixed solution of polyethylene glycol PEG8000 and alpha-glucan is calculated according to the mass ratio of 4-4.8:6.2-10.
2. The method according to claim 1, wherein the amount of the added α -glucanase is increased by 0.002 to 0.005U/mL per 1% increase in α -glucan in the total reaction system.
3. The method of claim 1, wherein the α -glucanase is derived from at least one of a bacterium and a commercial purchase; when the alpha-glucanase is derived from bacteria, the alpha-glucanase is derived from CCTCC No: m2013096 Pichia pastoris engineering bacteria.
4. The method of claim 1, wherein the enzyme activity of the α -glucanase is from 0.02 to 0.5U/mL.
5. The method of claim 1, wherein the temperature of the incubation is 40-60 ℃.
6. The method of claim 1, wherein the centrifugation conditions are: centrifuging 4000-6000 rpm to 4-6 min;
The alcohol is 80-95% v/v alcohol;
The final concentration of the alcohol after adding the alcohol downwards is 75% -95% v/v;
the conditions of centrifugation after adding alcohol are as follows: and (3) centrifuging the mixture at 1000-5000 rpm to 4-6 min.
7. The method of claim 1, wherein the step of determining the position of the substrate comprises,
The mass fraction of magnesium sulfate in the magnesium sulfate solution is 25% -36% w/w;
the adding amount of magnesium sulfate after adding the magnesium sulfate solution upwards is 10-14% w/w of the system;
the ultrafiltration desalination is performed by a polyethersulfone resin membrane.
8. Use of the method of precisely controlling the enzymatic hydrolysis of α -glucan and directionally extracting a specific molecular weight product according to any one of claims 1-7 for the preparation of a food and/or pharmaceutical product.
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