CN101693735A - Method for extracting protein and enzyme by aqueous two-phase extraction technology - Google Patents
Method for extracting protein and enzyme by aqueous two-phase extraction technology Download PDFInfo
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- 108090000790 Enzymes Proteins 0.000 title claims abstract description 47
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 47
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 42
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 42
- 238000000605 extraction Methods 0.000 title claims abstract description 23
- 238000000034 method Methods 0.000 title claims abstract description 20
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 17
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 14
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims description 6
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical class CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 5
- AMQJEAYHLZJPGS-UHFFFAOYSA-N N-Pentanol Chemical compound CCCCCO AMQJEAYHLZJPGS-UHFFFAOYSA-N 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 claims description 3
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 2
- 150000003863 ammonium salts Chemical class 0.000 claims description 2
- SZXQTJUDPRGNJN-UHFFFAOYSA-N dipropylene glycol Chemical compound OCCCOCCCO SZXQTJUDPRGNJN-UHFFFAOYSA-N 0.000 claims description 2
- 239000001103 potassium chloride Substances 0.000 claims description 2
- 235000011164 potassium chloride Nutrition 0.000 claims description 2
- 159000000000 sodium salts Chemical class 0.000 claims description 2
- 238000000926 separation method Methods 0.000 abstract description 11
- 239000000243 solution Substances 0.000 abstract description 10
- 150000003839 salts Chemical class 0.000 abstract description 8
- 238000002156 mixing Methods 0.000 abstract description 7
- 239000012460 protein solution Substances 0.000 abstract description 6
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract 1
- 229910052500 inorganic mineral Inorganic materials 0.000 abstract 1
- 239000011707 mineral Substances 0.000 abstract 1
- 150000005846 sugar alcohols Polymers 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 39
- 239000012071 phase Substances 0.000 description 20
- 238000005192 partition Methods 0.000 description 13
- 238000005303 weighing Methods 0.000 description 12
- 239000008346 aqueous phase Substances 0.000 description 10
- 230000000694 effects Effects 0.000 description 10
- PUPZLCDOIYMWBV-UHFFFAOYSA-N (+/-)-1,3-Butanediol Chemical compound CC(O)CCO PUPZLCDOIYMWBV-UHFFFAOYSA-N 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 7
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 7
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 7
- 235000011130 ammonium sulphate Nutrition 0.000 description 7
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 7
- 230000014759 maintenance of location Effects 0.000 description 6
- 230000008901 benefit Effects 0.000 description 5
- 239000003960 organic solvent Substances 0.000 description 5
- 229940058015 1,3-butylene glycol Drugs 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 4
- 108010019077 beta-Amylase Proteins 0.000 description 4
- 229940098773 bovine serum albumin Drugs 0.000 description 4
- 235000019437 butane-1,3-diol Nutrition 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 238000005070 sampling Methods 0.000 description 4
- 108010059892 Cellulase Proteins 0.000 description 3
- 239000004367 Lipase Substances 0.000 description 3
- 102000004882 Lipase Human genes 0.000 description 3
- 108090001060 Lipase Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 3
- 229940106157 cellulase Drugs 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 238000005265 energy consumption Methods 0.000 description 3
- 108010090785 inulinase Proteins 0.000 description 3
- 235000019421 lipase Nutrition 0.000 description 3
- 229960004063 propylene glycol Drugs 0.000 description 3
- 229910052938 sodium sulfate Inorganic materials 0.000 description 3
- 235000011152 sodium sulphate Nutrition 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- UXFQFBNBSPQBJW-UHFFFAOYSA-N 2-amino-2-methylpropane-1,3-diol Chemical compound OCC(N)(C)CO UXFQFBNBSPQBJW-UHFFFAOYSA-N 0.000 description 2
- 108010064696 N,O-diacetylmuramidase Proteins 0.000 description 2
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- WERYXYBDKMZEQL-UHFFFAOYSA-N butane-1,4-diol Chemical compound OCCCCO WERYXYBDKMZEQL-UHFFFAOYSA-N 0.000 description 2
- 238000001142 circular dichroism spectrum Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- -1 polyoxyethylene Polymers 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- YPFDHNVEDLHUCE-UHFFFAOYSA-N propane-1,3-diol Chemical compound OCCCO YPFDHNVEDLHUCE-UHFFFAOYSA-N 0.000 description 2
- 238000012827 research and development Methods 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 229920003169 water-soluble polymer Polymers 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 241001122315 Polites Species 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- SVOZWQVJNZEQFC-UHFFFAOYSA-M S(=O)(=O)([O-])[O-].Cl[NH3+].[Na+] Chemical compound S(=O)(=O)([O-])[O-].Cl[NH3+].[Na+] SVOZWQVJNZEQFC-UHFFFAOYSA-M 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- BVCZEBOGSOYJJT-UHFFFAOYSA-N ammonium carbamate Chemical compound [NH4+].NC([O-])=O BVCZEBOGSOYJJT-UHFFFAOYSA-N 0.000 description 1
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010364 biochemical engineering Methods 0.000 description 1
- BMRWNKZVCUKKSR-UHFFFAOYSA-N butane-1,2-diol Chemical compound CCC(O)CO BMRWNKZVCUKKSR-UHFFFAOYSA-N 0.000 description 1
- OWBTYPJTUOEWEK-UHFFFAOYSA-N butane-2,3-diol Chemical compound CC(O)C(C)O OWBTYPJTUOEWEK-UHFFFAOYSA-N 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- KXDHJXZQYSOELW-UHFFFAOYSA-N carbonic acid monoamide Natural products NC(O)=O KXDHJXZQYSOELW-UHFFFAOYSA-N 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 239000006084 composite stabilizer Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 230000002478 diastatic effect Effects 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229920001477 hydrophilic polymer Polymers 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229940045641 monobasic sodium phosphate Drugs 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 230000005501 phase interface Effects 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 235000007686 potassium Nutrition 0.000 description 1
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Substances [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 1
- 235000015320 potassium carbonate Nutrition 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 229940093916 potassium phosphate Drugs 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 235000017550 sodium carbonate Nutrition 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
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- Peptides Or Proteins (AREA)
Abstract
A method for extracting protein and enzyme by aqueous two-phase extraction technology belongs to the technical field of biological engineering, and is characterized in that polyhydric alcohol and mineral salt are directly added into protein solution or enzyme solution according to a definite proportion to form two phases through intimate mixing and stationary standing under room temperature, and the protein and the enzyme are primarily allocated in an upper phase and keep higher activeness, thereby realizing effective separation and extraction. The invention has the beneficial effects of developing the new method for extracting protein by aqueous two-phase extraction technology characterized by simple operation, temperate conditions, low production cost and easy industrialization.
Description
Technical field
The invention belongs to technical field of bioengineering, relate to proteinic extraction separating method, the method for particularly a kind of aqueous two-phase extraction isolated protein and enzyme.
Background technology
Protein and enzyme all play an important role in human daily life, disease prevention and treatment and industrial production, and its extraction separation is a necessary means of obtaining this class material.At present, the proteinic main method of industrial preliminary extraction has salting-out process, isoelectric point precipitation and organic solvent precipitation method.Though this several method can both reach isolating purpose to a certain extent, but still have many shortcomings, and comparatively loaded down with trivial details as operating process, operational condition is required also relatively harsher, and protein denaturation takes place in the sepn process easily, reduced yield and efficient that albumen sepn is extracted.Seek simple to operate, with short production cycle, energy consumption is little, operational condition is gentle, and the separation method that is easy to amplify be always the research and development important topic.
Aqueous two phase extraction technique is a kind of simple to operate, separation method of being easy to amplify, has obtained extensive concern since proposing the sixties in 20th century.As people such as N.L.P.Dallora utilize polyoxyethylene glycol/ammonium carbamate double-aqueous phase system respectively to the distribution of bovine serum albumin, Regular Insulin and N,O-Diacetylmuramidase carried out investigating (Biochemical Engineering Journal, 2007,34:92-97); Human such as Du Xuemin and Li Bo polyoxyethylene glycol/ammonium sulfate double-aqueous phase system respectively to saccharifying enzyme and diastatic extracting and separating carried out investigating (China brewages, 2007,1: 7～9; Foodstuffs industry science and technology, 2006,8:77-79).Traditional aqueous two-phase system is made up of hydrophilic two kinds of polymkeric substance or a kind of water-soluble polymers and a kind of inorganic salt, though but this kind system has advantages such as mild condition, easy amplification operate continuously, but because the water-soluble polymers viscosity in the component is big, target product needs to strip, and expensive superpolymer reclaims difficulty, has therefore limited its industrial application greatly.In fact, hydrophilic short chain alcohol and inorganic salt also can form double water-phase, cheap, advantages such as operation steps is simple, solvent recuperation is easy, phase-splitting time weak point that this system has.The double-aqueous phase system that the short chain alcohol of monohydroxy and inorganic salt form has obtained using widely in the extraction of bio-based chemical, amino acid, microbiotic, natural product, traditional Chinese medicine ingredients etc.The applicant has done a large amount of R﹠D works (Chinese patent: CN101012151A; Chinese patent: CN101531652A; Biotechnology Letters, 2008,30:2079-2084; Biotechnology Letters, 2009,31:371? 76).Aspect protein separation, A.Louwrier adopts ethanol/dipotassium hydrogen phosphate double-aqueous phase system that the partition characteristic of multiple proteins and enzyme is investigated, find this system to stability preferably albumen certain separating effect (Biotechnology Techniques is arranged, 1998,12 (5): 363-365), we also are applied to this system the double water-phase solid-liquid separation (Chinese patent: CN200910010281.8) of yeast source recombinant albumin fermented liquid.But for most protein, organic solvents such as the methyl alcohol of higher concentration, ethanol, Virahol cause the protein denaturation inactivation easily.Therefore, the short chain alcohol of monohydroxy/inorganic salt double-aqueous phase system is not too suitable concerning most protein separation.And the three-phase that the trimethyl carbinol and ammonium sulfate constitute distributes (Colloids andSurfaces A:Physicochemical and Engineering Aspects, 1998,142:295? 02), the characteristics of most of albumen precipitation have then been utilized in phase interface and reservation greater activity, acting in conjunction with organic solvent deposit comes down to saltout, shortage is to the selectivity of albumen sepn, and the practical application meeting is subject to many limitations.
Polyvalent alcohol is proteinic hydroxyl permeate agent, and protein is had provide protection, is often used as the stablizer and the additive of polypeptide, protein and enzyme.Scheme (the Chinese patent: CN101215555A) of the liquid basified protease composite stabilizer of propylene glycol has been proposed to contain as road good fortune equality people; (people such as polite moral. Ka Sijiade proposes to add polyol in substratum, waiting as 1,2 propylene glycol, 1,3 propylene glycol increases polypeptide and proteinic solubleness, reduces crystallization and precipitation (patent: WO2004/003187 English 2004.1.8); People such as Wang Wei have investigated the influence to N,O-Diacetylmuramidase of monohydroxy-alcohol, polyvalent alcohol, have confirmed that polyvalent alcohol still can keep stability (chemical industry journal, 2006,57 (1): 74-78) of enzyme preferably under higher concentration.
These polyvalent alcohols, especially hydrophilic binary propyl alcohol and butanols can form novel double-aqueous phase system with inorganic salt, and activity that can protected protein can reach the purpose of extracting and separating again, has the potential prospects for commercial application.
Summary of the invention
The technical problem to be solved in the present invention provides the method for a kind of aqueous two-phase extraction isolated protein and enzyme, be intended to low cost, high yield and realize proteinic separation and Extraction expeditiously, solve that the operating process that exists in present protein and the enzyme separation and Extraction is loaded down with trivial details, the production cycle is long, energy consumption is big, operational condition is harsh, and be prone to problems such as protein and enzyme denaturation inactivation in the operating process.
Technical scheme of the present invention is:
Polyvalent alcohol and inorganic salt are joined in protein or the enzyme solution by mass percentage, stir, leave standstill phase-splitting under the room temperature, the mass percent of described polyvalent alcohol is 10～40%, the inorganic salt mass percent is 10～40%, extraction temperature is 15～30 ℃, and stirring, leave standstill the phase-splitting time is 0.5 ~ 4.0h.Isolating target protein of institute or enzyme are distributed in phase (polyvalent alcohol phase) usually, and impurity is in following phase (inorganic salt face).Selected polyvalent alcohol can be the one or more combination of binary propyl alcohol, binary butanols, ternary butanols, binary amylalcohol, ternary amylalcohol, glycol ether, dipropylene glycol, as 1,2-propylene glycol, 1, ammediol, 1,4-butyleneglycol, 1,2-butyleneglycol, 1,3 butylene glycol, 2,3-butyleneglycol, 1, the one or more combination of 2-pentanediol; Selected inorganic salt can be the one or more combinations of sodium salt, sylvite, ammonium salt, as the one or more combination of sodium-chlor, ammonium sulfate, sodium sulfate, yellow soda ash, salt of wormwood, potassiumphosphate, potassium primary phosphate, dipotassium hydrogen phosphate, SODIUM PHOSPHATE, MONOBASIC, Sodium phosphate dibasic, primary ammonium phosphate, Secondary ammonium phosphate.
Benefit of the present invention is to compare with traditional extraction and separation method with effect, and the present invention has advantages such as operation steps is simple, the cycle is short, extraction conditions is gentle, energy consumption is low; Compare with hydrophilic polymer/inorganic salt or polymer/polymer double-aqueous phase system, have that organic solvent is cheap easily to be reclaimed, the phase-splitting of viscosity low mass transfer is fast, and one step of extracting and separating such as finishes at advantage; Compare with the small molecular organic solvent/inorganic salt system of monohydroxy, protein or enzyme can be extracted into phase, and keep higher activity, are more suitable for proteinic industrialization extraction separation.
Embodiment
Be described in detail the specific embodiment of the present invention below in conjunction with technical scheme.The all non-optimum of selected system and phase-splitting point should not be construed as limitation of the present invention.
The partition ratio of mentioning among the embodiment is to go up phase protein concentration and the following ratio of protein concentration mutually; Protein yield is to go up the protein content of phase and the per-cent of system total protein concentration; The ratio vigor of enzyme and the per-cent of extraction preferment during enzyme is meant mutually than retention rate alive than vigor.
Embodiment 1: the extracting and separating of bovine serum albumin
Bovine serum albumin is made into the protein solution of 1.0g/L.Adopt two kinds of double-aqueous phase systems that it is carried out extracting and separating: (one), take by weighing 2.4g dipotassium hydrogen phosphate and 2.7g 1, ammediol joins in the protein solution that 4.9g prepares; (2), take by weighing 2.0g dipotassium hydrogen phosphate and 2.5g 2, the 3-butyleneglycol joins in the protein solution that 5.5g prepares; (3), take by weighing 1.7g dipotassium hydrogen phosphate and 2.2g 1, the 2-pentanediol joins in the protein solution that 6.1g prepares; (4), taking by weighing 2.0g dipotassium hydrogen phosphate and 2.0g glycol ether joins in the protein solution that 6.0g prepares.With the system mixing, make the solid salt dissolving, leave standstill 2.0h under the room temperature.After the system for the treatment of is stable, sampling detect upper and lower mutually in proteic concentration, calculation of distribution coefficient, protein yield, and last albumen in mutually carried out the circular dichroism spectrum analysis.The result shows that bovine serum albumin all is distributed in phase in above-mentioned four double-aqueous phase systems, and partition ratio is respectively 4.92,7.84,3.85 and 5.47, and protein yield is 86.4%, 91.1%, 77.6% and 88.6%.Circular dichroism spectrum detects and shows that albuminous secondary structure remains unchanged.
Embodiment 2: the extracting and separating of α, beta-amylase
(1) extracting and separating of α-Dian Fenmei: the enzyme solution that α-Dian Fenmei (Ye Huamei) is made into about 1.0g/L.Adopt two kinds of systems that it is carried out extracting and separating: (one), take by weighing 1.8g sodium sulfate and 2.3g 1, the 4-butyleneglycol joins in the enzyme solution that 5.9g prepares; (2), taking by weighing 1.8g sodium sulfate and 2.3g 1,3 butylene glycol joins in the 5.9g enzyme liquid.With the system mixing, make the salt dissolving, leave standstill 3.0h under the room temperature.After the system for the treatment of is stable, measures upper and lower protein concentration and enzymic activity in mutually, the partition ratio of calculating albumen and enzyme, yield etc.The result shows that α-Dian Fenmei mainly is distributed in phase, and proteic partition ratio is respectively 4.54 and 7.67 in two individual system, and protein yield is 83.3% and 87.5%, and enzyme is 97.8% and 93.3% than retention rate alive.
(2) extracting and separating of beta-amylase: the enzyme solution that beta-amylase (saccharifying enzyme) is made into about 1.0g/L.Adopt two kinds of systems that it is carried out extracting and separating: (one), take by weighing 2.0g ammonium sulfate and 2.8g 1, the 4-butyleneglycol joins in the enzyme solution that 5.2g prepares; (2), taking by weighing 2.0g ammonium sulfate and 2.8g 1,3 butylene glycol joins in the 5.2g enzyme liquid.With the system mixing, make the salt dissolving, leave standstill 3.0h under the room temperature.After the system for the treatment of is stable, measures upper and lower protein concentration and enzymic activity in mutually, the partition ratio of calculating albumen and enzyme, yield etc.The result shows that beta-amylase mainly is distributed in the last phase of two individual system, and the albumen partition ratio is respectively 5.45 and 5.32, and protein yield is 85.6% and 85.3%, and enzyme is 98.3% and 90.0% than retention rate alive.
Embodiment 3: the extracting and separating of cellulase
Cellulase is made into the enzyme solution of about 0.5g/L.Take by weighing 2.8g 1,4-butyleneglycol and 2.0g ammonium sulfate join in the enzyme liquid that 5.2g prepares, and mixing is treated to leave standstill 3.5h after the salt dissolving.Upper and lower protein concentration and the enzymic activity in mutually of sampling and measuring calculated the indexs such as partition ratio, yield of albumen and enzyme.The result shows that cellulase mainly is distributed in 1, the last phase of 4-butyleneglycol enrichment, and the albumen partition ratio is 5.88, and protein yield is 86.0%, and enzyme is 74.8% than retention rate alive.
Embodiment 4: the extracting and separating of inulinase
Inulinase is made into the enzyme solution of about 0.5g/L.Adopt two kinds of systems that it is carried out extracting and separating: (one), take by weighing 2.0g ammonium sulfate and 2.8g 1, the 4-butyleneglycol joins in the enzyme solution that 5.2g prepares; (2), taking by weighing 2.0g ammonium sulfate and 2.8g 1,3 butylene glycol joins in the 5.2g enzyme liquid.With the system mixing, treat to leave standstill 2.5h after the salt dissolving.Upper and lower protein concentration and the enzymic activity in mutually of sampling and measuring calculated the indexs such as partition ratio, yield of albumen and enzyme.The result shows that inulinase mainly is distributed in the last phase of two double-aqueous phase systems, and the albumen partition ratio is respectively 11.05 and 6.94, and protein yield is 91.7% and 87.4%, and enzyme is 61.2% and 73.7% than retention rate alive.
Embodiment 5: the extracting and separating of alkaline lipase
Alkaline lipase is made into the enzyme solution of about 0.5g/L.Take by weighing 2.2g dipotassium hydrogen phosphate and 2.5g 1, the 4-butyleneglycol joins in the enzyme liquid that 5.3g prepares, and mixing is treated to leave standstill 4.0h after the salt dissolving.Upper and lower protein concentration and the enzymic activity in mutually of sampling and measuring calculated the indexs such as partition ratio, yield of albumen and enzyme.The result shows that alkaline lipase mainly is distributed in 1, the last phase of 4-butyleneglycol enrichment, and the albumen partition ratio is 4.92, and protein yield is 84.4%, and enzyme is 99.8% than retention rate alive.
Claims (3)
1. the method for aqueous two-phase extraction isolated protein and enzyme, it is characterized in that: polyvalent alcohol and inorganic salt are joined in protein or the enzyme solution by mass percentage, stir, leave standstill phase-splitting under the room temperature, the mass percent of described polyvalent alcohol is 10～40%, the inorganic salt mass percent is 10～40%, and extraction temperature is 15～30 ℃, and stirring, leave standstill the phase-splitting time is 0.5～4.0h.
2. the method for a kind of aqueous two-phase extraction isolated protein according to claim 1 and enzyme, its feature also is: selected polyvalent alcohol is the one or more combination of binary propyl alcohol, binary butanols, binary amylalcohol, ternary butanols, ternary amylalcohol, glycol ether, dipropylene glycol.
3. the method for a kind of aqueous two-phase extraction isolated protein according to claim 1 and enzyme, its feature also is: selected inorganic salt are one or more combinations of sodium salt, sylvite, ammonium salt.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910309154A CN101693735A (en) | 2009-10-30 | 2009-10-30 | Method for extracting protein and enzyme by aqueous two-phase extraction technology |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101962635A (en) * | 2010-10-20 | 2011-02-02 | 山东农业大学 | Three-step two aqueous phase extraction method of ginger protease |
| CN104474740A (en) * | 2014-12-01 | 2015-04-01 | 江苏远洋药业股份有限公司 | Purifying device of amino acid |
| CN105585430A (en) * | 2014-10-22 | 2016-05-18 | 中国石油化工股份有限公司 | Method of extracting 2,3-butanediol from fermentation liquid |
| CN106906200A (en) * | 2017-04-12 | 2017-06-30 | 山西新国大医药科技有限公司 | It is a kind of for synthesizing isolating and purifying and immobilization coupling process for the PA ase of Amoxicillin |
| CN111733104A (en) * | 2020-07-02 | 2020-10-02 | 上海澜海生物科技有限公司 | Novel preparation method and application of black tea fungus protein |
| CN111909234A (en) * | 2020-08-21 | 2020-11-10 | 南京康齐生物科技有限公司 | Preparation method of allium sativum fruit protein |
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2009
- 2009-10-30 CN CN200910309154A patent/CN101693735A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101962635A (en) * | 2010-10-20 | 2011-02-02 | 山东农业大学 | Three-step two aqueous phase extraction method of ginger protease |
| CN105585430A (en) * | 2014-10-22 | 2016-05-18 | 中国石油化工股份有限公司 | Method of extracting 2,3-butanediol from fermentation liquid |
| CN105585430B (en) * | 2014-10-22 | 2017-06-20 | 中国石油化工股份有限公司 | A kind of method that 2,3 butanediols are extracted from zymotic fluid |
| CN104474740A (en) * | 2014-12-01 | 2015-04-01 | 江苏远洋药业股份有限公司 | Purifying device of amino acid |
| CN106906200A (en) * | 2017-04-12 | 2017-06-30 | 山西新国大医药科技有限公司 | It is a kind of for synthesizing isolating and purifying and immobilization coupling process for the PA ase of Amoxicillin |
| CN106906200B (en) * | 2017-04-12 | 2018-06-15 | 山西新国大医药科技有限公司 | A kind of PA ase for being used to synthesize Amoxicillin isolates and purifies and immobilization coupling process |
| CN111733104A (en) * | 2020-07-02 | 2020-10-02 | 上海澜海生物科技有限公司 | Novel preparation method and application of black tea fungus protein |
| CN111733104B (en) * | 2020-07-02 | 2023-09-22 | 上海澜海生物科技有限公司 | New preparation method and application of black tea fungus protein |
| CN111909234A (en) * | 2020-08-21 | 2020-11-10 | 南京康齐生物科技有限公司 | Preparation method of allium sativum fruit protein |
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