CN101063114A - Method for pectic enzyme separating purification by double aqueous phase extraction system - Google Patents
Method for pectic enzyme separating purification by double aqueous phase extraction system Download PDFInfo
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Abstract
The invention discloses a method to separate and purify pectic enzyme with double water phase extractive system, which comprises the following steps: mixing carbowax, ammonia sulfate and water; producing mixed solution; shaking the mixed solution evenly; stewing; phase-splitting; adding into pectic enzyme liquid; shaking evenly; extracting and phase-splitting at room temperature; separating upper phase liquid carbowax to reserve; mixing with potassium sodium and water; back-extracting at room temperature; separating lower phase; filtering with film; getting pectic enzyme. This invention can optimize preparing craft, shorten operating period and increase receiving ratio of the product.
Description
Technical field
The present invention relates to a kind of separating and purifying technology of protein biological activity target product, particularly a kind of method of pectic enzyme separating purification by double aqueous phase extraction system.
Background technology
Biological products, the pharmacological action of especially protein-based biomacromolecule and its physiologically active are closely-related.Therefore, in the separation and purification process, must keep extracting operation under its bioactive prerequisite according to the characteristics of target product.Two phase aqueous extraction system is considered this present situation exactly, considers a kind of new separation technology that keeps biological activity to develop simultaneously based on the liquid-liquid extraction theory.
Aqueous two phase extraction technique is the effective ways of biomacromolecules such as separation and purification protein mixture.Aqueous two-phase system normally is made up of water miscible two kinds of polymkeric substance or a kind of water-soluble polymers and a kind of salt are formed, and compares with general separating and purifying technology, advantage such as the double water-phase technology has that the processing capacity is big, energy consumption is low, easy continuous operation and engineering amplification; In addition, biomacromolecule such as protein, enzyme, nucleic acid etc. directly do not contact with organic solvent, therefore, aspects such as separation and purification biomacromolecule are subjected to extensive attention, are a kind of bioseparation technology that is easy to industrial applications (P AAlbertsson.Partition of cells and Macromolecules 3 that development potentiality is arranged
Rd[M] .New York:John Wiley and Sons, 1986.).
The key of successfully utilizing aqueous two phase extraction technique separation and Extraction target protein is to select suitable double-aqueous phase system.The double-aqueous phase system of being used widely in biochemical engineering at present is polymer/polymer system and polymer/inorganic salt system, and polyoxyethylene glycol (PEG)/dextran (dextran) system and polyoxyethylene glycol (PEG)/phosphate system or polyoxyethylene glycol (PEG)/sulfate system are respectively the two representatives.
Polygalacturonase (Pectinases) is the general name of the class of enzymes of decompose pectin matter, is prozyme.All there is great application value (Kashyap D R the aspect in that food-processing, feed processing, weaving, papermaking, environment protection, inducing plant be disease-resistant etc.; Vohra P K; Chopra S; et al.Applicationsof pectinases in the commercial sector:a review[J] .BioresourceTechnology; 2001,77:215-227.).The method of polygalacturonase separation and purification has a lot, such as ammonium sulfate precipitation, organic solvent deposit, chromatography or the like (Jia Yue, bow love monarch, Qiu Lina, Wang Jiansen. the progress of polygalacturonase separation and purification and analytical procedure. industrial microorganism .2005,35 (1): 55-58).
The great majority of enzyme industry at present adopt salting-out process, and this method can only obtain comprising the crude enzyme liquid of thalline, salt, foreign protein and other solid substances, and this kind of enzyme is difficult to satisfy the specification of quality of food grade.In addition, present polygalacturonase extracting method organic solvent in operating process directly contacts the inactivation that causes enzyme easily with polygalacturonase, and this also is the low one of the main reasons of homemade pectinase activity.
Summary of the invention
The objective of the invention is to overcome the shortcoming of above-mentioned prior art, provide a kind of not only stable, and can keep the biological activity of product, and simplify the operational path of preparation polygalacturonase product, reduced the method for the pectic enzyme separating purification by double aqueous phase extraction system of production cost.
For achieving the above object, the technical solution used in the present invention is: at first under 20 ℃, polyoxyethylene glycol, ammonium sulfate and water are mixed and made into mixing solutions, wherein the quality percentage composition of polyoxyethylene glycol is 10%-30%, the quality percentage composition of ammonium sulfate is 10%-30%, and all the other are water; The mixing solutions vibration is shaken up, leave standstill the pectase liquid that adds mixing solutions 10% after the phase-splitting again, vibration shakes up, enzyme activity in measuring up and down mutually after the phase-splitting fully respectively, by up and down mutually in the size of enzyme activity can calculate the partition ratio of aqueous two-phase extraction, size according to partition ratio, can select optimum two phase aqueous extraction system, partition ratio is big more, distribution effects is good more, at room temperature leave standstill after 5-10 minute and extract phase-splitting, the polygalacturonase major part enter the phase polyoxyethylene glycol mutually in, phase liquid polyoxyethylene glycol is standby in the separation; Under 20 ℃, with the isolating phase liquid polyoxyethylene glycol of going up, Seignette salt and water mix, wherein the quality percentage composition of polyoxyethylene glycol is 10%-30%, the quality percentage composition of Seignette salt is 10%-30%, surplus is a water, at room temperature leave standstill and stripped in 5-10 minute, the enzyme activity in measuring up and down mutually after the phase-splitting fully respectively calculates the partition ratio that double water-phase is stripped, size according to partition ratio, can select optimum double water-phase reextraction system, partition ratio is big more, and distribution effects is good more, the polygalacturonase major part concentrates on down the phase aqueous phase, to be separated out down, adopt membrane filtration, filter out polygalacturonase.
Because the double water-phase reagent that the present invention adopts is polyoxyethylene glycol/ammonium sulfate, polyoxyethylene glycol has maintenance and stabilization to the biological activity of target substance, ammonium sulfate has provide protection to proteinic biological activity in the aqueous solution simultaneously, polygalacturonase is after the double-aqueous phase system separation and purification, the polygalacturonase partition ratio is higher than the report in the document, therefore, this double-aqueous phase system is specially adapted to the separation and Extraction of biologically active substance; Other biological such as the present invention and reextraction isolation technique is integrated, with optimizing the preparation technology of polygalacturonase, shortens the operational cycle, improves the yield of product.
Embodiment
The present invention is described in further detail below in conjunction with embodiment, but the present invention is not limited to following examples.
Embodiment 1, the pre-treatment of fermented liquid: because the polygalacturonase that aspergillus niger produces is an extracellular enzyme, at first adopt filtering method slightly to carry; Under 20 ℃, polyoxyethylene glycol, ammonium sulfate and water are mixed and made into mixing solutions, wherein the quality percentage composition of polyoxyethylene glycol is 27%, and the quality percentage composition of ammonium sulfate is 19%, and all the other are water; The mixing solutions vibration is shaken up, leave standstill the pectase liquid that adds mixing solutions 10% after the phase-splitting again, vibration shakes up, at room temperature leave standstill after 5-10 minute and extract phase-splitting, enzyme activity in measuring up and down mutually after the phase-splitting fully respectively, this moment, the partition ratio K of polygalacturonase can reach maximum value K=11.42; The polygalacturonase major part enter the phase polyoxyethylene glycol mutually in, phase liquid polyoxyethylene glycol is standby in the separation; Under 20 ℃, isolating phase liquid polyoxyethylene glycol, Seignette salt and the water gone up is mixed, wherein the quality percentage composition of polyoxyethylene glycol is 27%, the quality percentage composition of Seignette salt is 20%, surplus is a water, at room temperature leave standstill and stripped in 5-10 minute, the partition ratio of reextraction polyoxyethylene glycol/Seignette salt double-aqueous phase system can reach maximum value K=5.0, after stripping, the polygalacturonase major part concentrates on down in the phase (water), and its partition ratio is 5.0, will be separated out down, adopt membrane filtration, filter out polygalacturonase.
Embodiment 2, the pre-treatment of fermented liquid: because the polygalacturonase that aspergillus niger produces is an extracellular enzyme, at first adopt filtering method slightly to carry; Under 20 ℃, polyoxyethylene glycol, ammonium sulfate and water are mixed and made into mixing solutions, wherein the quality percentage composition of polyoxyethylene glycol is 25%, and the quality percentage composition of ammonium sulfate is 15%, and all the other are water; The mixing solutions vibration is shaken up, leave standstill the pectase liquid that adds mixing solutions 10% after the phase-splitting again, vibration shakes up, at room temperature leave standstill after 5-10 minute and extract phase-splitting, enzyme activity in measuring up and down mutually after the phase-splitting fully respectively, this moment polygalacturonase partition ratio K=2.15, the polygalacturonase major part enter the phase polyoxyethylene glycol mutually in, phase liquid polyoxyethylene glycol is standby in the separation; Under 20 ℃, isolating phase liquid polyoxyethylene glycol, Seignette salt and the water gone up is mixed, wherein the quality percentage composition of polyoxyethylene glycol is 25%, the quality percentage composition of Seignette salt is 20%, surplus is a water, at room temperature leave standstill and stripped in 5-10 minute, the partition ratio K=3.2 of reextraction polyoxyethylene glycol/Seignette salt double-aqueous phase system, after stripping, the polygalacturonase major part concentrates on down in the phase (water), and its partition ratio is 3.2, will be separated out down, adopt membrane filtration, filter out polygalacturonase.
Embodiment 3, the pre-treatment of fermented liquid: because the polygalacturonase that aspergillus niger produces is an extracellular enzyme, at first adopt filtering method slightly to carry; Under 20 ℃, polyoxyethylene glycol, ammonium sulfate and water are mixed and made into mixing solutions, wherein the quality percentage composition of polyoxyethylene glycol is 27%, and the quality percentage composition of ammonium sulfate is 13%, and all the other are water; The mixing solutions vibration is shaken up, leave standstill the pectase liquid that adds mixing solutions 10% after the phase-splitting again, vibration shakes up, at room temperature leave standstill after 5-10 minute and extract phase-splitting, enzyme activity in measuring up and down mutually after the phase-splitting fully respectively, this moment polygalacturonase partition ratio K=6.67, the polygalacturonase major part enter the phase polyoxyethylene glycol mutually in, phase liquid polyoxyethylene glycol is standby in the separation; Under 20 ℃, isolating phase liquid polyoxyethylene glycol, Seignette salt and the water gone up is mixed, wherein the quality percentage composition of polyoxyethylene glycol is 27%, the quality percentage composition of Seignette salt is 20%, surplus is a water, at room temperature leave standstill and stripped in 5-10 minute, the partition ratio K=4.9 of reextraction polyoxyethylene glycol/Seignette salt double-aqueous phase system, after stripping, the polygalacturonase major part concentrates on down in the phase (water), and its partition ratio is 4.9, will be separated out down, adopt membrane filtration, filter out polygalacturonase.
Embodiment 4, the pre-treatment of fermented liquid: because the polygalacturonase that aspergillus niger produces is an extracellular enzyme, at first adopt filtering method slightly to carry; Under 20 ℃, polyoxyethylene glycol, ammonium sulfate and water are mixed and made into mixing solutions, wherein the quality percentage composition of polyoxyethylene glycol is 10%, and the quality percentage composition of ammonium sulfate is 30%, and all the other are water; Mixing solutions vibration is shaken up, leave standstill the pectase liquid that adds mixing solutions 10% after the phase-splitting again, vibration shakes up, at room temperature leave standstill after 5-10 minute and extract phase-splitting, the polygalacturonase major part enter the phase polyoxyethylene glycol mutually in, phase liquid polyoxyethylene glycol is standby in the separation; Under 20 ℃, isolating phase liquid polyoxyethylene glycol, Seignette salt and the water gone up is mixed, wherein the quality percentage composition of polyoxyethylene glycol is 30%, the quality percentage composition of Seignette salt is 10%, surplus is a water, at room temperature leaves standstill to strip in 5-10 minute, will be separated out down, adopt membrane filtration, filter out polygalacturonase.
Embodiment 5, the pre-treatment of fermented liquid: because the polygalacturonase that aspergillus niger produces is an extracellular enzyme, at first adopt filtering method slightly to carry; Under 20 ℃, polyoxyethylene glycol, ammonium sulfate and water are mixed and made into mixing solutions, wherein the quality percentage composition of polyoxyethylene glycol is 30%, and the quality percentage composition of ammonium sulfate is 10%, and all the other are water; Mixing solutions vibration is shaken up, leave standstill the pectase liquid that adds mixing solutions 10% after the phase-splitting again, vibration shakes up, at room temperature leave standstill after 5-10 minute and extract phase-splitting, the polygalacturonase major part enter the phase polyoxyethylene glycol mutually in, phase liquid polyoxyethylene glycol is standby in the separation; Under 20 ℃, isolating phase liquid polyoxyethylene glycol, Seignette salt and the water gone up is mixed, wherein the quality percentage composition of polyoxyethylene glycol is 10%, the quality percentage composition of Seignette salt is 30%, surplus is a water, at room temperature leaves standstill to strip in 5-10 minute, will be separated out down, adopt membrane filtration, filter out polygalacturonase.
The main feature of aqueous two-phase extraction pectase of the present invention:
1. have preferably biocompatibility owing to polyethylene glycol, and do not have toxicity, enzyme is difficult for inactivation;
2. polyethylene glycol is the most cheap in various high polymers, and polyethylene glycol/the salt system price just simultaneously Suitable, running cost is lower;
3. simple to operate, the processing capacity is big, energy consumption is low, easy continuous operation and engineering are amplified.
Claims (6)
1, a kind of method of pectic enzyme separating purification by double aqueous phase extraction system is characterized in that:
1) at first under 20 ℃, polyoxyethylene glycol, ammonium sulfate and water are mixed and made into mixing solutions, wherein the quality percentage composition of polyoxyethylene glycol is 10%-30%, and the quality percentage composition of ammonium sulfate is 10%-30%, and all the other are water;
2) the mixing solutions vibration is shaken up, leave standstill the pectase liquid that adds mixing solutions 10% after the phase-splitting again, vibration shakes up, and at room temperature leaves standstill after 5-10 minute and extracts phase-splitting, the polygalacturonase major part enter the phase polyoxyethylene glycol mutually in, phase liquid polyoxyethylene glycol is standby in the separation;
3) under 20 ℃, isolating phase liquid polyoxyethylene glycol, Seignette salt and the water gone up is mixed, wherein the quality percentage composition of polyoxyethylene glycol is 10%-30%, the quality percentage composition of Seignette salt is 10%-30%, and surplus is a water, at room temperature leaves standstill and strips in 5-10 minute, the polygalacturonase major part concentrates on down the phase aqueous phase, to be separated out down, adopt membrane filtration, filter out polygalacturonase.
2, the method for pectic enzyme separating purification by double aqueous phase extraction system according to claim 1, it is characterized in that: under 20 ℃, polyoxyethylene glycol, ammonium sulfate and water are mixed and made into mixing solutions, wherein the quality percentage composition of polyoxyethylene glycol is 27%, the quality percentage composition of ammonium sulfate is 19%, and all the other are water; Mixing solutions vibration is shaken up, leave standstill the pectase liquid that adds mixing solutions 10% after the phase-splitting again, vibration shakes up, at room temperature leave standstill after 5-10 minute and extract phase-splitting, the polygalacturonase major part enter the phase polyoxyethylene glycol mutually in, phase liquid polyoxyethylene glycol is standby in the separation; Under 20 ℃, isolating phase liquid polyoxyethylene glycol, Seignette salt and the water gone up is mixed, wherein the quality percentage composition of polyoxyethylene glycol is 27%, and the quality percentage composition of Seignette salt is 20%, and surplus is a water, at room temperature leave standstill and stripped in 5-10 minute, after stripping, during the polygalacturonase major part concentrates on down mutually, will be separated out down, adopt membrane filtration, filter out polygalacturonase.
3, the method for pectic enzyme separating purification by double aqueous phase extraction system according to claim 1, it is characterized in that: under 20 ℃, polyoxyethylene glycol, ammonium sulfate and water are mixed and made into mixing solutions, wherein the quality percentage composition of polyoxyethylene glycol is 25%, the quality percentage composition of ammonium sulfate is 15%, and all the other are water; Mixing solutions vibration is shaken up, leave standstill the pectase liquid that adds mixing solutions 10% after the phase-splitting again, vibration shakes up, at room temperature leave standstill after 5-10 minute and extract phase-splitting, the polygalacturonase major part enter the phase polyoxyethylene glycol mutually in, phase liquid polyoxyethylene glycol is standby in the separation; Under 20 ℃, isolating phase liquid polyoxyethylene glycol, Seignette salt and the water gone up is mixed, wherein the quality percentage composition of polyoxyethylene glycol is 25%, the quality percentage composition of Seignette salt is 20%, and surplus is a water, at room temperature leaves standstill and strips in 5-10 minute, during the polygalacturonase major part concentrates on down mutually, to be separated out down, adopt membrane filtration, filter out polygalacturonase.
4, the method for pectic enzyme separating purification by double aqueous phase extraction system according to claim 1, it is characterized in that: under 20 ℃, polyoxyethylene glycol, ammonium sulfate and water are mixed and made into mixing solutions, wherein the quality percentage composition of polyoxyethylene glycol is 27%, the quality percentage composition of ammonium sulfate is 13%, and all the other are water; Mixing solutions vibration is shaken up, leave standstill the pectase liquid that adds mixing solutions 10% after the phase-splitting again, vibration shakes up, at room temperature leave standstill after 5-10 minute and extract phase-splitting, the polygalacturonase major part enter the phase polyoxyethylene glycol mutually in, phase liquid polyoxyethylene glycol is standby in the separation; Under 20 ℃, isolating phase liquid polyoxyethylene glycol, Seignette salt and the water gone up is mixed, wherein the quality percentage composition of polyoxyethylene glycol is 27%, and the quality percentage composition of Seignette salt is 20%, and surplus is a water, at room temperature leave standstill and stripped in 5-10 minute, after stripping, during the polygalacturonase major part concentrates on down mutually, will be separated out down, adopt membrane filtration, filter out polygalacturonase.
5, the method for pectic enzyme separating purification by double aqueous phase extraction system according to claim 1, it is characterized in that: under 20 ℃, polyoxyethylene glycol, ammonium sulfate and water are mixed and made into mixing solutions, wherein the quality percentage composition of polyoxyethylene glycol is 10%, the quality percentage composition of ammonium sulfate is 30%, and all the other are water; Mixing solutions vibration is shaken up, leave standstill the pectase liquid that adds mixing solutions 10% after the phase-splitting again, vibration shakes up, at room temperature leave standstill after 5-10 minute and extract phase-splitting, the polygalacturonase major part enter the phase polyoxyethylene glycol mutually in, phase liquid polyoxyethylene glycol is standby in the separation; Under 20 ℃, isolating phase liquid polyoxyethylene glycol, Seignette salt and the water gone up is mixed, wherein the quality percentage composition of polyoxyethylene glycol is 30%, the quality percentage composition of Seignette salt is 10%, surplus is a water, at room temperature leaves standstill to strip in 5-10 minute, will be separated out down, adopt membrane filtration, filter out polygalacturonase.
6, the method for pectic enzyme separating purification by double aqueous phase extraction system according to claim 1, it is characterized in that: under 20 ℃, polyoxyethylene glycol, ammonium sulfate and water are mixed and made into mixing solutions, wherein the quality percentage composition of polyoxyethylene glycol is 30%, the quality percentage composition of ammonium sulfate is 10%, and all the other are water; Mixing solutions vibration is shaken up, leave standstill the pectase liquid that adds mixing solutions 10% after the phase-splitting again, vibration shakes up, at room temperature leave standstill after 5-10 minute and extract phase-splitting, the polygalacturonase major part enter the phase polyoxyethylene glycol mutually in, phase liquid polyoxyethylene glycol is standby in the separation; Under 20 ℃, isolating phase liquid polyoxyethylene glycol, Seignette salt and the water gone up is mixed, wherein the quality percentage composition of polyoxyethylene glycol is 10%, the quality percentage composition of Seignette salt is 30%, surplus is a water, at room temperature leaves standstill to strip in 5-10 minute, will be separated out down, adopt membrane filtration, filter out polygalacturonase.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101824070A (en) * | 2010-04-14 | 2010-09-08 | 山东汇盛天泽环境工程有限公司 | Method for separating and purifying protein from corn starch wastewater |
| CN102224160A (en) * | 2008-11-25 | 2011-10-19 | 通用电气健康护理生物科学股份公司 | Aqueous two phase extraction augmented precipitation process for purification of therapeutic proteins |
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| CN106148304A (en) * | 2016-06-22 | 2016-11-23 | 瑞安市智造科技有限公司 | A kind of method of modified poly (ethylene glycol) sodium tartrate Bi-aqueous extraction purifying velardon |
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| CN112194721A (en) * | 2019-10-29 | 2021-01-08 | 西北农林科技大学 | Double-solution mixed extraction system and application thereof in ostrich liver |
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- 2007-05-11 CN CNB2007100178199A patent/CN100529071C/en not_active Expired - Fee Related
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| CN102224160A (en) * | 2008-11-25 | 2011-10-19 | 通用电气健康护理生物科学股份公司 | Aqueous two phase extraction augmented precipitation process for purification of therapeutic proteins |
| CN101824070A (en) * | 2010-04-14 | 2010-09-08 | 山东汇盛天泽环境工程有限公司 | Method for separating and purifying protein from corn starch wastewater |
| CN103275941A (en) * | 2013-06-14 | 2013-09-04 | 齐鲁工业大学 | Method for preparing pyruvic oxidase |
| CN103275941B (en) * | 2013-06-14 | 2014-12-31 | 齐鲁工业大学 | Method for preparing pyruvic oxidase |
| CN106148304A (en) * | 2016-06-22 | 2016-11-23 | 瑞安市智造科技有限公司 | A kind of method of modified poly (ethylene glycol) sodium tartrate Bi-aqueous extraction purifying velardon |
| CN109206539A (en) * | 2018-08-10 | 2019-01-15 | 昆明理工大学 | A kind of method of microwave-assisted aqueous two-phase separation and Extraction bagasse hemicellulose |
| CN109206539B (en) * | 2018-08-10 | 2021-03-02 | 昆明理工大学 | Method for extracting bagasse hemicellulose by microwave-assisted double-aqueous-phase separation |
| CN111363024A (en) * | 2018-12-26 | 2020-07-03 | 深圳翰宇药业股份有限公司 | Method for purifying polypeptide |
| CN112194721A (en) * | 2019-10-29 | 2021-01-08 | 西北农林科技大学 | Double-solution mixed extraction system and application thereof in ostrich liver |
| CN112194721B (en) * | 2019-10-29 | 2022-12-23 | 西北农林科技大学 | Double-solution mixed extraction system and application thereof in ostrich liver |
| CN112626041A (en) * | 2020-12-24 | 2021-04-09 | 齐鲁工业大学 | Process for extracting lipoxygenase from soybean whey protein wastewater by two aqueous phases |
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