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A method for optimizing the conditions of two-phase extraction and its application

An optimization method, a two-phase technology, which is applied in the preparation methods of peptides, chemical instruments and methods, biochemical equipment and methods, etc., can solve the problems of large experimental workload, inconsistency, and difficulty in quickly finding a two-phase system, etc., To achieve the effect of high economic efficiency, not easy to denature inactivation

Active Publication Date: 2022-01-14
XIAMEN MEDICAL COLLEGE
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  • Description
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  • Application Information

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Problems solved by technology

Therefore, the simple method of single-factor variable or multi-factor variable method is a huge amount of experimental work, and methods such as orthogonal test and response surface are not comprehensive enough, and the results of comprehensive single-factor experiments are not consistent.
Most studies use single-factor or multi-factor variables of the system, orthogonal experiments or response surface experiments to optimize the conditions, but because the extraction rules have not been systematically explored, and because the extracted raw materials are different, the aqueous two-phase system will change accordingly. changes, it is difficult to quickly find the best two-phase system to achieve the best extraction effect, and seldom carry out further research and application of secondary extraction methods

Method used

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  • A method for optimizing the conditions of two-phase extraction and its application
  • A method for optimizing the conditions of two-phase extraction and its application
  • A method for optimizing the conditions of two-phase extraction and its application

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Effect test

Embodiment 1

[0044] Embodiment 1: the optimization method summary of two-phase extraction condition

[0045] A method for optimizing the conditions of two-phase extraction, also known as "5 2 *3+1 aqueous two-phase experiment method”, taking PEG / salt two-phase aqueous phase as an example, includes the following steps:

[0046] 1) Determine the two-phase type used in two-phase extraction, which is the first phase and the second phase; among the present embodiment, the first phase is PEG, and the second phase is salt (such as sodium dihydrogen phosphate, ammonium sulfate, etc. );

[0047]2) Take the mass fraction of the first phase PEG as the vertical axis and the mass fraction of the second phase salt as the horizontal axis to draw a phase diagram;

[0048] 3) Based on the two-node line of the phase diagram, select five critical phase-forming points located at the center of the two-node line, and the PEG mass fraction interval between two adjacent points among the five critical phase-form...

Embodiment 2

[0054] Example 2: Taking the separation and purification of β-glucosidase from abalone viscera as an example to illustrate the optimization method of two-phase extraction conditions

[0055] 1. Raw materials and reagents

[0056] The viscera of wrinkled abalone, provided by the School of Ocean and Earth Sciences, Xiamen University, was stored frozen at -20°C until use; p-nitrophenyl-β-D-glucopyranoside (p-NPG): Beijing Suolaibao Technology Co., Ltd.; p-nitrophenol, sodium carbonate, sodium dihydrogen phosphate (NaH 2 PO 4 ), citric acid, acetic acid, sodium acetate, sodium hydroxide (all of analytical grade), polyethylene glycol (Polyethylene glycol, PEG) 600, 1000, 1500, 2000, 4000: Sinopharm Chemical Reagent Co., Ltd.

[0057] 2. Instruments and equipment

[0058] High-speed tissue masher: A25 emulsifier, Shanghai Ouhe; ultrasonic cell crusher: Scientz-ⅡD, Ningbo Xinzhi Biotechnology Co., Ltd.; high-speed refrigerated centrifuge: sigma 3-18K, Germany; ice machine: AF100, ...

Embodiment 3

[0087] Example 3: Explore the optimal conditions and rules for the separation and purification of β-glucosidase from abalone viscera by optimizing the conditions of two-phase extraction

[0088] 1. The effect of the composition of the two-phase system on the extraction effect of β-glucosidase

[0089] Reference Example 2, using abalone viscera homogenate as raw material, using the optimization method of two-phase extraction conditions "5 2 *3+1 two-phase aqueous phase experiment method", using PEG / salt two-phase aqueous system to separate and purify β-glucosidase from abalone viscera, and explore PEG600 / 1000 / 1500 / 2000 / 4000 (PEG400 is too small due to molecular weight, The extraction effect is not good; PEG6000 has too large a molecular weight and is too viscous, and the measurement effect of the extraction effect is not good, so there is no comparison significance, so the experiment has been excluded) After the construction of the optimal two-phase system for extracting β-gluc...

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Abstract

The invention discloses a method for optimizing two-phase extraction conditions and its application. The design method of the two-phase aqueous phase experiment process of the present invention can be applied to the experimental design of the two-phase aqueous extraction target substance to explore its optimal two-phase aqueous system composition. This optimization method is based on the two-node line of the phase diagram of each two-phase system, and at the same time makes a more comprehensive comparison of the horizontal and vertical points, breaking the mass fraction of traditional comparison methods such as the single-factor variable method and the orthogonal method. Choices are limited and incomplete.

Description

technical field [0001] The invention belongs to the technical field of bioseparation engineering, and in particular relates to an optimization method of two-phase extraction conditions and its application. Background technique [0002] Most of the existing separation and extraction methods use traditional and common separation and extraction methods, such as salting out, dialysis, column chromatography, ultrafiltration, etc. These methods have complex processes, long time consumption, large loss of target enzymes during the separation and purification process, In the separation process, the cost of various types of materials is huge, and the economic benefits are not high. [0003] Although aqueous two-phase extraction is often used as a biological separation technique, the regularity of its separation and extraction has not been explored in detail, and due to the particularity of phase formation in two-phase aqueous phase, the composition and extraction effect of the system...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C07K14/765C07K1/14B01D11/04
CPCC12N9/2445C07K14/765B01D11/04C12Y302/01021
Inventor 曾颖张亚楠陈仲巍曾臻余莉莉
Owner XIAMEN MEDICAL COLLEGE
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