Method for regulating and controlling crystal form of lysozyme by using ionic liquid

A technology of ionic liquid and lysozyme, which is applied in the direction of biochemical equipment and methods, enzymes, hydrolytic enzymes, etc., can solve problems such as poor crystal form consistency, crystallization rate needs to be further improved, and solid-liquid separation is not easy to achieve, etc. Achieve the effects of convenient operation, easy process control, and easy solid-liquid separation

Active Publication Date: 2021-08-13
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the consistency of crystal forms prepared by traditional crystal form control methods is not good, the crystallization rate needs to be further improved, and solid-liquid separation is not easy to achieve

Method used

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  • Method for regulating and controlling crystal form of lysozyme by using ionic liquid
  • Method for regulating and controlling crystal form of lysozyme by using ionic liquid
  • Method for regulating and controlling crystal form of lysozyme by using ionic liquid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] A method utilizing ionic liquid to regulate lysozyme crystal form, comprising the following steps:

[0040] (1) The water-soluble imidazolium-based ionic liquid 1-ethyl-3-methylimidazolium tetrafluoroborate [Emim]BF 4 Join in solution I, make water-soluble imidazolium-based ionic liquid [Emim] BF 4 The mass concentration is 1%, and the solution I has a pH value of 5.0 and contains 0.1M (NH 4 ) 2 SO 4 Potassium dihydrogen phosphate buffer;

[0041] (2) Add excess lysozyme to the liquid obtained in step (1), and stir the lysozyme at 20° C. with a stirring rate of 200 rpm for 20 min;

[0042] (3) the suspension obtained in step (2) is filtered through a sand core funnel under reduced pressure rapidly to obtain a saturated solution of lysozyme;

[0043] (4) Cool the lysozyme-saturated solution obtained in step (3) to 0°C at a cooling rate of 5°C / h, maintain stirring at 150rpm during this period, and maintain crystal growth for 1h;

[0044](5) The crystal suspension ob...

Embodiment 2

[0046] A method utilizing ionic liquid to regulate lysozyme crystal form, comprising the following steps:

[0047] (1) Water-soluble imidazolium-based ionic liquid 1-ethyl-3-methylimidazolium bromide [Emim]Br is added in solution I, the mass concentration that makes water-soluble imidazolium-based ionic liquid [Emim]Br is 3%, The solution I is 6.0 with a pH value of 5% containing 0.1M (NH 4 ) 2 SO 4 dipotassium hydrogen phosphate buffer;

[0048] (2) Add excess lysozyme to the liquid obtained in step (1), and stir the lysozyme at 30° C. at a stirring rate of 250 rpm for 30 min;

[0049] (3) the suspension obtained in step (2) is filtered through a sand core funnel under reduced pressure rapidly to obtain a saturated solution of lysozyme;

[0050] (4) Cool the lysozyme-saturated solution obtained in step (3) to 5°C at a cooling rate of 10°C / h, maintain stirring at 250rpm during this period, and maintain crystal growth for 3h;

[0051] (5) The crystal suspension obtained in...

Embodiment 3

[0053] A method utilizing ionic liquid to regulate lysozyme crystal form, comprising the following steps:

[0054] (1) Water-soluble imidazolium-based ionic liquid 1,3-dimethylimidazolium iodonium salt [dmim] I is added in solution I, the mass concentration that makes water-soluble imidazolium-based ionic liquid [dmim] I is 1%, described Solution I is pH 5.0, containing 0.1M (NH 4 ) 2 SO 4 Potassium dihydrogen phosphate buffer;

[0055] (2) Add excess lysozyme to the liquid obtained in step (1), and stir the lysozyme at 20° C. with a stirring rate of 200 rpm for 20 min;

[0056] (3) the suspension obtained in step (2) is filtered through a sand core funnel under reduced pressure rapidly to obtain a saturated solution of lysozyme;

[0057] (4) Cool the lysozyme-saturated solution obtained in step (3) to 0°C at a cooling rate of 5°C / h, maintain stirring at 150rpm during this period, and maintain crystal growth for 1h;

[0058] (5) The crystal suspension obtained in step (4)...

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Abstract

The invention discloses a method for regulating and controlling the crystal form of lysozyme by using an ionic liquid. The method comprises the following steps: (1) adding the ionic liquid into a buffer solution to ensure that the mass concentration of the ionic liquid is 1%-3%; (2) adding the lysozyme into the liquid obtained in the step (1), and performing stirring to obtain turbid liquid; (3) filtering the turbid liquid obtained in the step (2) to obtain a lysozyme saturated solution; (4) cooling the lysozyme saturated solution obtained in the step (3) to 0-5 DEG C, and keeping stirring for 1-3 hours during the period; and (5) centrifuging the crystal turbid liquid obtained in the step (4), discarding the supernatant, taking the precipitate, drying to obtain lysozyme crystals with different crystal forms, and performing preserving at a low temperature of 4 DEG C. According to the method, the crystal form of the lysozyme is regulated and controlled through the variety and the adding amount of the ionic liquid; process control is easy to achieve; operation is convenient; and the crystallization rate is high.

Description

technical field [0001] The invention relates to the production field of lysozyme, in particular to a method for regulating the crystal form of lysozyme by using ionic liquid. Background technique [0002] As a non-specific immune factor existing in normal body fluids and tissues of the human body, lysozyme has a variety of pharmacological effects, and it has antibacterial, antiviral, and antitumor effects. Lysozyme has the function of destroying the bacterial cell wall structure, and is an essential tool enzyme for cell fusion operations in genetic engineering and cell engineering. Lysozyme is also a non-toxic, non-side-effect protein, and has a certain bacteriolytic effect, which can be used as a food preservative. [0003] The crystallization of protein is a complex physical and chemical process, which is much more difficult than the crystallization of general small molecular compounds. There are many factors that affect the protein crystallization process. First, protei...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/36C12N9/98
CPCC12N9/2462C12Y302/01017C12N9/98Y02P20/54
Inventor 王占忠党乐平昝明阳李娜
Owner TIANJIN UNIV
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