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A preparation method and device for a polypeptide with a controllable molecular weight range

A molecular weight and molecular weight cut-off technology, applied in the biological field, can solve problems such as difficult peptides, difficult to prepare peptides, and protease residues in enzymatic hydrolysis products, and achieve the effects of increasing unit loading, improving enzymatic hydrolysis efficiency, and reducing steric hindrance

Active Publication Date: 2020-09-08
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the preparation process of these polypeptides based on enzymatic hydrolysis and further membrane separation, on the one hand, the polypeptides released during the protein enzymatic hydrolysis process will continue to be enzymatically hydrolyzed in the solution. Polypeptides with molecular weight cut-offs lead to a too broad molecular weight distribution range of the prepared polypeptides; on the other hand, traditional methods are difficult to prepare polypeptides with a higher molecular weight range, especially difficult to prepare polypeptides with an average molecular weight higher than that of the enzyme, and it is easy to cause enzymatic hydrolysis products. Some protease residues

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  • A preparation method and device for a polypeptide with a controllable molecular weight range

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Embodiment 1

[0063] Example 1 A preparation device for a polypeptide with a controllable molecular weight range

[0064] A preparation device for polypeptides with a controllable molecular weight range, such as figure 1 As shown, the device includes a protein solution storage bottle 1, a fluid delivery pump 2, a heat exchanger 3 and a tubular ceramic membrane 6 connected in sequence;

[0065] Porous microspheres 7 loaded with immobilized enzymes are filled in the tubular ceramic membrane 6;

[0066] The filling volume of the porous microsphere 7 loaded with immobilized enzyme is 60-100% of the inner pore volume of the tubular ceramic membrane 6, preferably 70-90%;

[0067] The inner hole diameter of the tubular ceramic membrane 6 is 1-30mm, preferably 3-20mm;

[0068] The molecular weight cut-off range of the tubular ceramic membrane 6 is 1-800kDa, preferably 3-500kDa, more preferably 5-300kDa;

[0069] Both the inlet and the outlet of the tubular ceramic membrane 6 are provided with sc...

Embodiment 2

[0078] The raw material is soybean protein isolate with an average molecular weight greater than 23kDa, and the protein content exceeds 93% (dry basis). Weigh 5kg of soybean protein isolate and prepare 80L of a solution with a concentration of 6.25%:

[0079] (1) Enzyme immobilization: Pepsin is covalently coupled to porous ceramic microspheres with a diameter of 0.5 mm. The loading capacity of the pepsin is 10 mg / g. The diameter range is 100nm-20μm;

[0080] (2) Membrane loading: The ceramic microspheres loaded with pepsin are loaded into a tubular ceramic membrane with an inner diameter of 5 mm. The molecular weight cut-off of the ceramic membrane is 5 kDa. Microsphere encapsulation, the filling volume of porous ceramic microspheres loaded with pepsin in the ceramic membrane is 80% of the total volume of the pores in the ceramic membrane;

[0081] (3) Synchronous enzymatic hydrolysis-ultrafiltration process: put 80L soybean protein isolate solution with a concentration of 5...

Embodiment 3

[0085] The raw material is gelatin with an average molecular weight greater than 80kDa, and the protein content exceeds 96% (dry basis). Take 10kg of the gelatin and configure it into 100L of a 10% solution:

[0086] (1) Enzyme immobilization: Trypsin is covalently coupled to porous glass microspheres with a diameter of 1 mm. Pore ​​diameter ranges from 200nm to 10μm;

[0087] (2) Membrane loading: the glass microspheres loaded with trypsin are packed into a tubular ceramic membrane with an inner hole diameter of 10 mm, the molecular weight cut-off of the ceramic membrane is 30 kDa, and the inlet and outlet of the ceramic membrane are provided with screens with a pore size of 0.02 mm. The filling volume of the porous glass microspheres loaded with trypsin in the ceramic membrane is 75% of the total volume of the pores in the ceramic membrane;

[0088] (3) Synchronous enzymolysis-ultrafiltration process: put 100L gelatin solution with a concentration of 10% in a 200L stainless...

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Abstract

The invention relates to a preparation method and device of polypeptide with a controllable molecular weight range. The method comprises the following steps: (1) carrying out enzyme immobilization: covalently coupling proteinase on porous microspheres; (2) filling into a membrane: filling the porous microspheres loaded with an immobilized enzyme into a tubular ceramic membrane, wherein a membrane molecular weigh cutoff range is 1kDa-800kDa; (3) carrying out simultaneous enzymolysis-ultrafiltration: preparing protein liquid and adjusting the pH (Potential of Hydrogen); adjusting the temperature of the protein liquid and introducing the protein liquid into the tubular ceramic membrane of step (2); collecting tubular ceramic membrane permeated liquid; and (4) concentrating and drying: concentrating and drying the tubular ceramic membrane permeated liquid collected by step (3). According to the preparation method provided by the invention, the polypeptide with the uniform quality can be prepared and the molecular weight of the polypeptide can be accurately controlled; the method provided by the invention is simple in process, few in steps and easy to operate; the molecular weight of the prepared polypeptide can be greater than that of the proteinase and the polypeptide does not contain impurities including the proteinase and the like; and the polypeptide is convenient for subsequent utilization and can be industrially produced in a large scale.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a preparation method of a polypeptide, in particular to a preparation method and device of a polypeptide with a controllable molecular weight range. Background technique [0002] Polypeptides are compounds formed by connecting amino acids with peptide bonds. They can be directly extracted from animals or plants, or can be obtained by chemical or biological degradation of proteins as raw materials. Peptides have the characteristics of easy digestion, fast absorption and high utilization in the human body. Its absorption mechanism is mainly based on the transmembrane transport of peptide transport carriers. It is an important mechanism of the traditional metabolic model that believes that proteins must be hydrolyzed into free amino acids to be absorbed. Replenish. The peptide absorption process is mainly divided into three types, including H-dependent + Concentration or Ca 2+ Concentra...

Claims

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Application Information

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IPC IPC(8): C12P21/06C07K1/34C07K1/14
CPCC07K1/14C07K1/34C12P21/06
Inventor 张贵锋孔英俊苏志国
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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