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Method for preparing sericin polypeptides with aqueous enzymatic method

The technology of sericin and water enzymatic method is applied in the field of biotechnology polypeptide preparation, which can solve the problems of polluted protein, amino acid damage, difficult control of hydrolyzed molecular weight, etc., and achieve the effects of low cost, simple process and high quality.

Inactive Publication Date: 2016-02-17
ZHEJIANG ESIGMA BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When preparing sericin polypeptide by acid and alkali hydrolysis, the hydrolysis molecular weight is not easy to control, and it will cause racemization to amino acids, some amino acids will be destroyed, and protein will be polluted

Method used

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  • Method for preparing sericin polypeptides with aqueous enzymatic method
  • Method for preparing sericin polypeptides with aqueous enzymatic method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Add 6g of sericin protein to 94g of water, adjust the pH of the reaction solution to 8 with 1mol / L sodium hydroxide aqueous solution, add 0.02g of Alcalase2.4L alkaline protease, raise the temperature to 60°C, and maintain the pH=8 for 1 hour after enzymolysis, Use 1mol / L hydrochloric acid to adjust the pH of the reaction solution to 7, add 0.015g AS1398 neutral protease, and enzymolyze at 50°C, pH=7 for 1 hour; then inactivate at 85°C for 20 minutes, centrifuge, take the supernatant, and concentrate , freeze-drying to obtain 5.39g sericin polypeptide. The amino acid composition before and after enzymolysis is shown in Table 1. The relative molecular mass distribution of the enzymolysis products is shown in Table 2:

[0016] Amino acid composition (g / 100g protein) before and after enzymolysis of table 1

[0017] amino acid

Sericin

Enzyme hydrolyzate

Ser

25.3

32.5

Asp

18.3

20.5

Gly

10.9

9.9

Thr

7.4...

Embodiment 2

[0021] Add 4g of sericin to 96g of water, adjust the pH of the reaction solution to 8.5 with 1mol / L sodium hydroxide aqueous solution, add 0.03g of Alcalase2.4L alkaline protease, raise the temperature to 55°C, and maintain the pH = 8.5. After 1.5 hours of enzymolysis, Use 1mol / L hydrochloric acid to adjust the pH of the reaction solution to 7, add 0.016g AS1398 neutral protease, and enzymolyze at 45°C, pH=7 for 1.5 hours; then inactivate at 80°C for 30 minutes, centrifuge, take the supernatant, and concentrate , freeze-drying to obtain 3.36g sericin polypeptide. The amino acid composition before and after enzymolysis is shown in Table 3, and the relative molecular mass distribution of the enzymolysis products is shown in Table 4:

[0022] Amino acid composition (g / 100g protein) before and after enzymolysis of table 3

[0023] amino acid

Sericin

Enzyme hydrolyzate

Ser

25.3

33.2

Asp

18.3

20.1

Gly

10.9

10.3

Th...

Embodiment 3

[0027] Add 7g of sericin to 93g of water, adjust the pH of the reaction solution to 9 with 1mol / L sodium hydroxide aqueous solution, add 0.04g of Alcalase2.4L alkaline protease, raise the temperature to 65°C, and maintain the pH=9 for 2 hours after enzymatic hydrolysis, Use 1mol / L hydrochloric acid to adjust the pH of the reaction solution to 7, add 0.018g AS1398 neutral protease, enzymolyze at 55°C, pH=7 for 2 hours; then inactivate at 90°C for 40 minutes, centrifuge, take the supernatant, and concentrate 1. Freeze-drying to obtain 6.43g sericin polypeptide. The amino acid composition before and after enzymolysis is shown in Table 5, and the relative molecular mass distribution of the enzymolysis product is shown in Table 6:

[0028] Amino acid composition (g / 100g protein) before and after enzymolysis of table 5

[0029]

[0030]

[0031] Table 6 Relative molecular mass distribution of enzymatic hydrolysis products

[0032] relative molecular mass

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Abstract

The invention provides a method for preparing sericin polypeptides with an aqueous enzymatic method. According to the method, sericin is taken as a raw material, a 4%-7% sericin aqueous solution is prepared, the PH is adjusted to range from 8 to 9 by the aid of a 1 mol / L sodium hydroxide solution, Alcalase2.4L alkaline protease is added, enzymolysis is performed at the temperature of 55-65 DEG C for 1-2 h, then the PH is adjusted to 7 by the aid of 1 mol / L hydrochloric acid, AS1398 neutral protease is added, enzymolysis is performed at the temperature of 45-55 DEG C for 1-2 h, inactivation is performed at the temperature of 80-90 DEG C for 20-40 min, centrifugation is performed, a supernate is taken, concentrated and freeze-dried, and the sericin polypeptides are obtained. The sericin is degraded twice by the aid of the Alcalase2.4L alkaline protease and the AS1398 neutral protease, so that the enzymolysis rate is greatly increased. Meanwhile, the technology is simple, the method is safe and environment-friendly, the cost is low, the obtained sericin polypeptides have high quality, and industrial production conditions are provided.

Description

technical field [0001] The invention relates to the technical field of biotechnology polypeptide preparation, in particular to a method for preparing sericin polypeptide from sericin protein. Background technique [0002] Sericin is a globular protein that is coated on the periphery of silk fibroin in silk. Its properties are similar to animal glue. It protects and glues silk fibroin. It has a relative molecular weight of 1.4-314,000 Da and is composed of 18 kinds of amino acids. Glycine, serine, alanine and tyrosine have pharmacological effects, health care functions and high nutritional value. At the same time, sericin also has good moisture absorption and desorption, anti-oxidation, ultraviolet absorption, adhesion, etc., so Sericin has good application prospects in cosmetics, medical biomaterials, functional materials, food and feed additives and other fields. At the same time, in the production of modern silk industry, sericin is largely discarded during silk processin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/06
Inventor 吴中华张小朋陈贵才徐天华何奇雷闻鸣张丽佳
Owner ZHEJIANG ESIGMA BIOTECH CO LTD
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