Separation method for silk fibroin peptides with small molecular weights

Abstract

The invention provides a separation method for silk fibroin peptides with small molecular weights. The separation method comprises the steps: degumming and dissolving silkworm silk to obtain a solution; adding the solution into a glucan G10 column, carrying out elution by using sterile water, and carrying out collection to obtain a desalted silk fibroin protein aqueous solution; freeze-drying thedesalted silk fibroin protein aqueous solution to obtain freeze-dried silk fibroin proteins; adding a freeze-dried silk fibroin protein solution into a glucan G50 column, eluting the silk fibroin proteins by using the sterile water, and collecting the silk fibroin proteins by using different tubes; screening the silk fibroin peptides of which the molecular weights are smaller than 1500Da in collecting tubes in the step (d), adding the silk fibroin peptides into a glucan G15 column again, eluding the silk fibroin peptides by using the sterile water, collecting the silk fibroin peptides by usingdifferent collecting tubes, and carrying out separation to obtain multilevel silk fibroin peptides with small molecular weights being 1500Da or smaller. The multilevel silk fibroin peptides with small molecular weights being 1500Da or smaller are obtained through dissolution with neutral salts and gradient separation of different molecular sieve columns. Meanwhile, the hydrophilicity of the silkfibroin peptides is made better by using a specific freeze-drying-dissolving method.

Description

technical field

[0001] The invention relates to the technical field of textiles, in particular to a method for separating silk fibroin with small molecular weight. Background technique

[0002] Bombyx mori silk is a natural animal protein synthesized and secreted by silkworms. It has a wide range of sources. Its silk fibroin has good biocompatibility and is composed of 20 kinds of amino acids that can be absorbed by the human body. The final degradation products are amino acids or small peptides, which are easily absorbed by cells. Absorption or phagocytosis will not cause obvious immune response. A large number of researches at home and abroad have shown that silk fibroin materials can support the growth of various cells, so silk fibroin is an ideal raw material for the development of skin care products. However, as a skin care product, whether it can be absorbed by the skin is very important, which has certain requirements for the molecular weight of silk protein. Before ...

Examples

Embodiment 1

[0081] (1) Put the raw silkworm silk into 0.2% sodium carbonate aqueous solution at a bath ratio of 1:50 (g / mL), and treat it three times at 98-100°C for 30 minutes each time, and then deionized water The silk is fully cleaned, loosened, and dried in an oven at 60°C to obtain degummed silkworm silk fiber.

[0082] (2) Weigh the degummed silkworm silk fibroin and dissolve it in an aqueous solution of calcium chloride-ethanol with a molar ratio of 1:2 at a bath ratio of 1:20 (g / mL), and dissolve at 70°C for 5 to 7 hours to obtain silk fibroin Dissolve the mixed solution.

[0083] (3) Weigh dextran G-10 and put it into sufficient deionized water, and put it in a boiling water bath for 1.5 hours. After settling for 2 hours, pressurize the column with a peristaltic pump and equilibrate the column with 5 times column volume of deionized water three times.

[0084] (4) When the equilibrium water drops to the surface of the column material for the last time, add 5-15ml (10-50mg / ml) ...

Embodiment 2

[0087] (1) The silk fibroin collected and freeze-dried in Example 1 was prepared into an aqueous solution of 30-80 mg / ml, and filtered with a 0.45 μm membrane to remove microparticles.

[0088] (2) Weigh dextran G-50 and put it into sufficient deionized water, and put it in a boiling water bath for 1.5 hours. After settling for 2 hours, pressurize the column with a peristaltic pump and equilibrate the column with 5 times column volume of deionized water three times.

[0089](3) When the equilibrium water drops to the surface of the column material for the last time, add 5-15ml silk fibroin aqueous solution. When the silk fibroin solution completely enters the surface of the column material, carefully add sterilized deionized water to elute, and the peristaltic pump controls The flow rate of the eluent is 100-120mL / h; the effluent is collected in separate tubes until the silk fibroin is exhausted to obtain silk fibroin aqueous solutions with different molecular weight distribut...

Embodiment 3

[0095] (1) Put the raw silkworm silk into 0.2% sodium carbonate aqueous solution at a bath ratio of 1:50 (g / mL), and treat it three times at 98-100°C for 30 minutes each time, and then deionized water The silk is fully cleaned, loosened, and dried in an oven at 60°C to obtain degummed silkworm silk fiber.

[0096] (2) Weigh the degummed silkworm silk fibroin and dissolve it in 9M lithium bromide aqueous solution at a bath ratio of 1:10-30 (g / mL), and dissolve it at 65°C for 5-7 hours until the silk fiber is completely dissolved to obtain silk fibroin protein mixture solution.

[0097] (3) Weigh dextran G-10 and put it into sufficient deionized water, and put it in a boiling water bath for 1.5 hours. After settling for 2 hours, pressurize the column with a peristaltic pump and equilibrate the column with 5 times column volume of deionized water three times.

[0098] (4) When the equilibrium water drops to the surface of the column material for the last time, add 5-15ml (10-50...

technical field

[0001] The invention relates to the technical field of textiles, in particular to a method for separating silk fibroin with small molecular weight. Background technique

[0002] Bombyx mori silk is a natural animal protein synthesized and secreted by silkworms. It has a wide range of sources. Its silk fibroin has good biocompatibility and is composed of 20 kinds of amino acids that can be absorbed by the human body. The final degradation products are amino acids or small peptides, which are easily absorbed by cells. Absorption or phagocytosis will not cause obvious immune response. A large number of researches at home and abroad have shown that silk fibroin materials can support the growth of various cells, so silk fibroin is an ideal raw material for the development of skin care products. However, as a skin care product, whether it can be absorbed by the skin is very important, which has certain requirements for the molecular weight of silk protein. Before ...

Examples

Embodiment 1

[0081] (1) Put the raw silkworm silk into 0.2% sodium carbonate aqueous solution at a bath ratio of 1:50 (g / mL), and treat it three times at 98-100°C for 30 minutes each time, and then deionized water The silk is fully cleaned, loosened, and dried in an oven at 60°C to obtain degummed silkworm silk fiber.

[0082] (2) Weigh the degummed silkworm silk fibroin and dissolve it in an aqueous solution of calcium chloride-ethanol with a molar ratio of 1:2 at a bath ratio of 1:20 (g / mL), and dissolve at 70°C for 5 to 7 hours to obtain silk fibroin Dissolve the mixed solution.

[0083] (3) Weigh dextran G-10 and put it into sufficient deionized water, and put it in a boiling water bath for 1.5 hours. After settling for 2 hours, pressurize the column with a peristaltic pump and equilibrate the column with 5 times column volume of deionized water three times.

[0084] (4) When the equilibrium water drops to the surface of the column material for the last time, add 5-15ml (10-50mg / ml) ...

Embodiment 2

[0087] (1) The silk fibroin collected and freeze-dried in Example 1 was prepared into an aqueous solution of 30-80 mg / ml, and filtered with a 0.45 μm membrane to remove microparticles.

[0088] (2) Weigh dextran G-50 and put it into sufficient deionized water, and put it in a boiling water bath for 1.5 hours. After settling for 2 hours, pressurize the column with a peristaltic pump and equilibrate the column with 5 times column volume of deionized water three times.

[0089](3) When the equilibrium water drops to the surface of the column material for the last time, add 5-15ml silk fibroin aqueous solution. When the silk fibroin solution completely enters the surface of the column material, carefully add sterilized deionized water to elute, and the peristaltic pump controls The flow rate of the eluent is 100-120mL / h; the effluent is collected in separate tubes until the silk fibroin is exhausted to obtain silk fibroin aqueous solutions with different molecular weight distribut...

Embodiment 3

[0095] (1) Put the raw silkworm silk into 0.2% sodium carbonate aqueous solution at a bath ratio of 1:50 (g / mL), and treat it three times at 98-100°C for 30 minutes each time, and then deionized water The silk is fully cleaned, loosened, and dried in an oven at 60°C to obtain degummed silkworm silk fiber.

[0096] (2) Weigh the degummed silkworm silk fibroin and dissolve it in 9M lithium bromide aqueous solution at a bath ratio of 1:10-30 (g / mL), and dissolve it at 65°C for 5-7 hours until the silk fiber is completely dissolved to obtain silk fibroin protein mixture solution.

[0097] (3) Weigh dextran G-10 and put it into sufficient deionized water, and put it in a boiling water bath for 1.5 hours. After settling for 2 hours, pressurize the column with a peristaltic pump and equilibrate the column with 5 times column volume of deionized water three times.

[0098] (4) When the equilibrium water drops to the surface of the column material for the last time, add 5-15ml (10-50...