阅读说明：本技术 一种大分子量角蛋白的制备方法及其应用 (Preparation method and application of high molecular weight keratin ) 是由 史劲松 龚劲松 叶金鹏 许正宏 蒋敏 钱建瑛 于 2020-06-02 设计创作，主要内容包括：本发明公开了一种大分子量角蛋白的制备方法及其应用,本发明通过角蛋白酶与谷氨酰胺转氨酶联合使用,通过角蛋白酶来水解羊毛得到可溶性角蛋白水解液,通过离心,在上清液中加入Tgase来交联角蛋白,经过透析和冷冻干燥即可得到大分子角蛋白粉末。本发明的方法可以从废弃羊毛提取羊毛角蛋白并将其交联,羊角蛋白的分子量可以达到120kDa。该大分子量角蛋白的制备方法工艺简单,条件易控,原料为废弃的羊毛,废物再利用,不引入化学试剂,绿色环保,节约能源；本发明还提供所述大分子量角蛋白在生物材料上的应用,该纳米材料垫具有生物相容性以及促进伤口愈合的作用。(The invention discloses a preparation method and application of high molecular weight keratin. The method can extract and crosslink the wool keratin from the waste wool, and the molecular weight of the wool keratin can reach 120 kDa. The preparation method of the high molecular weight keratin has the advantages of simple process and easily controlled conditions, the raw material is waste wool, the waste is recycled, no chemical reagent is introduced, the method is green and environment-friendly, and the energy is saved; the invention also provides application of the high molecular weight keratin to a biological material, and the nano material pad has the effects of biocompatibility and wound healing promotion.)

1. A method for producing high molecular weight keratin, comprising the steps of:

(1) cleaning wool fibers, degreasing for 12-24 hours by using a degreasing solvent, cleaning to remove the degreasing solvent remained on the surface, drying, and shearing to obtain degreased wool fibers;

(2) adding the degreased wool fibers obtained in the step (1) into water, adjusting the pH value to 9-10, adding keratinase, hydrolyzing at 45-55 ℃ for 12-24 h, and centrifuging to obtain keratin hydrolysate;

(3) adjusting the pH value of the keratin hydrolysate obtained in the step (2) to 6-8, adding glutamine transaminase, reacting at 45-55 ℃ for 24-48 h, and inactivating enzyme to obtain a keratin solution;

(4) and (4) dialyzing, freezing and drying the keratin solution obtained in the step (3) to obtain the high molecular weight keratin.

2. The method according to claim 1, wherein the amount of keratinase added is 15,000 to 20,000U/g wool.

3. The method according to claim 1, wherein said transglutaminase is added in an amount of 15-20U/g keratin.

4. The method according to claim 1, wherein in step (2), the bath ratio of the degreased wool fibers to the water is 4-6: 100.

5. The method according to claim 1, wherein in step (1), the degreasing solvent is one or more of ether, benzene, carbon disulfide, acetone, and chloroform.

6. The method according to claim 1, wherein in the step (1), the degreasing is performed at 65-75 ℃ for 12-24 h.

7. The method of claim 1, wherein in the step (4), the dialysis is performed for 2-4 days by using a dialysis bag with a molecular cut-off of 8,000-14,000, and the water is changed for 4-6 h during the dialysis.

8. The method according to claim 1, wherein in the step (2), the centrifugation is carried out at 6000 to 10000rpm for 5 to 10 min.

9. A keratin produced by the method of any one of claims 1 to 8.

10. Use of the keratin of claim 9 in a bio-nanomaterial pad.

Technical Field

The invention relates to a preparation method and application of high molecular weight keratin, and belongs to the technical field of biology.

Background

China is a big country for producing and using wool, a large amount of non-spinnable short fibers and thick fibers exist every year, and damaged wool in the pretreatment process is discarded, so that resources are wasted, and the environmental pressure is increased. How to reuse the waste wool is always a hot point of research.

The processing and hydrolyzing process of the wool keratin comprises a chemical treatment method, a physical processing method and a biological enzyme hydrolysis method. The chemical treatment method adopts strong acid and strong base for matching treatment, the nutrition value of the hydrolysis product is damaged more, and the environmental pollution is serious and the application is minimum. The physical processing method, i.e. the high-temperature high-pressure hydrolysis method, has high requirements on equipment and energy consumption, so the production cost is high. Chemical treatment and physical processing can only obtain polypeptide with very low molecular weight; the biological enzyme hydrolysis method is to degrade wool into soluble wool by adopting high-activity proteolytic enzyme or microorganism under mild and proper conditions, the method has the advantages of less pollution, low energy consumption and mild reaction conditions, but the problems of difficult reaction control, poor enzyme action specificity and the like exist in the use process of the keratinase, and the prepared keratin has lower molecular weight (<60kDa) and poor mechanical performance.

Disclosure of Invention

In order to solve the technical problems, the invention uses the combination of keratinase and glutamine transaminase, hydrolyzes wool by the keratinase to obtain soluble keratin hydrolysate, adds Tgase into supernatant fluid by centrifugation to crosslink keratin, and obtains macromolecular keratin powder by dialysis and freeze drying. The method provided by the invention is simple to operate, and the molecular weight of the obtained wool keratin is 120 kDa.

The first object of the present invention is to provide a process for the preparation of high molecular weight keratin, comprising the steps of:

(1) cleaning wool fibers, degreasing for 12-24 hours by using a degreasing solvent, cleaning to remove the degreasing solvent remained on the surface, drying, and shearing to obtain degreased wool fibers;

(2) adding the degreased wool fibers obtained in the step (1) into water, adjusting the pH value to 9-10, adding keratinase, hydrolyzing at 45-55 ℃ for 12-24 h, and centrifuging to obtain keratin hydrolysate;

(3) adjusting the pH value of the keratin hydrolysate obtained in the step (2) to 6-8, adding glutamine transaminase, reacting at 45-55 ℃ for 24-48 h, and inactivating enzyme to obtain a keratin solution;

(4) and (4) dialyzing, freezing and drying the keratin solution obtained in the step (3) to obtain the high molecular weight keratin.

Furthermore, the addition amount of the keratinase is 15,000-20,000U/g wool.

Further, the amount of the transglutaminase added is 15-20U/g keratin.

Further, in the step (2), the bath ratio of the degreased wool fibers to the water is 4-6: 100.

Further, in the step (1), the degreasing solvent is one or more of ether, benzene, carbon disulfide, acetone and chloroform.

Further, in the step (1), the degreasing is carried out for 12-24 hours at 65-75 ℃.

Further, in the step (4), the dialysis is carried out for 2-4 days by adopting a dialysis bag with the molecular interception amount of 8,000-14,000, and water is changed for 4-6 h in the dialysis process.

Further, in the step (2), the centrifugation is carried out at 6000-10000 rpm for 5-10 min.

The second purpose of the invention is to provide the keratin prepared by the method.

The third purpose of the invention is to provide the application of the keratin in the biological nano-material pad.

Further, the application specifically includes: weighing wool keratin powder, dissolving the wool keratin powder in hexafluoroisopropanol under a room temperature condition, stirring the solution under a sealed condition until the wool keratin powder is uniformly dissolved, weighing poly (beta-hydroxybutyrate valerate) and adding the poly (beta-hydroxybutyrate valerate) into the solution to prepare macromolecular keratin/poly (beta-hydroxybutyrate valerate) spinning solution, sealing the solution for 3 hours, and stirring the solution for later use, wherein the mass fraction of the macromolecular keratin/poly (beta-hydroxybutyrate valerate) spinning solution is 4-6 wt%, and the mass ratio of the macromolecular keratin to the poly (beta-hydroxybutyrate valerate) is 3:7-5: 5; extracting the dissolved macromolecular keratin/poly (beta-hydroxybutyrate valerate) spinning solution by using an injector, and fixing the macromolecular keratin/poly (beta-hydroxybutyrate valerate) spinning solution on a constant flow pump, wherein the flow rate of the constant flow pump is 0.5-1.0 mL/h; then connecting the positive electrode of the high-voltage electrostatic generator to the needle head, connecting the negative electrode of the high-voltage electrostatic generator to a receiving plate, paving a layer of aluminum foil paper on the receiving plate for receiving the electrostatic spinning fiber, starting electrostatic spinning, and performing under the condition of room temperature, wherein the humidity is lower than 75%, the distance is controlled to be 15-25cm, and the voltage is controlled to be 10-20 kV; spinning for 4 hours each time, taking down the aluminum foil paper after the spinning is finished, and storing under a dry condition.

The invention has the beneficial effects that:

(1) the keratin prepared by the method has a molecular weight of up to 120kDa, belongs to soluble protein, and has improved heat stability.

(2) The invention utilizes keratinase and TGase when preparing the keratin with large molecular weight, has mild reaction condition and small pollution to the environment.

(3) The high molecular weight keratin prepared by the invention improves the survival rate of cells and promotes cell migration.

(4) The high molecular weight keratin prepared by the method can improve the mechanical property of the keratin nano fiber, wherein the Young modulus is improved by 41 percent.

Drawings

FIG. 1 is an SDS-PAGE pattern of wool keratin before and after crosslinking

FIG. 2 is an XRD diffractogram of wool and wool keratin before and after crosslinking

FIG. 3 is an infrared spectrum of wool and wool keratin before and after crosslinking

FIG. 4 is a circular dichroism chart of wool keratin before and after crosslinking

FIG. 5 shows the distribution of the structural content of wool keratin before and after crosslinking

FIG. 6 shows the results of the thermodynamic analysis of wool and wool keratin before and after crosslinking

FIG. 7 shows the effect of crosslinked keratin on cell viability

FIG. 8 is a graph showing the effect of cross-linking keratin on cell migration

FIG. 9 is SEM image and diameter distribution of nanofibers prepared by composite electrospinning of keratin and poly (beta-hydroxybutyrate valerate) (PHBV) before and after crosslinking, AB, CD and EF are PHBV, PHBV/30% hydrolyzed keratin and PHBV/30% crosslinked keratin respectively

FIG. 10 is an infrared spectrum of nanofibers obtained by electrospinning

FIG. 11 is an XRD diffractogram of electrospun nanofibers

FIG. 12 is a stress-strain curve of nanofibers prepared by composite electrospinning of keratin and poly (β -hydroxybutyrate valerate) before and after crosslinking

Fig. 13 shows the effect of electrospun nanofibers on wound healing in mice.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.