CN105088540A - Method for preparing nano fibroin material on basis of tyrosinase/polyphenol medium - Google Patents

Abstract

The invention discloses a method for preparing nano-silk fibroin material based on tyrosinase/polyphenolic mediator, which utilizes tyrosinase to catalyze the oxidation of tyrosine in silk fibroin and phenolic hydroxyl group in polyphenolic mediator to form a reaction The active quinone structure promotes the reaction between silk fibroin and exogenous functional amino compounds to prepare functional nano silk fibroin materials. The specific steps are as follows: (1) preparation of silk fibroin solution; (2) catalyzing the cross-linking of oxidized silk fibroin and amino compounds with tyrosinase, and adding polyphenol mediators to the system to promote the reaction after a period of reaction; (3) nano Forming of silk fibroin material: after the reaction of silk fibroin and amino compounds, the solution is spread through flow extension, air-dried or freeze-dried at room temperature to form a film, and the spinning solution is prepared with formic acid, and the nano silk fibroin material is spun by electrospinning technology. Compared with the preparation of functional silk fibroin materials by the traditional chemical cross-linking method, the invention has high enzyme catalysis efficiency, mild enzyme treatment conditions, and obvious improvement in performance of nano silk fibroin materials.

Description

A method for preparing nano silk fibroin material based on tyrosinase/polyphenol mediator

technical field

The present invention relates to a method for preparing nano-silk fibroin material based on tyrosinase/polyphenolic mediator, in particular to a method of using tyrosinase to catalyze the oxidation of silk fibroin and polyphenolic mediator to promote the interaction between silk fibroin and external The invention relates to a method for preparing a functional nano silk fibroin material by combining source functional amino compounds, which belongs to the field of textile biotechnology.

Background technique

Silk fibroin has good biocompatibility, and the silk fibroin film processed from it is widely used in the construction of tissue engineering materials. Natural silk fibroin materials have poor toughness and high water dissolution rate. In order to improve the mechanical properties and functionality of regenerated silk fibroin membrane materials, relevant research has been carried out on the modification of silk fibroin and its composite materials at home and abroad. Among them, chemical methods are widely used. Generally, glutaraldehyde, polycarboxylic acid, etc. are used to cross-link silk fibroin or graft with exogenous functional compounds. While improving the properties of silk fibroin materials, these methods also have certain deficiencies, including problems such as the easy generation of chemical harmful substance residues. Biological methods have the advantages of safety, high efficiency, specificity, and environmental friendliness. Enzymatically modifying silk fibroin materials not only maintains the original excellent properties of silk fibroin, but also endows silk fibroin materials with new application properties. The impact on the environment is also small, so it has certain application prospects.

The modification effect of biological enzymes on silk fibroin materials is related to the structure of silk fibroin and the selectivity of enzymes. The residues of hydrophobic glycine and alanine in silk fibroin are relatively high, followed by serine (containing hydroxymethyl) and tyrosine (containing phenolic hydroxyl) residues with polar groups. Tyrosinase is a polyphenol oxidase with catalytic oxidation activity, which can catalyze the oxidation of tyrosine residues containing phenolic hydroxyl groups to successively generate o-phenol and dopaquinone, and can further react with functional compounds containing amino groups to produce silk A method is provided for the functional modification of the protein. The research results show that because the tyrosine residues in silk fibroin are distributed in the macromolecular chain composed of alanine and glycine with strong hydrophobicity, it has a certain impact on the accessibility of enzymatic reactions. Therefore, based on The efficiency of functional modification of silk fibroin by tyrosinase method needs to be improved.

Polyphenolic compounds include tea polyphenols, caffeic acid, ferulic acid, etc. These compounds also have a phenolic hydroxyl structure similar to tyrosine. Polyphenolic compounds can also be catalyzed and oxidized by tyrosinase to form a highly reactive active quinone structure (A). The active quinone molecules can not only polymerize with each other (A-A-A), but also react with two molecules of amino compounds (B). Reaction to form cross-links (B-A-B) between amino compound molecules. According to this characteristic, in the reaction of oxidized silk fibroin (SF) catalyzed by tyrosinase, silk fibroin can not only directly react with exogenous amino compounds to form a direct cross-link (SF-B); An appropriate amount of polyphenolic medium system, on the basis of oxidizing polyphenolic mediators to generate quinone active groups, forms the indirect grafting (SF-A-B) of amino compounds on the surface of silk fibroin with the active quinone structure as a bridge, thereby improving silk fibroin. The efficiency of enzymatic functional modification, on this basis to prepare functional nano-silk fibroin materials.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for preparing nano-silk fibroin materials based on tyrosinase/polyphenol mediator, aiming at improving the grafting of silk fibroin and exogenous amino compounds under tyrosinase-catalyzed conditions Reaction efficiency, on this basis to prepare functional silk fibroin materials.

In order to solve the above-mentioned technical problems, the present invention uses tyrosinase to catalyze the oxidation of silk fibroin and polyphenolic mediators, promotes the cross-linking between silk fibroin and exogenous functional amino compounds, and combines electrospinning to prepare functional nano-silk fibroin The method of the material, the specific process and steps are as follows:

(1) Preparation of silk fibroin solution: dissolving mulberry silk with lithium bromide or calcium chloride to prepare silk fibroin solution;

Treatment process prescription and conditions: Add mulberry silk to the aqueous solution system of lithium bromide or calcium chloride, dissolve it at 30-80°C, put the silk fibroin solution into a dialysis bag, dialyze it in deionized water for 8-36 hours, and then filter it to produce The obtained concentration is 10～50g/L silk fibroin solution;

(2) Tyrosinase/polyphenolic mediator catalyzes the crosslinking of silk fibroin and amino compounds: add tyrosinase and amino compounds to the silk fibroin solution in step (1), and add polyphenols after reacting for 0.5 to 8 hours Mediator, continue to process under the same temperature conditions for 0.5 to 24 hours;

Treatment process prescription and conditions: tyrosinase 1～100U/ml, amino compound 1～50g/L, polyphenol mediator 1～10g/L, temperature 20～60℃, pH range 6.0～8.0;

(3) Forming of nano-silk fibroin material: spread the silk fibroin solution reacted in step (2) smoothly in the forming mold by casting, air-dry at room temperature to form a film or under the condition of -50°C ~ -20°C Freeze-drying to form a film; dissolve the silk film with 98% formic acid, prepare spinning solution, and spin nano silk material by electrospinning technology;

Spinning process and conditions: the concentration of silk fibroin in the spinning solution is 10-45%, the spinning voltage is 10-40kV, the spinning speed is 0.3-2ml/h, and the spinning distance between the needle tip of the syringe and the surface of the metal roller is 5 ~20cm.

A method for preparing nano-silk fibroin material based on tyrosinase/polyphenols mediator, wherein lithium bromide solution, lithium bromide-ethanol aqueous solution or calcium chloride-ethanol aqueous solution are selected for preparation of the silk fibroin solution; the tyrosinase Varieties are derived from animals, plants or microorganisms; the polyphenolic mediators include catechin, epicatechin, caffeic acid, ferulic acid and chlorogenic acid; the amino compounds include chemical finishing agents containing amino groups, functional Peptides, chitosan, polylysine, elastin and gelatin.

The invention utilizes tyrosinase to catalyze the reaction between oxidized silk fibroin and exogenous amino compounds, and improves the efficiency of enzymatic modification of silk fibroin on the basis of oxidizing polyphenol mediators to generate quinone active groups, thereby constructing functional nano silk fibroin materials . Compared with the preparation of silk fibroin composite film by traditional chemical cross-linking method, the present invention has the following advantages:

(1) Enzyme catalytic efficiency is high, utilize tyrosinase to catalyze the oxidation of silk fibroin and polyphenols mediator, promote the enzymatic catalytic efficiency in the cross-linking reaction of silk fibroin and amino compound, and the dosage of enzyme preparation is less;

(2) Enzyme treatment conditions are moderate, and silk fibroin functional modification is carried out under low temperature and near neutral conditions, which has the advantages of low energy consumption and safe treatment process, and avoids environmental pollution caused by chemical cross-linking reactions. Defects in many aspects such as low material safety;

(3) The properties of nano-silk fibroin materials have been significantly improved. Modification of silk fibroin based on tyrosinase/polyphenol mediator method not only has low water solubility, but also has specific functionalities. Compatibility is also good.

Detailed ways

Using tyrosinase and polyphenolic mediators to catalyze the crosslinking of silk fibroin and exogenous amino compounds to prepare functional nano silk fibroin materials, specific examples are as follows:

Example 1

(1) Preparation of silk fibroin solution: add mulberry silk to lithium bromide-ethanol-water (mass ratio 45:45:10) solution, dissolve at 60°C, put the silk fibroin solution into a dialysis bag, and dialyze in deionized water for 24 hours. Change the water once an hour during this period, and filter to obtain 20g/L silk fibroin aqueous solution after dialysis;

(2) Add 10U/ml tyrosinase and 5g/L elastin to the silk fibroin solution in step (1), treat at 30°C and pH7 for 4 hours, then add 2g/L catechin, and continue the treatment for 12 hours ;

(3) Forming of nano-silk fibroin material: Spread the silk fibroin solution after the reaction in step (3) flatly in a polytetrafluoroethylene forming mold, and air-dry to form a film at room temperature; dissolve the silk fibroin film with 98% formic acid, and prepare a concentration of 15% silk fibroin spinning solution, using electrospinning technology to spin nano silk fibroin material, wherein the spinning voltage is 15kV, the spinning speed is 0.6ml/h, and the spinning distance between the needle tip of the syringe and the surface of the metal roller is 10cm.

Silk fibroin nanomaterial sample 1: tyrosinase was added in the treatment, and catechin was not added;

Silk fibroin nanomaterial sample 2: adding tyrosinase and catechin in the treatment;

After the above process, the survival rate of NIH/3T3 cells immersed in the silk fibroin/elastin composite membrane medium for 24 hours was evaluated, and the breaking strength and elongation at break of the nano silk fibroin material were measured. The results showed that the NIH/3T3 cell survival rate of sample 1 was 80%, the breaking strength and elongation at break were 2.1MPa and 1.9% respectively; the cell survival rate of sample 2 was 83%, the breaking strength and elongation at break Can respectively reach 2.8MPa, 2.8%.

Example 2

(1) Preparation of silk fibroin solution: add mulberry silk to calcium chloride-ethanol-water (molar ratio 1:1:7), dissolve at 70°C, put the silk fibroin solution into a dialysis bag, and dialyze in deionized water for 24 hours , during which the water was changed once an hour, and filtered to obtain 18g/L silk fibroin aqueous solution after dialysis;

(2) Add 8U/ml tyrosinase and 2g/L chitosan to the silk fibroin solution in step (1), add 1g/L caffeic acid after processing at 30°C and pH7 for 3 hours, and continue processing for 15 hours ;

(3) Forming of nano-silk fibroin material: spread the solution after the reaction in step (3) in a polytetrafluoroethylene molding mold, and air-dry to form a film at room temperature; dissolve the silk fibroin film with 98% formic acid, and prepare a 15% The silk fibroin spinning liquid adopts the electrospinning technology to spin the nano silk fibroin material, wherein the spinning voltage is 20kV, the spinning speed is 0.8ml/h, and the spinning distance between the needle tip of the syringe and the surface of the metal roller is 12cm.

Silk fibroin nanomaterial sample 3: no tyrosinase and caffeic acid were added in the treatment;

Silk fibroin nanomaterial sample 4: tyrosinase was added in the treatment, but caffeic acid was not added;

Silk fibroin nanomaterial sample 5: add tyrosinase and caffeic acid in the treatment;

After the above process, samples 3, 4, and 5 were treated with 50% ethanol solution and immersed in 30°C aqueous solution for 6 hours to measure the water dissolution rate and antibacterial rate of the silk film material to Staphylococcus aureus. Wherein, the water dissolution loss rate of sample 3 is 12.2%, and the bacteriostasis rate is 85%; the water dissolution loss rate of sample 4 is 6.2%, and the bacteriostasis rate is 91%; The bacteria rate is 95%.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

Claims (2)

1. prepare the method for nano silk cellulosic material based on tyrosinase/Polyphenols amboceptor for one kind, particularly one utilizes tyrosinase catalysis to be oxidized fibroin and Polyphenols amboceptor, promote that fibroin albumen is combined with outer source functional amino-compound, prepare the method for functional nano fibroin membrane material, concrete technology and step as follows:

(1) silk fibroin solution preparation: dissolve mulberry silk with lithium bromide or calcium chloride, prepare silk fibroin solution;

Treatment process prescription and condition: mulberry silk is joined in the water solution system of lithium bromide or calcium chloride, after dissolving at 30 ~ 80 DEG C, silk fibroin solution is loaded bag filter, filter after dialysing 8 ~ 36 hours in deionized water, obtained concentration is 10 ~ 50g/L silk fibroin solution;

(2) tyrosinase/Polyphenols amboceptor catalysis fibroin and amino-compound are cross-linked: in step (1) silk fibroin solution, add tyrosinase and amino-compound, react and add Polyphenols amboceptor after 0.5 ~ 8 hour, under identical temperature conditions, continue process 0.5 ~ 24 hour;

Treatment process prescription and condition: tyrosinase 1 ~ 100U/ml, amino-compound 1 ~ 50g/L, Polyphenols amboceptor 1 ~ 10g/L, temperature 20 ~ 60 DEG C, pH scope 6.0 ~ 8.0;

(3) nano silk cellulosic material is shaping: by step (2) reacted silk fibroin solution, makes it smoothly in formation mould sprawl by prolonging stream, at room temperature air-dry film forming or under-50 DEG C ~-20 DEG C conditions freeze drying film forming; Carry out fibroin membrane dissolving with 98% formic acid, preparation spinning solution, adopt Static Spinning technology spinning nano silk cellulosic material;

Spinning technique and condition: in spinning solution, fibroin concentration is 10 ~ 45%, spinning voltage is 10 ~ 40kV, and spinning speed is 0.3 ~ 2ml/h voltage, and the spinning spacing of syringe needle point and metal roller surface is 5 ~ 20cm.

2. method according to claim 1, is characterized in that: select lithium-bromide solution, lithium bromide-ethanol water or calcium chloride-ethanol water time prepared by described silk fibroin solution; Described tyrosinase cultivar origin is in animal, plant or microorganism; Described Polyphenols amboceptor comprises catechin, epicatechin, caffeic acid, forulic acid and chlorogenic acid; Described amino-compound comprises containing amino chemical finishing agent, functional polypeptide, shitosan, polylysine, elastin laminin and gelatin.