CN103421201A - Preparation method for antibacterial silk fibroin membrane by biological enzyme method - Google Patents
Preparation method for antibacterial silk fibroin membrane by biological enzyme method Download PDFInfo
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Abstract
The invention discloses a preparation method for an antibacterial silk fibroin membrane by a biological enzyme method, belongs to the field of textile biological technology, and aims to solve the disadvantages that a silk fibroin membrane prepared by a conventional method has a low strength and a hard and brittle membrane body, while avoiding the defects of large pollution and low biological safety of the silk fibroin membrane in a membrane preparing process by a chemical crosslink method. A catalytic oxidation function of tyrosinase is utilized, while achieving an intermolecular cross-linking of silk fibroin, a polypeptide antibacterial agent (such as epsilon-polylysine and L-polyarginine) having a primary amine structure is grafted onto the surface of the silk fibroin, and thus application performance of the silk fibroin membrane is improved; the specific technological process includes: preparing a silk fibroin solution, carrying out catalytic oxidation of the silk fibroin by tyrosinase, carrying out enzymatic grafting by the polypeptide antibacterial agent, drying and forming the membrane. The silk fibroin membrane prepared by the preparation method of the invention not only enables antibacterial performance to be improved, but also enables the strength, the elasticity and other mechanical properties of the silk fibroin membrane material to be improved. Compared with the silk fibroin membrane preparation method based on chemical crosslink, the tyrosinase method used for preparation of the silk fibroin membrane has the advantages of low energy consumption, high efficiency and less pollution, and is beneficial for environmental protection.
Description
Technical field
The present invention relates to the antibiotic silk fibroin membrane preparation method of a kind of biological enzyme, is a kind of tyrosinase catalysis oxygenizement of utilizing, and improves the mechanical property of fibroin protein film, gives the method for fibroin protein film germ resistance simultaneously, belongs to field of textile biotechnology.
Background technology
Fibroin has good biocompatibility and environment friendly, with the mould material of silk fibroin protein solution processing, has nontoxic, nonirritant and antigenicity, characteristics that water-permeable and air permeable is good, is a kind of natural biological goods and slow releasing carrier of medication of excellent property.The performance of fibroin membrane depends on the macromolecular form of silk fibroin and structure, the fibroin membrane aggregated structure made after drying under general condition take that amorphous film body is hard and crisp as main, and physical and mechanical properties is poor, in water, dissolve-loss ratio is high, has limited the application of fibroin protein film.For improving the performance of fibroin membrane material, expand its application in fields such as biological medicines, need to carry out modification to silk fibroin, to fibroin membrane, the preparation method is optimized.
The fibroin protein film method of modifying comprises wet heat treatment, chemically crosslinked, polymer blended etc. with other.Wherein, the chemically crosslinked application is more, ultimate principle be utilize in silk fibroin molecular there is hydroxyl, the characteristics of amino, carboxyl isoreactivity group, make covalent cross-linking between the fibroin macromole by means of linking agents such as glutaraldehyde, polycarboxylic acids, or with functional finish (active monomer), graft polymerization reaction occurs.Also there is certain deficiency in these class methods when improving the fibroin membrane performance, comprise that the crosslinking reaction condition is harsh, easily produce the problem such as the residual and environmental pollution of chemical objectionable impurities.Utilize biotechnology to silk fibroin material carry out functional modification have efficient, single-minded, action condition is gentle and eco-friendly advantage, day by day becomes the focus of material subject area research.
Tyrosine oxidase (Tyrosinase) is a kind of polyphenoloxidase with catalytic oxidation activity, can comprise that the phenolic hydroxyl structure substrate of TYR, L-3,4 dihydroxyphenylalanine forms reactive stronger active group (DOPA quinone) by catalyzed oxidation, cause substrate and react with primary amine.According to this principle, can produce the quinones active group by means of tyrosine residues in tyrosinase catalysis oxidation silk fibroin, cause self-crosslinking between the silk fibroin macromole, promote film mechanics mechanical property and the chemical stability of silk fibroin; But simultaneously also the catalysis fibroin with there is the functional finishing agent grafting containing the primary amine structure, realize the biological enzyme functionalization preparation of fibroin protein film.In silk fibroin difference in functionality modification processing, antibacterial finish owing to can reduce microbial growth, alleviate the body bacterium and infect and there is positive effect.The polypeptide antiseptic-germicides such as ε-poly-lysine, L-poly arginine have the antibiotic and antiinflammation same with chemical antiseptic-germicides such as nano inorganic silver, quaternary ammonium salts, the insoluble mucopolysaccharide of hydrolyzable germ cell walls, also can be directly multiple virally inactivated in conjunction with making with electronegative viral protein, and this class antiseptic peptide stability is better, to the almost non-toxic evil effect of the normal cell of higher animal.In the silk fibroin film preparation, can be according to thering is the characteristic of primary amine structure in the antibacterial peptide structure, by means of its grafting on silk fibroin of tyrosinase catalysis, preparation has the fibroin protein film of antibacterial.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of environmental friendliness, ecological safety, the antibiotic silk fibroin membrane preparation method of biological enzyme that energy consumption is low.The fibroin protein film prepared with present method has mechanics mechanical property preferably, has solved common fibroin membrane brute force low, and the defect that the film body is hard and crisp has been expanded the application category of fibroin protein film in conjunction with grafting polypeptide antiseptic-germicide.
For solving the problems of the technologies described above, the present invention utilizes the catalysed oxidn of tyrosine oxidase, will have tyrosine residues grafting in amino polypeptide antibacterial finishing agent and silk fibroin, improves the fibroin membrane application performance.
Concrete technology is as follows:
(1) preparation of silk fibroin solution: dissolve fibroin fiber with lithiumbromide or calcium chloride, prepare silk fibroin solution.
Treatment process prescription and condition: the rear domestic silkworm silk that will come unstuck joins in the aqueous solution (ethanolic soln) system of lithiumbromide or calcium chloride, constantly is stirred to dissolving (bath raio 1: 10-50), obtain the silk union cellulose solution 70-80 ℃ of left and right.By the silk fibroin solution dialysis tubing of packing into, with dialysis 24-48 hour in deionized water, during per hour change water one time, after dialysis, filter, make silk fibroin aqueous solution.
(2) tyrosine oxidase is processed: add tyrosine oxidase and carry out catalytic oxidation treatment in silk fibroin solution.
Treatment process prescription and condition: the tyrosine oxidase consumption is the 1-200U/g silk fibroin, temperature 20-40 ℃, and pH scope 6.0-8.0, process 1-24 hour.
(3) polypeptide antiseptic-germicide grafting: after tyrosine oxidase is processed for some time, add the polypeptide antiseptic-germicide in fibroin is bathed, carry out silk fibroin antibacterial modified.
Treatment process prescription and condition: polypeptide antiseptic-germicide consumption 0.05-5.0g/L, temperature 20-40 ℃, pH scope 6.0-8.0, process 2-24 hour.
(4) dry: the enzymatic graft reaction is got quantitative silk fibroin solution and is poured in the polystyrene surface ware after finishing, and by prolonging stream, makes its smooth sprawling, and dries 1-12 hour under 25-85 ℃ of condition, obtains antibiotic fibroin protein film.
Silk fibroin membrane preparation method based on the grafting of tyrosinase catalysis natural antibacterial finishing composition, characteristics are lithium-bromide solution, lithiumbromide-aqueous ethanolic solution or calcium chloride-aqueous ethanolic solutions that the silk fibroin solution preparation can be selected different concns; The tyrosine oxidase of application has comprised the enzyme kind of the different sourcess such as animal, plant and microorganism; The polypeptide antibacterial finishing agent kind of application has comprised the polypeptide antiseptic-germicides such as the epsilon-polylysine, L-poly arginine of different molecular weight; The antibacterial modified middle tyrosine oxidase of silk fibroin and polypeptide antiseptic-germicide both can be added in silk fibroin solution step by step, also can adopt the method for simultaneously adding to carry out.
Useful consequence of the present invention
The present invention utilizes tyrosinase catalysis oxidation fibroin, has not only improved the mechanics mechanical property of fibroin membrane, and has improved the germ resistance of mould material, with traditional silk fibroin membrane preparation method, compares, and the present invention has the following advantages:
(1) enzyme catalysis efficiency is high, utilizes tyrosine oxidase to make the catalyzer of silk fibroin modification, and between the catalysis silk fibroin molecular, in crosslinked and enzymatic polypeptide antiseptic-germicide graft reaction, catalytic efficiency is high, and the zymin consumption is few.
(2) reaction conditions relaxes, completed the preparation of the crosslinked and antibiotic fibroin membrane of silk fibroin under the nearly neutrallty condition of low temperature, have advantages of that energy consumption is low, the treatment process safety and environmental protection, avoided the chemical crosslink technique severe reaction conditions, easily caused the defect of the aspects such as environmental pollution and fibroin membrane biological safety are low.
(3) the fibroin membrane improved performance is obvious, utilize tyrosinase catalysis oxidation silk fibroin molecular, not only can promote on fibroin covalent bonding between tyrosine residues and silk peptide, improve the mechanics mechanical property of fibroin protein film, by catalysis and containing amino allogenic polypeptide antiseptic-germicide grafting, also strengthened the germ resistance of fibroin membrane.After wet treatment, the antibacterial effect of fibroin membrane material is still better.
Embodiment
Adopt tyrosinase catalysis oxidation silk fibroin, realize silk fibroin self-crosslinking and grafting polypeptide antiseptic-germicide, preparation has the fibroin protein film of better mechanics and anti-microbial property, and specific embodiment is as follows:
Embodiment 1
(1) preparation of silk fibroin solution: the domestic silkworm silk after coming unstuck is joined in lithiumbromide-alcohol-water (mass ratio 45: 44: 11) solution, at 70 ℃, constantly be stirred to dissolving (bath raio 1: 50), obtain mixing solutions.By the silk fibroin solution dialysis tubing of packing into, with dialysis in deionized water 24 hours, during per hour change water one time, filter and obtain silk fibroin aqueous solution after dialysis.
(2) tyrosine oxidase is processed: add tyrosine oxidase (200U/g fibroin) in silk fibroin solution, 30 ℃ of temperature, process 6 hours under the pH=7.0 condition.
(3) polypeptide antiseptic-germicide grafting: after tyrosine oxidase is processed, add epsilon-polylysine (2g/L) and carry out the enzymatic grafting in enzyme is bathed, 30 ℃ of temperature, process 12 hours under the pH=7.0 condition.
(4) dry: reaction is got the watch-glass that 40 milliliters of silk fibroin solutions are poured polystyrene into after finishing, and under 50 ℃ of conditions, dries 6 hours, forms the antibiotic fibroin protein film of flat transparent.
Sample 1: process without tyrosine oxidase, only add the epsilon-polylysine treatment samples;
Sample 2: respectively through tyrosine oxidase, epsilon-polylysine combined treatment sample;
After above-mentioned art breading, sample 1 breaking tenacity is 25.5MPa, and elongation at break is 1.2%, and initial modulus is 5.6N/mm, and the dissolve-loss ratio in 37 ℃ of hot water is 8.8%, and after lower 3 washings of room temperature, fibroin membrane is 47% to the inhibiting rate of streptococcus aureus; Sample 2 breaking tenacitys are 32.3MPa, and elongation at break is 1.8%, and initial modulus is 3.2N/mm, and the dissolve-loss ratio in 37 ℃ of hot water is 5.9%, and after lower 3 washings of room temperature, fibroin membrane is 85% to the inhibiting rate of streptococcus aureus.
Embodiment 2
(1) preparation of silk fibroin solution: the domestic silkworm silk after coming unstuck is joined in calcium chloride-alcohol-water (mol ratio 1: 2: 8) solution, at 70 ℃, constantly be stirred to dissolving (bath raio 1: 50), obtain mixing solutions.By the silk fibroin solution dialysis tubing of packing into, with the 24h that dialyses in deionized water, during per hour change water one time, after dialysis, filter, obtain silk fibroin aqueous solution.
(2) tyrosine oxidase and polypeptide antiseptic-germicide are processed: add tyrosine oxidase (100U/g fibroin), L-poly arginine (1g/L) simultaneously in silk fibroin solution, 30 ℃ of temperature, process 12 hours with bathing under the pH=7.0 condition.
(3) dry: reaction is got 40 milliliters of silk fibroin solutions and is poured in polyacrylic container after finishing, and under 50 ℃ of conditions, dries 6 hours, forms antibiotic fibroin protein film.
Sample 3: only add L-poly arginine treatment samples;
Sample 4: through tyrosine oxidase, L-poly arginine treatment samples;
After above-mentioned art breading, sample 3 breaking tenacitys are 20.7MPa, and elongation at break is 1.3%, and initial modulus is 6.2N/mm, and 37 ℃ of hot water dissolve-loss ratios are 10.2%, and after lower 3 washings of room temperature, fibroin membrane is 45% to the inhibiting rate of streptococcus aureus; Sample 4 breaking tenacitys are 28.1MPa, and elongation at break is 1.9%, and initial modulus is 3.5N/mm, and 37 ℃ of hot water dissolve-loss ratios are 6.1%, and after lower 3 washings of room temperature, fibroin membrane is 74% to the inhibiting rate of streptococcus aureus.
Claims (6)
1. the preparation method of the antibiotic fibroin protein film of biological enzyme, is characterized in that utilizing tyrosinase catalysis oxidation fibroin to carry out fibroin molecule self-crosslinking, the polypeptide antiseptic-germicide is grafted on the fibroin surface simultaneously, improves mechanics mechanical property and the germ resistance of fibroin membrane.
2. method according to claim 1 is characterized in that concrete steps are as follows:
(1) the rear domestic silkworm silk that will come unstuck joins the aqueous solution (ethanolic soln) system of lithiumbromide or calcium chloride, constantly is stirred to dissolving (bath raio 1: 10-50), obtain the silk union cellulose solution 70-80 ℃ of left and right.By the silk fibroin solution dialysis tubing of packing into, with dialysis 24-48 hour in deionized water, during per hour change water one time, after dialysis, filter, make silk fibroin aqueous solution.
(2) add tyrosine oxidase in the silk fibroin aqueous solution after step (1) is processed and carry out catalytic oxidation treatment, treatment process prescription and condition: the tyrosine oxidase consumption is the 1-200U/g silk fibroin, temperature 20-40 ℃, pH scope 6.0-8.0, process 1-24 hour.
(3) add the polypeptide antiseptic-germicide in the mixed solution of step (2), treatment process prescription and condition: polypeptide antiseptic-germicide consumption is 0.05-5.0g/L, temperature 20-40 ℃, and pH scope 6.0-8.0, process 2-24 hour.
(4) after step (3) reaction finishes, get quantitative silk fibroin solution and pour in the polystyrene surface ware, by prolonging stream, make its smooth sprawling, dry 1-12 hour under 25-85 ℃ of condition, obtain antibiotic fibroin protein film.
3. method according to claim 2, is characterized in that when prepared by described silk fibroin solution selecting lithium-bromide solution, lithiumbromide-aqueous ethanolic solution or the calcium chloride-aqueous ethanolic solution of different concns.
4. method according to claim 2, is characterized in that described tyrosine oxidase can derive from animal, plant and microorganism.
5. method according to claim 2, is characterized in that described polypeptide antiseptic-germicide kind has comprised the multiple antibacterial peptides such as the epsilon-polylysine of different molecular weight size, L-poly arginine.
6. method according to claim 2, is characterized in that step (2), (3) can carry out or proceed step by step simultaneously.
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Effective date of registration: 20200521 Address after: Room 1208, building 1, No. 58, Chongchuan Road, Chongchuan District, Nantong City, Jiangsu Province Patentee after: Jiangsu Zixia Biotechnology Co., Ltd Address before: 1800 No. 214122 Jiangsu city of Wuxi Province Li Lake Avenue Patentee before: Jiangnan University |