CN105088540B - A kind of method that nanometer silk fibroin material is prepared based on tryrosinase/polyphenol amboceptoid - Google Patents
A kind of method that nanometer silk fibroin material is prepared based on tryrosinase/polyphenol amboceptoid Download PDFInfo
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Abstract
The invention discloses a kind of method for preparing nanometer silk fibroin material based on tryrosinase/polyphenol amboceptoid, using phenolic hydroxyl group in L-Tyrosine in tyrosinase catalysis oxidation fibroin and polyphenol amboceptoid, generation of reactive activity quinone structure, promote fibroin to react with outer source functional amino-compound, prepare functional nano silk fibroin material.Comprise the following steps that：(1) prepared by silk fibroin solution；(2) it is crosslinked with tyrosinase catalysis oxidation fibroin and amino-compound, adds polyphenol amboceptoid in system after reaction a period of time and promote reaction；(3) nanometer silk fibroin material molding：After fibroin is reacted with amino-compound, solution is sprawled by prolonging stream, and Jing room temperatures air-dry film forming or lyophilization film forming, prepares spinning liquid with formic acid, spins nanometer silk fibroin material using electrostatic spinning technique.Compared with traditional chemical cross-linking method prepares functional type silk fibroin material, enzyme catalysiss efficiency high of the present invention, ferment treatment condition relax, and the performance improvement of nanometer silk fibroin material is obvious.
Description
Technical field
The present invention relates to a kind of method that nanometer silk fibroin material is prepared based on tryrosinase/polyphenol amboceptoid, particularly one
Plant and fibroin and polyphenol amboceptoid are aoxidized using tyrosinase catalysis, promote fibroin albumen to tie with outer source functional amino-compound
Close, the method for preparing functional nano silk fibroin material belongs to field of textile biotechnology.
Background technology
Fibroin has good biocompatibility, and the fibroin protein film with which as Raw material processing is in tissue engineering material structure
There is wide purposes.Natural fibroin albumen toughness of material is poor, and water dissolve-loss ratio is high, is the mechanics for improving regenerated silk membrane material
Performance and feature, have carried out correlational study both at home and abroad in terms of fibroin albumen and its composite modification.Wherein chemical method
It is using more, general fibroin albumen to be crosslinked with glutaraldehyde, polybasic carboxylic acid etc. more or be grafted with external source functional compounds.This
Class method there is also certain deficiency while silk fibroin material performance is improved, and ask including chemical noxious material residual etc. is also easy to produce
Topic.Biological method has safe efficient, single-minded and advantages of environment protection, fibroin material is modified with enzyme process, no
But the original good characteristic of fibroin albumen is maintained, and gives fibroin material new application performance, effect on environment
It is less, therefore with certain application prospect.
Enzyme is related to fibroin albumen structure, the selectivity of enzyme etc. to modified effect of fibroin material.Fibroin egg
In white, the surplus base content of hydrophobicity glycine, alanine is higher, is secondly the serine (containing methylol) and cheese ammonia of polar functionalities
Sour (phenolic hydroxy group) residue.Tryrosinase is the polyphenol oxidase with catalytic oxidation activity, energy catalysis oxidation phenolic hydroxy group
The surplus base of L-Tyrosine generates o-phenyl phenol and DOPA quinone in succession, and further can react with the functional compound containing amino, is fibroin
The functional modification of albumen provides method.Result of study shows, as in fibroin albumen, tyrosine residue is distributed in by hydrophobic
Property stronger alanine and glycine composition macromolecular chain in, certain impact is generated on the accessibility of enzymatic reaction, therefore,
Had much room for improvement based on the fibroin albumen functional modification efficiency of L-Tyrosine enzyme process.
Polyphenol compound includes tea polyphenols, caffeic acid, ferulic acid etc., and this kind of compound is also with similar to L-Tyrosine
Phenolic hydroxyl structure.Polyphenol compound also can be aoxidized by tyrosinase catalysis, the stronger active quinone structure (A) of generation of reactive,
Active quinone molecule not only mutually can be polymerized (A-A-A), it is also possible to while react with two molecule amino-compounds (B), shape
Into the intermolecular crosslinking of amino-compound (B-A-B).According to this characteristic, the reaction of fibroin (SF) is aoxidized in tyrosinase catalysis
In, fibroin not only directly can be reacted with external source amino-compound, form both directly crosslinkings (SF-B)；Can also add in system suitable
Amount Polyphenols medium system, on the basis of oxidation polyphenol amboceptor generates quinones active group, forms with active quinone structure as bridge
Amino-compound fibroin surface indirect grafting (SF-A-B), so as to lift the efficiency of fibroin enzyme process functional modification,
Functional nano silk fibroin material is prepared on the basis of this.
The content of the invention
The technical problem to be solved is to provide a kind of based on tryrosinase/polyphenol amboceptoid preparation nanometer fibroin
The method of material, it is intended to improve the graft reaction efficiency of fibroin and external source amino-compound under the conditions of tyrosinase catalysis, here
On the basis of prepare feature silk fibroin material.
To solve above-mentioned technical problem, the present invention aoxidizes fibroin and polyphenol amboceptoid using tyrosinase catalysis, promotes silk
Crosslinking between fibroin and outer source functional amino-compound, prepares the side of functional nano silk fibroin material with reference to electrostatic spinning
Method, concrete technology are as follows with step：
(1) prepared by silk fibroin solution：With lithium bromide or calcium chloride dissolving mulberry silk, silk fibroin solution is prepared；
Handling process prescription and condition：Mulberry silk is added in the water solution system of lithium bromide or calcium chloride, 30～
80 DEG C dissolving after silk fibroin solution is loaded into bag filter, with deionized water dialyse 8～36 hours after filter, be obtained concentration be 10～
50g/L silk fibroin solutions；
(2) tryrosinase/polyphenol amboceptoid catalysis fibroin is crosslinked with amino-compound：Add in step (1) silk fibroin solution
Enter tryrosinase and amino-compound, reaction added polyphenol amboceptoid after 0.5～8 hour, at continuation under identical temperature conditionss
Reason 0.5～24 hour；
Handling process prescription and condition：1～100U/ml of tryrosinase, 1～50g/L of amino-compound, polyphenol amboceptoid 1
～10g/L, 20～60 DEG C of temperature, pH scopes 6.0～8.0；
(3) nanometer silk fibroin material molding：By step (2) reacted silk fibroin solution, which is made in mould by prolonging stream
It is interior it is smooth sprawl, at room temperature air-dry film forming or under the conditions of -50 DEG C～-20 DEG C lyophilization film forming；Carried out with 98% formic acid
Fibroin membrane dissolves, and prepares spinning liquid, spins nanometer silk fibroin material using electrostatic spinning technique；
Spinning technique and condition：In spinning liquid fibroin concentration be 10～45%, spinning voltage be 10～40kV, spinning speed
For 0.3～2ml/h, syringe needle point is 5～20cm with the spinning spacing of metal roller surface.
A kind of method that nanometer silk fibroin material is prepared based on tryrosinase/polyphenol amboceptoid, when prepared by the silk fibroin solution
From lithium-bromide solution, lithium bromide-ethanol water or calcium chloride-ethanol water；The tryrosinase cultivar origin is in dynamic
Thing, plant or microorganism；The polyphenol amboceptoid includes catechin, epicatechin, caffeic acid, ferulic acid and chlorogenic acid；It is described
Amino-compound includes the chemical finishing agent containing amino, functional polypeptide, shitosan, polylysine, elastin laminin and gelatin.
The present invention is reacted using tyrosinase catalysis oxidation fibroin and external source amino-compound, is generated in oxidation polyphenol amboceptor
On the basis of quinones active group, the efficiency of fibroin enzyme modification, constructing function nanometer silk fibroin material are lifted.Hand over traditional chemical
Connection method prepares silk fibroin composite membrane coating and compares, and the present invention has advantages below：
(1) enzyme catalysiss efficiency high, is aoxidized using tyrosinase catalysis fibroin and polyphenol amboceptoid, promotes fibroin and amination
Enzyme catalysiss efficiency high in the reaction of compound crosslinking, enzyme preparation consumption are less；
(2) ferment treatment condition relaxes, and carries out fibroin albumen functional modification, with energy consumption under low temperature and near-neutral sulfite deinking
Low, handling process safe advantage, it is to avoid it is low all that chemical crosslink technique reaction easily causes environmental pollution, silk fibroin material safety
Many defects；
(3) nanometer silk fibroin material performance improvement substantially, based on tryrosinase/polyphenol amboceptoid method modified fibroin albumen, no
But obtained fibroin nano material water solublity is low, with specific feature, the biocompatibility of product is also preferable.
Specific embodiment
Using tryrosinase and polyphenol amboceptoid catalysis fibroin and the crosslinking of external source amino-compound, functional nano silk is prepared
Cellulosic material, specific embodiment are as follows：
Embodiment 1
(1) silk fibroin solution prepares：Mulberry silk is added to lithium bromide-alcohol-water (mass ratio 45:45:10) in solution, 60 DEG C
Silk fibroin solution is loaded into bag filter after dissolving, is dialysed 24 hours in deionized water, period changes 1 water, mistake after dialysis per hour
Filter obtains the silk fibroin aqueous solution of 20g/L；
(2) 10U/ml tryrosinases and 5g/L elastin laminins are added in step (1) silk fibroin solution, in 30 DEG C, pH7 conditions
Lower process added 2g/L catechins after 4 hours, continued with 12 hours；
(3) nanometer silk fibroin material molding：Step (3) reacted silk fibroin solution is put down in molded polytetrafluoroethylarticle mould
It is whole to sprawl, film forming is air-dried under room temperature；Fibroin membrane dissolving is carried out with 98% formic acid, compound concentration is 15% fibroin spinning liquid, is adopted
Nanometer silk fibroin material is spinned with electrostatic spinning technique, wherein spinning voltage is 15kV, and spinning speed is 0.6ml/h, syringe needle point
Spinning spacing with metal roller surface is 10cm.
Fibroin nano material sample 1：Add tryrosinase in process, be not added with catechin；
Fibroin nano material sample 2：Add tryrosinase, addition catechin in process；
Jing after above-mentioned PROCESS FOR TREATMENT, it is little that evaluation NIH/3T3 cells impregnate 24 in fibroin/elastin laminin composite membrane culture medium
When after survival rate, and determine the fracture strength and elongation at break of nanometer silk fibroin material.As a result show, the NIH/3T3 of sample 1
Cell survival rate be 80%, fracture strength and elongation at break be respectively 2.1MPa, 1.9%；The cell survival rate of sample 2 is
83%th, fracture strength and elongation at break can respectively reach 2.8MPa, 2.8%.
Embodiment 2
(1) silk fibroin solution prepares：Mulberry silk is added to calcium chloride-alcohol-water (mol ratio 1:1:7), after 70 DEG C of dissolvings
Silk fibroin solution is loaded into bag filter, is dialysed 24 hours in deionized water, period changes 1 water per hour, is filtrated to get after dialysis
The silk fibroin aqueous solution of 18g/L；
(2) 8U/ml tryrosinases and 2g/L shitosans are added in step (1) silk fibroin solution, under the conditions of 30 DEG C, pH7
1g/L caffeic acids are added after processing 3 hours, 15 hours are continued with；
(3) nanometer silk fibroin material molding：Solution smooth paving in the molded polytetrafluoroethylarticle mould after step (3) is reacted
Exhibition, air-dries film forming under room temperature；Fibroin membrane dissolving is carried out with 98% formic acid, compound concentration is 15% fibroin spinning liquid, using quiet
Electrospinning spins nanometer silk fibroin material, and wherein spinning voltage is 20kV, and spinning speed is 0.8ml/h, syringe needle point and gold
The spinning spacing of category roller surface is 12cm.
Fibroin nano material sample 3：Tryrosinase and caffeic acid are not added with process；
Fibroin nano material sample 4：Add tryrosinase in process, be not added with caffeic acid；
Fibroin nano material sample 5：Add tryrosinase and caffeic acid in process；
Jing after above-mentioned PROCESS FOR TREATMENT, by sample 3,4,5 so that after the process of 50% ethanol solution, in 30 DEG C of aqueous solutions, dipping 6 is little
When, determine the water dissolve-loss ratio and the bacteriostasis rate to staphylococcus aureuses of fibroin membrane material.Wherein, the water dissolve-loss ratio of sample 3 is
12.2%, bacteriostasis rate is 85%；The water dissolve-loss ratio of sample 4 is 6.2%, and bacteriostasis rate is 91%；The water dissolve-loss ratio of sample 5 is
4.3%, bacteriostasis rate is 95%.
Although the present invention is disclosed as above with preferred embodiment, which is not limited to the present invention, any to be familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore the protection model of the present invention
Enclosing should be by being defined that claims are defined.
Claims (2)
1. a kind of method for preparing nanometer silk fibroin material based on tryrosinase/polyphenol amboceptoid, is characterized in that one kind utilizes L-Tyrosine
Enzymatic oxidation fibroin and polyphenol amboceptoid, are promoted fibroin albumen to be combined with outer source functional amino-compound, prepare feature
The method of nanometer fibroin membrane material, concrete technology are as follows with step：
(1) prepared by silk fibroin solution：With lithium bromide or calcium chloride dissolving mulberry silk, silk fibroin solution is prepared；
Handling process prescription and condition：Mulberry silk is added in the water solution system of lithium bromide or calcium chloride, at 30～80 DEG C
Silk fibroin solution is loaded into bag filter after dissolving, to filter after dialysing 8～36 hours in deionized water, it is 10～50g/ that concentration is obtained
L silk fibroin solutions；
(2) tryrosinase/polyphenol amboceptoid catalysis fibroin is crosslinked with amino-compound：Cheese is added in step (1) silk fibroin solution
Propylhomoserin enzyme and amino-compound, reaction added polyphenol amboceptoid after 0.5～8 hour, under identical temperature conditionss continued with 0.5
～24 hours；
Handling process prescription and condition：1～100U/ml of tryrosinase, 1～50g/L of amino-compound, polyphenol amboceptoid 1～
10g/L, 20～60 DEG C of temperature, pH scopes 6.0～8.0；
(3) nanometer silk fibroin material molding：By step (2) reacted silk fibroin solution, which is made to put down in mould by prolonging stream
It is whole to sprawl, film forming is air-dried at room temperature or the lyophilization film forming under the conditions of -50 DEG C～-20 DEG C；Fibroin is carried out with 98% formic acid
Film dissolves, and prepares spinning liquid, spins nanometer silk fibroin material using electrostatic spinning technique；
Spinning technique and condition：In spinning liquid, fibroin concentration is 10～45%, and spinning voltage is 10～40kV, and spinning speed is
0.3～2ml/h voltages, syringe needle point are 5～20cm with the spinning spacing of metal roller surface.
2. method according to claim 1, it is characterised in that：Lithium-bromide solution, bromine are selected when prepared by the silk fibroin solution
Change lithium-ethanol water or calcium chloride-ethanol water；The tryrosinase cultivar origin is in animal, plant or microorganism；Institute
Stating polyphenol amboceptoid includes catechin, epicatechin, caffeic acid, ferulic acid and chlorogenic acid；The amino-compound include containing
The chemical finishing agent of amino, functional polypeptide, shitosan, polylysine, elastin laminin and gelatin.
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| CN106149195B (en) * | 2016-08-18 | 2018-05-15 | 华南理工大学 | Nano fibrous membrane that is a kind of while embedding fish oil and forulic acid and preparation and application |
| CN107050511A (en) * | 2017-04-11 | 2017-08-18 | 江南大学 | A kind of biological enzyme fibroin albumen/calcium phosphate composite materials preparation method |
| CN107099046B (en) * | 2017-04-25 | 2020-03-06 | 江南大学 | Method for preparing fibroin-based biomimetic mineralized material by grafting vinyl monomer |
| CN107594068A (en) * | 2017-09-25 | 2018-01-19 | 中国海洋大学 | A kind of be crosslinked using tyrosinase reduces knife volume newly to the method for shrimp allergen tropomyosin allergenicity |
| CN108744023B (en) * | 2018-06-15 | 2021-04-27 | 福州大学 | Silk fibroin medical biological adhesive and preparation method thereof |
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Effective date of registration: 20201229 Address after: 215200 Badu Industrial Park, Zhenze Town, Wujiang District, Suzhou City, Jiangsu Province Patentee after: Wujiang Aolinte Artware Co.,Ltd. Address before: No. 1800 road 214122 Jiangsu Lihu Binhu District City of Wuxi Province Patentee before: Jiangnan University |