CN102824654B - Double-bioenzyme modified blending biological material containing gelatin and chitosan and preparation method and application thereof - Google Patents
Double-bioenzyme modified blending biological material containing gelatin and chitosan and preparation method and application thereof Download PDFInfo
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- CN102824654B CN102824654B CN201210332803.8A CN201210332803A CN102824654B CN 102824654 B CN102824654 B CN 102824654B CN 201210332803 A CN201210332803 A CN 201210332803A CN 102824654 B CN102824654 B CN 102824654B
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Abstract
The invention provides a double-bioenzyme modified blending biological material containing gelatin and chitosan and a preparation method and application of the double-bioenzyme modified blending biological material. The blending biological material comprises gelatin and chitosan which are used a blending main body and microbial transglutaminase and tyrosinase which are used as a modifier. The stability of the blending biological material is enhanced, and the mechanical property thereof is improved; furthermore, the material has antimicrobial activity, and has good application prospect and excessively wide application in the fields of biomedical materials and a biomedicine, such as the reduction of infection and inflammatory response, antibiosis, wound protection, a biologically degradable skin dressing, a wound healing agent, artificial skin, and the like; and meanwhile, the material is simple in preparation technology, easy to control, free from side and toxic effects and temperate in preparation condition.
Description
Technical field
The invention belongs to bio-medical material, polymer chemistry and biochemical field, particularly relate to a kind of gelatin that adopts twin thing enzyme modification and chitosan blend biomaterial and preparation method thereof and application.
Background technology
Gelatin is as natural polymer and the biological macromolecule material of nature source rich in protein class, it has good biocompatibility, nontoxic, biodegradable, the advantage such as can be absorbed by the body and cheap is widely used in biomedical materials field, especially the aminoacid composition of gelatin and ratio and human body skin approach, therefore at biological dressing, the skin tissue engineering such as Wound-healing agent and artificial skin aspect has good application prospect [functional material, 2004, 35:2310-2313], but gelatin film fragility is large, easily broken, its application of easily having grown the drawbacks limit such as bacterial infection.Therefore the mechanical performance and the anti-microbial property that, improve gelatin are of great significance to expand its application tool at biomedical sector.Chitosan is the deacetylated product of the N-of chitin; also be natural polymer and the biological macromolecule material that a kind of occurring in nature originates biological polyoses class abundant; there is good film property, biocompatibility, degradability; wide material sources also have unique broad spectrum antibacterial performance [International Journal of Food Microbiology; 2000,62 (1-2): 139-148].Therefore, gelatin and chitosan blend can be formed to the mutual supplement with each other's advantages between the each component of material, not only be conducive to improve the mechanical performance of gelatin-based material, particularly be conducive to improve gelatin-based material and easily grow and the shortcoming of bacterial infection.
In the preparation process of gelatin and chitosan blend material, often need to be cross-linked to improve mechanical performance and the stability [Food Chemistry, 2007,103:295 – 300] of intermingling material.But, although traditional chemical crosslinking can effectively improve mechanical performance and the stability of material, but due to for example aldehydes (formaldehyde of conventional chemical cross-linking agent, glutaraldehyde and glyceraldehyde), epoxide component and carbodiimides etc. often have cytotoxicity, this will cause biomaterial prepared by Chemical Crosslinking Methods after implanting, and affects the growth of receptor normal structure.In view of the foregoing, in the preparation process of material, use natural and avirulent dressing agent, be of great significance for preparing the biomaterial tool that biocompatibility is good.Enzyme, as biocatalyzer, has a series of advantage: enzyme is a kind of biocatalyzer, there is no side reaction, can not produce toxicity problem; Reaction condition gentleness, reactions steps is simple; Catalytic efficiency is high, and specificity is strong, and is widely used in the industries such as food, medical inspection, biological manufacture.Therefore, whether can find suitable enzyme to modify to gelatin and chitosan blend material the study hotspot that becomes this area.
Summary of the invention
The technical problem to be solved in the present invention is: a kind of gelatin that adopts twin thing enzyme modification and chitosan blend biomaterial and preparation method thereof and application are provided, gelatin and chitosan blend material are after twin thing enzyme modification, the stability of material and mechanics mechanical performance improve, and this material has good antibacterial activity.
The present invention for solving the problems of the technologies described above taked technical scheme is:
The gelatin and the chitosan blend biomaterial that adopt twin thing enzyme modification, is characterized in that, described pair of enzyme is respectively microbial transglutaminase and tryrosinase.
The preparation method that adopts gelatin and the chitosan blend biomaterial of twin thing enzyme modification, is characterized in that, it comprises following concrete steps:
1) preparation of solution:
Press gelatin: deionized water=2～6g:50～100mL, choose gelatin and deionized water, for subsequent use; Gelatin is joined in deionized water, within 1～2 hour, after gelatin fully dissolves, obtain gelatin solution the stirred in water bath of 40～60 DEG C;
Press chitosan: 1%(percent by volume) acetic acid solution=1～2g:50～100mL, choose chitosan and acetic acid solution, for subsequent use; Chitosan is joined in acetic acid solution, stir and fully dissolve to chitosan for 3～5 hours under room temperature, then in solution, drip 0.5～1.0mol/L sodium hydroxide solution, the pH of regulator solution is 5.0～6.0, obtains chitosan solution;
Press microbial transglutaminase: deionized water=10～20U:5～10mL, choose microbial transglutaminase and deionized water, for subsequent use; Microbial transglutaminase is joined in deionized water, under room temperature, stir and fully dissolve for 15～30 minutes, refilter and obtain microbial transglutaminase solution;
PBS buffer solution=5000～10000U:5～the 10mL that is 7.0 by tryrosinase: pH, chooses tryrosinase and pH and is 7.0 PBS buffer solution, for subsequent use; It is in 7.0 PBS buffer solution that tryrosinase is dissolved in to described pH, stirs and after tryrosinase fully dissolves, obtain tryrosinase solution in 2～5 minutes under room temperature;
2) blend of gelatin and chitosan:
Press gelatin solution: chitosan solution=50～100mL:50～100mL, choose gelatin solution and the chitosan solution of above-mentioned preparation, for subsequent use; Gelatin solution and chitosan solution equal-volume are mixed, stir 15～30 minutes at 40～60 DEG C, make solution mix homogeneously, obtain gelatin and chitosan blend solution;
3) preparation of the gelatin of twin thing enzyme modification and chitosan blend material:
Press gelatin and chitosan blend solution: microbial transglutaminase: tryrosinase=50～100mL:3～8U:3000～7000U, choose above-mentioned gelatin and chitosan blend solution, microbial transglutaminase and tryrosinase, for subsequent use; In gelatin and chitosan blend solution, add mentioned microorganism T-5398 and tryrosinase, stirring reaction 10～20 minutes at 28～30 DEG C, reactant liquor is poured in culture dish, at room temperature aeration-drying 48～72 hours, obtain gelatin and the chitosan blend material diaphragm of twin thing enzyme modification, then diaphragm is soaked 0.5～1 hour in 0.1～0.5mol/L sodium hydroxide solution, use again deionized water flushing membrane sheet, at 30～40 DEG C, be dried 12～24 hours, obtain gelatin and the chitosan blend biomaterial of the twin thing enzyme modification of described employing.
Adopt the gelatin of twin thing enzyme modification and chitosan blend biomaterial at dressing for skin, Wound-healing agent, or the application of artificial skin aspect.
The invention has the beneficial effects as follows:
1) the present invention adopts two enzyme (microbial transglutaminase and tryrosinase) to modify gelatin and chitosan blend material, not only there is enzyme method of modifying, and due to the synergistically modified effect of two kinds of enzymes of this material employing, with a kind of enzyme of independent use as compared with dressing agent also tool have the following advantages: form cross-linked network thereby microbial transglutaminase can make, between the epsilon-amino on hydroxyl amide groups and the lysine on the gamma-glutamic acid in gelatin molecule, association reaction occurs, and tryrosinase is by being oxidized to the tyrosine residue in gelatin molecule quinones residue, then with chitosan molecule in amino reaction form covalently bound.Therefore, by the synergism of two kinds of enzyme, gelatin molecule and chitosan molecule not only can be formed stable covalently boundly, also form stable cross-linked network, thereby greatly improve the stability of material simultaneously.In addition, this covalently bound and cross-linked network forming by the synergistically modified effect of two enzyme, mechanics mechanical performance that can also reinforcing material, and the stability of material and mechanics mechanical performance improve, just greatly expansion material in purposes and the range of application of bio-medical material and biomedical sector.And, because microbial transglutaminase is cheap and easy to get, therefore adopt two enzyme (microbial transglutaminase and tryrosinase) can, in improving material settling out performance and mechanical property, can also effectively reduce the production cost of material as dressing agent.
2) the prepared biomaterial of the present invention is all made up of biomacromolecule, what wherein blend material of main part adopted is gelatin and chitosan, what dressing agent adopted is enzyme, and therefore this biomaterial being all made up of biomacromolecule has good biocompatibility, and no cytotoxicity.
3) gelatin is nature source rich in protein class biological macromolecule material, chitosan is the polysaccharide biological macromolecule material that nature is originated abundant, all there is good biocompatibility, nontoxic, biodegradable, the advantage such as can be absorbed by the body and cheap, chitosan also has unique broad spectrum antibacterial performance, this material of being prepared by protein and biological polyoses blend, can form the mutual supplement with each other's advantages between the each component of material, not only be conducive to improve the mechanical performance of gelatin-based material, particularly being conducive to improve gelatin-based material easily grows and the shortcoming of bacterial infection.
4) enzyme, as bio-modification agent, has avoided adopting traditional chemical dressing agent problem, the problem includes: the problem that biocompatibility is poor, toxicity is large; Do not need complicated instrument and equipment, reaction condition gentleness, reactions steps is simple, and enzyme has high efficiency and specificity.
5) preparation method of the present invention is simple, is easy to control, have no side effect, and preparation condition gentleness.
6) the present invention adopts gelatin and the chitosan blend biomaterial of twin thing enzyme modification; in the purposes of bio-medical material and biomedical sector; particularly reducing infection and inflammatory reaction, antibacterial, protection wound, biodegradable dressing for skin and Wound-healing agent, and the bio-medical material such as artificial skin and biomedical sector has a good application prospect and purposes very widely.
Brief description of the drawings
Fig. 1 does not adopt enzyme to modify, adopt separately a kind of enzyme modification, and the comparison diagram of the employing microbial transglutaminase of embodiment 1 gained and the gelatin of tryrosinase modification and the hot strength of chitosan blend material, gelatin and chitosan blend material that wherein G/CS representative does not adopt enzyme to modify, m-G/CS represents gelatin and the chitosan blend material that microbial transglutaminase is modified, t-G/CS represents gelatin and the chitosan blend material that tryrosinase is modified, gelatin and chitosan blend material that the two enzyme (microbial transglutaminase and tryrosinase) of mt-G/CS representative are modified.
Fig. 2 is gelatin materials, and the gelatin of the twin thing enzyme modification of employing that obtains of embodiment 1 and chitosan blend material are to colibacillary fungistatic effect comparison diagram, wherein Control representative does not add the blank group of sample, G represents the gelatin materials of unmodified, gelatin and chitosan blend material that the two enzyme (microbial transglutaminase and tryrosinase) of mt-G/CS representative are modified.
Fig. 3 is gelatin materials, and gelatin and the fungistatic effect comparison diagram of chitosan blend material to staphylococcus aureus of the twin thing enzyme modification of employing that obtains of embodiment 1, wherein Control representative does not add the blank group of sample, G represents the gelatin materials of unmodified, gelatin and chitosan blend material that the two enzyme (microbial transglutaminase and tryrosinase) of mt-G/CS representative are modified.
Detailed description of the invention
Below in conjunction with drawings and Examples, the invention will be further described, and certain following embodiment should not be construed as limitation of the present invention.
The invention provides a kind of gelatin and chitosan blend biomaterial that adopts twin thing enzyme modification, it adopts biological macromolecule material to form as intermingling material and dressing agent completely, what blend material of main part adopted is gelatin and chitosan, then adopt two kinds of enzyme simultaneously, be microbial transglutaminase and tryrosinase as dressing agent, after gelatin and chitosan blend biomaterial are modified, obtain.Below in conjunction with specific embodiment, preparation method of the present invention is described further, but does not limit the present invention.
Embodiment 1:
A preparation method that adopts twin thing enzyme modification gelatin and chitosan blend biomaterial, it comprises following concrete steps:
1) preparation of solution:
Take 2g gelatin and join in 50mL deionized water, within 1 hour, dissolve obtain the gelatin solution that concentration is 0.04g/mL completely to gelatin the stirred in water bath of 50 DEG C; Take 1g chitosan and join 50mL 1%(percent by volume v/v%) in acetic acid solution, under room temperature, stir and fully dissolve to chitosan for 3 hours, obtain the chitosan solution that concentration is 0.02g/mL, to slowly drip in chitosan solution 1mol/L sodium hydroxide solution to the pH of solution be 5.5; Microbial transglutaminase is joined in deionized water and under room temperature, stirred 10 minutes, refilter the microbial transglutaminase solution that obtains 2U/mL; Tryrosinase is joined to PBS(pH7.0) in buffer solution, under room temperature, stir 2 minutes, obtain the tryrosinase solution of 1000U/mL.
2) blend of gelatin and chitosan:
Measure respectively the gelatin solution of the above-mentioned preparation of 50 mL and chitosan solution equal-volume and mix, stir 30 minutes at 50 DEG C, make solution mix homogeneously, obtain gelatin and chitosan blend solution.
3) preparation of the gelatin of twin thing enzyme modification and chitosan blend material:
In the blend solution of 100mL gelatin and chitosan, add 4U microbial transglutaminase and 5000U tryrosinase, stirring reaction 15 minutes at 28 DEG C, reactant liquor is poured in culture dish, at room temperature aeration-drying 72 hours, obtain gelatin and the chitosan blend material diaphragm of twin thing enzyme modification, then diaphragm is soaked 0.5 hour in 0.1mol/L sodium hydroxide solution, use again a large amount of deionized water rinsing diaphragms, at 30 DEG C, be dried 12 hours, obtain adopting gelatin and the chitosan blend biomaterial of twin thing enzyme modification.
Fig. 1 does not adopt enzyme to modify, adopt separately the employing microbial transglutaminase of a kind of enzyme modification and embodiment 1 gained and the comparison diagram of the gelatin of tryrosinase modification and the hot strength of chitosan blend material.
Test process is: choose smooth, clean, flawless material film, be cut into the horizontal stripe of 70mm × 1Omm.Measure film thickness, each sample is measured six points in gauge length, gets its meansigma methods, as the thickness of film.Sample is placed in to relative humidity and is about 50% exsiccator, be positioned under 25 DEG C of conditions balance 24 hours.Then adopt universal testing machine (CMT-6503, material tests company limited is newly thought carefully in Shenzhen) to carry out mechanics performance determining, initial folder is apart from being set as 30mm, and rate of extension is set as 40mm/min.The mechanical property of different samples is all parallel makees 6 samples, gets the meansigma methods of 6 results.As can be seen from Figure 1, not adopting the gelatin of enzyme modification and the hot strength of chitosan blend material G/CS is 30.75MPa, be 43.09MPa and adopt separately the gelatin of microbial transglutaminase enzyme modification and the hot strength of chitosan blend material m-G/CS, the gelatin that employing tryrosinase is modified separately and the hot strength of chitosan blend material t-G/CS are 39.97MPa, and this explanation adopts separately the gelatin of a kind of enzyme (microbial transglutaminase or tryrosinase) modification and the hot strength of chitosan blend material to be improved.Adopting the common gelatin of modifying of two enzyme (microbial transglutaminase and tryrosinase) and the hot strength of chitosan blend material mt-G/CS is 50.06MPa, improve 62.80% with G/CS phase specific tensile strength, above result shows, the twin thing enzyme modification method that the present invention adopts can obviously improve the mechanical property of gelatin and chitosan blend material.
Fig. 2 is gelatin materials, and the gelatin of the twin thing enzyme modification of employing that obtains of embodiment 1 and chitosan blend material are to colibacillary fungistatic effect comparison diagram.
Its test process is with reference to People's Republic of China's light industry standard: QB/T 2591-2003 " antibiotic plastic-anti-microbial property method of testing and antibacterial effect ".Concrete test process is: by fresh bacterial cultures (transferring in 24 hours), add in culture fluid, take turns doing 10 times and increase progressively dilution, selecting bacterial concentration is (5.0～10.0) × 10
5the diluent of CFU/mL is as test bacterium liquid.In each culture tube, add 2mL bacterium liquid, add respectively gelatin and the chitosan blend diaphragm of 1 gelatin diaphragm and the twin thing enzyme modification of employing, and do not add the culture tube of diaphragm as blank group, 37 DEG C of constant-temperature shaking culture after 24 hours, calculate bacterium colony number with colony counting method.Every group of data are done twice parallel laboratory test, and each sample does three parallel antibacterial tests.Wherein antibiotic rate (%)=(blank sample average bacterial count recovered-antibacterial sample average bacterial count recovered)/blank sample average bacterial count recovered × 100%.
As can be seen from Figure 2, the blank culture medium that does not add sample colibacillary concentration after incubated overnight of Control representative is 6.48 × 10
7cFU/mL, and the e. coli concentration of the gelatin materials that adds unmodified of G representative after incubated overnight is 8.22 × 10
7cFU/mL, the gelatin-based material of this explanation unmodified does not only have colibacillary antibacterial activity, and it can promote colibacillary Growth and reproduction on the contrary.The gelatin that adds twin thing enzyme modification and the e. coli concentration of chitosan blend material after incubated overnight of mt-G/CS representative are 5.70 × 10
7cFU/mL, compared with the gelatin materials G of unmodified, antibiotic rate has improved 30.66%.Above result shows, gelatin and the chitosan blend material of the twin thing enzyme modification that the present invention obtains have antibacterial activity to escherichia coli.
Fig. 3 is gelatin materials, and gelatin and the fungistatic effect comparison diagram of chitosan blend material to staphylococcus aureus of the twin thing enzyme modification of employing that obtains of embodiment 1.
Its test process is with reference to People's Republic of China's light industry standard: QB/T 2591-2003 " antibiotic plastic-anti-microbial property method of testing and antibacterial effect ".Concrete test process is: by fresh bacterial cultures (transferring in 12 hours), add in culture fluid, take turns doing 10 times and increase progressively dilution, selecting bacterial concentration is (1.0～5.0) × 10
9the diluent of CFU/mL is as test bacterium liquid.In each culture tube, add 3mL bacterium liquid, add respectively gelatin and the chitosan blend diaphragm of 1 gelatin diaphragm and the twin thing enzyme modification of employing, and do not add the culture tube of diaphragm as blank group, 37 DEG C of constant-temperature shaking culture after 12 hours, calculate bacterium colony number with colony counting method.Every group of data are done twice parallel laboratory test, and each sample does three parallel antibacterial tests.Wherein antibiotic rate (%)=(blank sample average bacterial count recovered-antibacterial sample average bacterial count recovered)/blank sample average bacterial count recovered × 100%.
As can be seen from Figure 3, the concentration of the blank culture medium staphylococcus aureus after incubated overnight that does not add sample of Control representative is 2.79 × 10
11cFU/mL, and the staphylococcus aureus concentration of the gelatin materials that adds unmodified of G representative after incubated overnight is 3.71 × 10
11cFU/mL, the gelatin-based material of this explanation unmodified does not only have the antibacterial activity to staphylococcus aureus, and it can promote the Growth and reproduction of staphylococcus aureus on the contrary.The gelatin that adds twin thing enzyme modification and the staphylococcus aureus concentration of chitosan blend material after incubated overnight of mt-G/CS representative are 2.26 × 10
11cFU/mL, compared with the gelatin materials G of unmodified, antibiotic rate has improved 39.08%.Above result shows, gelatin and the chitosan blend material of the twin thing enzyme modification that the present invention obtains have antibacterial activity to staphylococcus aureus.
Embodiment 2:
A preparation method that adopts twin thing enzyme modification gelatin and chitosan blend biomaterial, it comprises following concrete steps:
1) preparation of solution:
Take 3g gelatin and join in 50mL deionized water, within 1.5 hours, dissolve obtain the gelatin solution that concentration is 0.06g/mL completely to gelatin the stirred in water bath of 40 DEG C; Take 1.5g chitosan and join 50mL 1%((percent by volume v/v%) in acetic acid solution, under room temperature, stir and fully dissolve to chitosan for 4 hours, obtain the chitosan solution that concentration is 0.03g/mL, to slowly drip in chitosan solution 0.5mol/L sodium hydroxide solution to the pH of solution be 5.5; Microbial transglutaminase is added in deionized water and stirred 15 minutes under temperature, refilter the microbial transglutaminase solution that obtains 2U/mL; Tryrosinase is added to PBS(pH7.0) in buffer solution, under room temperature, stir 3 minutes, obtain the tryrosinase solution of 1000U/mL.
2) blend of gelatin and chitosan:
Measure respectively the gelatin solution of the above-mentioned preparation of 50 mL and chitosan solution equal-volume and mix, stir 30 minutes at 40 DEG C, make solution mix homogeneously, obtain gelatin and chitosan blend solution.
3) preparation of the gelatin of twin thing enzyme modification and chitosan blend material:
In the blend solution of 100mL gelatin and chitosan, add 6U microbial transglutaminase and 3000U tryrosinase, stirring reaction 20 minutes at 28 DEG C, reactant liquor is poured in culture dish, at room temperature aeration-drying 48 hours, obtain gelatin and the chitosan blend material diaphragm of twin thing enzyme modification, then diaphragm is soaked 45 minutes in 0.2mol/L sodium hydroxide solution, use again a large amount of deionized water rinsing diaphragms, at 35 DEG C, be dried 18 hours, obtain a kind of gelatin and chitosan blend biomaterial that adopts twin thing enzyme modification.
Embodiment 3:
A preparation method that adopts twin thing enzyme modification gelatin and chitosan blend biomaterial, it comprises following concrete steps:
1) preparation of solution:
Take 6g gelatin and join in 100mL deionized water, within 1.5 hours, dissolve obtain the gelatin solution that concentration is 0.06g/mL completely to gelatin the stirred in water bath of 60 DEG C; Take 2g chitosan and join 100mL 1%((percent by volume v/v%) in acetic acid solution, under room temperature, stir and fully dissolve to chitosan for 5 hours, obtain the chitosan solution that concentration is 0.02g/mL, to slowly drip in chitosan solution 0.8mol/L sodium hydroxide solution to the pH of solution be 5.5; Microbial transglutaminase is added under deionized water room temperature and stirred 20 minutes, refilter the microbial transglutaminase solution that obtains 2U/mL; Tryrosinase is added to PBS(pH7.0) in buffer solution, under room temperature, stir 5 minutes, obtain 1000U/mL tryrosinase solution.
2) blend of gelatin and chitosan:
Measure respectively the gelatin solution of the above-mentioned preparation of 100mL and chitosan solution equal-volume and mix, stir 30 minutes at 60 DEG C, make solution mix homogeneously, obtain gelatin and chitosan blend solution.
3) preparation of the gelatin of twin thing enzyme modification and chitosan blend material:
In the blend solution of 100 mL gelatin and chitosan, add 8U microbial transglutaminase and 4000U tryrosinase, stirring reaction 10 minutes at 30 DEG C, reactant liquor is poured in culture dish, at room temperature aeration-drying 60 hours, obtain gelatin and the chitosan blend material diaphragm of twin thing enzyme modification, then diaphragm is soaked 1 hour in 0.5mol/L sodium hydroxide solution, use again a large amount of deionized water rinsing diaphragms, at 40 DEG C, be dried 24 hours, obtain adopting gelatin and the chitosan blend biomaterial of twin thing enzyme modification.
It should be noted that, those of ordinary skill in the art should be appreciated that and can modify or be equal to replacement technical scheme of the present invention, and do not depart from aim and the scope of technical solution of the present invention, and it all should be encompassed in the middle of claim scope of the present invention.
Claims (1)
1. adopt the gelatin of twin thing enzyme modification and a preparation method for chitosan blend biomaterial, it is characterized in that, it comprises following concrete steps:
1) preparation of solution:
Press gelatin: deionized water=2～6g:50～100mL, choose gelatin and deionized water, for subsequent use; Gelatin is joined in deionized water, within 1～2 hour, after gelatin fully dissolves, obtain gelatin solution the stirred in water bath of 40～60 DEG C;
By chitosan: acetic acid solution=1～2g:50～100mL that percent by volume is 1%, choose chitosan and acetic acid solution, for subsequent use; Chitosan is joined in acetic acid solution, stir and fully dissolve to chitosan for 3～5 hours under room temperature, then in solution, drip 0.5～1.0mol/L sodium hydroxide solution, the pH of regulator solution is 5.0～6.0, obtains chitosan solution;
Press microbial transglutaminase: deionized water=10～20U:5～10mL, choose microbial transglutaminase and deionized water, for subsequent use; Microbial transglutaminase is joined in deionized water, under room temperature, stir and fully dissolve for 15～30 minutes, refilter and obtain microbial transglutaminase solution;
PBS buffer solution=5000～10000U:5～the 10mL that is 7.0 by tryrosinase: pH, chooses tryrosinase and pH and is 7.0 PBS buffer solution, for subsequent use; It is in 7.0 PBS buffer solution that tryrosinase is dissolved in to described pH, stirs and after tryrosinase fully dissolves, obtain tryrosinase solution in 2～5 minutes under room temperature;
2) blend of gelatin and chitosan:
Press gelatin solution: chitosan solution=50～100mL:50～100mL, choose gelatin solution and the chitosan solution of above-mentioned preparation, for subsequent use; Gelatin solution and chitosan solution equal-volume are mixed, stir 15～30 minutes at 40～60 DEG C, make solution mix homogeneously, obtain gelatin and chitosan blend solution;
3) preparation of the gelatin of twin thing enzyme modification and chitosan blend material:
Press gelatin and chitosan blend solution: microbial transglutaminase: tryrosinase=50～100mL:3～8U:3000～7000U, choose above-mentioned gelatin and chitosan blend solution, microbial transglutaminase and tryrosinase, for subsequent use; In gelatin and chitosan blend solution, add mentioned microorganism T-5398 and tryrosinase, stirring reaction 10～20 minutes at 28～30 DEG C, reactant liquor is poured in culture dish, at room temperature aeration-drying 48～72 hours, obtain gelatin and the chitosan blend material diaphragm of twin thing enzyme modification, then diaphragm is soaked 0.5～1 hour in 0.1～0.5mol/L sodium hydroxide solution, use again deionized water flushing membrane sheet, at 30～40 DEG C, be dried 12～24 hours, obtain gelatin and the chitosan blend biomaterial of the twin thing enzyme modification of described employing.
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| 周小华.壳聚糖-明胶共聚物的酶促合成及抑菌性质.《应用化学》.2008,第25卷(第3期),第334-339页. |
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