CN103849616A - Fiber carrier based immobile laccase and preparation method and application thereof - Google Patents
Fiber carrier based immobile laccase and preparation method and application thereof Download PDFInfo
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- CN103849616A CN103849616A CN201410112485.3A CN201410112485A CN103849616A CN 103849616 A CN103849616 A CN 103849616A CN 201410112485 A CN201410112485 A CN 201410112485A CN 103849616 A CN103849616 A CN 103849616A
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Abstract
The invention relates to fiber carrier based immobile laccase and a preparation method and application thereof. The fiber carrier based immobile laccase comprises fiber serving as a carrier, active molecules distributed on the surface of the fiber, a mesoporous silica layer covering the fiber and the active molecules and laccase embedded in the mesoporous silica layer, wherein the mesoporous silica layer is formed by depositing active molecule catalyzed organosilane on the surface of the fiber. The immobile laccase disclosed by the invention is high in enzyme activity recovery rate, high in immobilization efficiency, good in reusability and good in operability. The preparation method is moderate in reaction conditions and greatly protects activity of the laccase. The immobile laccase disclosed by the invention is applicable to such fields as food, textile, papermaking from pulp or sewage treatment.
Description
Technical field
The present invention relates to enzyme immobilization technology field, relate in particular to a kind of immobilization laccase based on fibrous carrier and preparation method thereof and the application in dye decolored.
Background technology
Enzyme is catalytic specie important in the vital movement of organism, and it is controlling the whole metabolism of organism.At in vitro, enzyme is widely used in pharmacy, food and the field such as brewages because of its efficient single-minded feature.But, when enzyme has left this environment relatively stable and mild condition of organism, just become extremely fragile.In the environment of high temperature, strongly-acid or alkalescence, organic solvent, easy especially deactivation, is restricted the widespread use of enzyme in industry.And because enzyme is difficult for separatedly in reaction system, cause enzyme repeated multiple timesly not use, increased cost, return product and purify and brought difficulty.In order to address these problems, risen the immobilization technology of enzyme in the eighties of last century sixties, open up another research direction of enzyme industry.Enzyme immobilization material, by the surface interaction of its unique surface property and enzyme, is controlled the space conformation of enzyme, makes enzyme be fixed on material surface and effectively stablize its conformation, allows enzyme relatively under exacting terms, bring into play katalysis.
Laccase is the polyphenoloxidase of a class cupric, has confirmed that it can be widely used in the industrial circles such as the synthetic and bio-sensing of papermaking, weaving, filth disposal, chemical industry.Compare other physico-chemical processes that extensively adopt now, use laccase can significantly reduce the input of energy and the use of the unfriendly compound of environment.But the real application requiring laccase in similar industrial field has enough stability, good operability, can reuse and be convenient to the performances such as separation.
Therefore, the research of immobilization laccase becomes one of means that realize laccase industrial applications, and is widely studied.The technology of the immobilization laccase of having developed at present, is roughly divided three classes: physisorphtion, chemical bond-linking connection and entrapping method.Physisorphtion generally adopts micro Nano material that specific surface area is large as carrier, utilizes static, hydrophilic and hydrophobic etc. that laccase is adsorbed on to material surface.This method is simple, and the fixing condition being usually directed to is gentleer, but because this method itself is can design degree low, is difficult to ensure immobilization efficiency, and the laccase that has been adsorbed on carrier surface in use is also easy to desorption again and leaves carrier.The chemical bond that chemical bond-linking connection utilizes chemical reaction to generate is conventionally fixed on laccase on solid support material.This method designability is high, and chemical bond has ensured to connect reliably between laccase and material, but in chemical ligation, may relate to for the damaging larger reaction conditions of free laccase activity and chemical molecular.Therefore, the shortcoming of this method is that the enzyme rate of recovery alive is low.Entrapping method utilizes porous material to do carrier conventionally, and laccase is constrained in to material internal, thereby realizes the fixing and protection of solid support material to laccase of laccase.Conventionally, this method has higher immobilization efficiency, and also better to the protection of laccase activity in immobilization process, in use procedure, the leakage of enzyme is few; But owing to being wrapped in the laccase of carrier inside, be difficult to participate in follow-up reaction, its enzyme rate of recovery alive may not be certain very high.
Summary of the invention
For the deficiencies in the prior art, the invention provides a kind of immobilization laccase based on fibrous carrier and preparation method thereof and the application in dye decolored.The enzyme of described immobilization laccase is lived, and the rate of recovery is high, immobilization efficiency is high, reusability good and operability is good; Described preparation method's reaction conditions gentleness, has greatly protected the activity of laccase.
In first aspect, the invention provides a kind of immobilization laccase based on fibrous carrier, comprising: as the mesopore silicon oxide layer of the fiber of carrier, the bioactive molecule that is distributed in described fiber surface, coated described fiber and bioactive molecule and be embedded in the laccase in described mesopore silicon oxide layer; Wherein said mesopore silicon oxide layer is that described bioactive molecule catalysis organosilane is at described fiber surface formation of deposits.
In the present invention, the outstanding feature of the bioactive molecule of described fiber surface is to have for example, near physiological condition (normal temperature, normal pressure, pH value are neutral and water condition) time catalysis organosilane characteristic at fiber surface formation of deposits mesopore silicon oxide layer.Therefore, any bioactive molecule with this catalysis characteristics all can be used in the present invention.The bioactive molecule adopting in embodiment of the present invention has amido.Therefore, as preferred version of the present invention, described bioactive molecule has amido, and its amido has the characteristic of catalysis organosilane at fiber surface formation of deposits mesopore silicon oxide layer.
Preferably, described bioactive molecule is the molecule that amido content is very high, and for example described bioactive molecule is selected from polymethyl acrylic acid N, N-dimethylamino ethyl ester (PDMAEMA), polyetherimide (PEI) or their multipolymer.
In the present invention, can described bioactive molecule be modified to described fiber surface by self-assembly layer by layer, surface grafting and/or surperficial initiated polymerization.The expertise that those skilled in the art can possess according to it and existing techniques in realizing, such as Marc R.Knecht etc. utilizes the dendrimer of polyamines base to do template and catalyzer, mineralising silicon oxide bead (Langmuir2004 thereon, 20 (11), pp4728 – 4732), the porous microgel that amido is rich in the utilizations such as Fen Zhou is as template, Catalytic Oxygen SiClx in-situ deposition (Langmuir2007, 23 (19), pp9737 – 9744), Dong Jin Kim etc. are rich in the PDMAEMA of amido on gold surface is modified, catalytic deposition silicon oxide layer (Langmuir2004, 20 (19), pp7904-7906).
In the present invention, described fiber can be various macromolecular fibres prepared by various processing modes, and for example described fiber can be selected from styroflex, pet fiber, polyethylene fibre, polypropylene fibre, polyether-imide fiber, nylon fiber, cellulosic fibre and/or fibroin fiber.Preferably, the diameter of described fiber is 50nm～50 μ m, for example 80nm, 120nm, 200nm, 300nm, 500nm, 800nm, 900nm, 1.2 μ m, 2 μ m, 5 μ m, 8 μ m, 10 μ m, 20 μ m, 35 μ m, 40 μ m, 45 μ m or 48 μ m, and the scope between any two in these concrete point values.
In the present invention, can obtain described fiber by techniques such as direct spinning, spinning-machinery (chemistry) stripping method, co-blended spinning-dissolving (hydrolysis) stripping methods.The expertise that those skilled in the art can possess according to it and existing techniques in realizing, as magnify (Beijing Institute of Clothing Tech's journals 2004 such as province, 24 (2), pp62-68.) summarize direct spinning and prepared super fine polyester, ultra-fine nylon, superfine polypropylene nitrile fiber; Spinning-mechanically peel method, or spinning-chemical stripping legal system is for nylon/polyethylene, nylon/polystyrene composite ultrafine fiber; And ultra-fine nylon/polyethylene " island " shape structural fibers of preparing of co-blended spinning-dissolving stripping method.
As preferred version of the present invention, the thickness of described mesopore silicon oxide layer is 10nm～10 μ m, for example 12nm, 20nm, 50nm, 80nm, 150nm, 200nm, 400nm, 700nm, 900nm, 1.2 μ m, 1.5 μ m, 2 μ m, 3 μ m, 5 μ m, 8 μ m, 9 μ m or 9.5 μ m, and the scope between any two in these concrete point values.The thickness of described mesopore silicon oxide layer is determined by immobilized reactant time and silane molecule concentration.Wherein, the silicon oxide in mesopore silicon oxide layer can be silicon-dioxide, can be also the silicon oxide settling with some active group.
As preferred version of the present invention, described organosilane is can in water, be hydrolyzed and the silane of condensation.Preferably, described organosilane is selected from tetramethoxy-silicane, tetraethoxysilane, Trimethoxy silane, 3-mercaptopropyltriethoxysilane, chloropropyl triethoxysilane, 3-(2,3-epoxy the third oxygen) propyl trimethoxy silicane, 3-aminopropyl triethoxysilane and/or vinyltrimethoxy silane.Can use separately above-mentioned any organosilane, also can mix and use two or more above-mentioned organosilane, preferably use separately.
In the present invention, described laccase can be any any kind of plant, microorganism and/or animal and laccase of form of deriving from.
In second aspect, the invention provides a kind of method of preparing the immobilization laccase described in first aspect, comprise: fiber surface to described bioactive molecule is soaked in the solution that contains described organosilane and laccase, treat that described organosilane is at described fiber surface formation of deposits mesopore silicon oxide layer, and described laccase is embedded in described mesopore silicon oxide layer, obtain described immobilization laccase.
Wherein, the fiber that described surface has a described bioactive molecule can be modified fiber surface by bioactive molecule by self-assembly layer by layer, surface grafting and/or surperficial initiated polymerization and obtain, and also can directly prepare the ultra-fine fibre that is rich in amido; Described bioactive molecule has for example, near physiological condition (normal temperature, normal pressure, pH value are neutral and water condition) time catalysis organosilane characteristic at fiber surface formation of deposits mesopore silicon oxide layer.The concrete selection of described bioactive molecule, fiber and organosilane, as described in first aspect, does not repeat them here.
The described solution that contains described organosilane and laccase, can first prepare respectively organosilane solution and laccase solution, is then mixed to get mixing solutions; Also can be simultaneously organosilane and laccase be dissolved in and in solvent, obtain mixing solutions; Preferably first prepare respectively remix and obtain mixing solutions.Wherein, solvent can be ortho-water, deionized water or distilled water etc., preferably deionized water.
As preferred version of the present invention, described method comprises: surface is had to the fiber of described bioactive molecule with the amount of 0.05-5mg fiber/mL solution, be soaked in and contain in 0.01-1mM organosilane, 0.1-10mg/mL laccase and the hydrionic solution of 0.001-1mM, at 4-30 DEG C, soak 0.1-48 hour, obtain described immobilization laccase.
Preferably, the amount of described fiber is 0.1-1mg fiber/mL solution, for example 0.11mg fiber/mL solution, 0.12mg fiber/mL solution, 0.15mg fiber/mL solution, 0.2mg fiber/mL solution, 0.4mg fiber/mL solution, 0.7mg fiber/mL solution, 0.9mg fiber/mL solution, 0.95mg fiber/mL solution or 0.98mg fiber/mL solution, and the scope between any two in these concrete point values; Be preferably 0.2-0.8mg fiber/mL solution, more preferably 0.3-0.6mg fiber/mL solution.
Preferably, in described solution, the concentration of organosilane is 0.02-0.8mM, for example 0.025mM, 0.03mM, 0.05mM, 0.08mM, 0.09mM, 0.11mM, 0.15mM, 0.18mM, 0.25mM, 0.4mM, 0.6mM, 0.7mM, 0.75mM or 0.78mM, and the scope between any two in these concrete point values; Be preferably 0.05-0.5mM, more preferably 0.1-0.3mM.
Preferably, in described solution, the concentration of laccase is 0.2-8mg/mL, for example 0.25mg/mL, 0.4mg/mL, 0.5mg/mL, 0.7mg/mL, 0.9mg/mL, 1.1mg/mL, 1.5mg/mL, 1.9mg/mL, 2.1mg/mL, 4mg/mL, 5mg/mL, 6.5mg/mL, 6.8mg/mL, 7.2mg/mL, 7.5mg/mL or 7.9mg/mL, and the scope between any two in these concrete point values; Be preferably 0.5-5mg/mL, more preferably 0.8-2mg/mL.
Preferably, in described solution, hydrionic concentration is 0.001-0.8mM, for example 0.003mM, 0.005mM, 0.007mM, 0.01mM, 0.03mM, 0.05mM, 0.11mM, 0.15mM, 0.18mM, 0.25mM, 0.4mM, 0.48mM, 0.52mM, 0.60mM, 0.75mM or 0.78mM, and the scope between any two in these concrete point values; Be preferably 0.005-0.5mM, more preferably 0.05-0.1mM.
Preferably, in described solution, hydrionic source is hydrochloric acid, sulfuric acid and/or nitric acid, can be also other strong acid or weak acid.
Preferably, at 8-25 DEG C, soak 0.5-24 hour, for example 9 DEG C, 10 DEG C, 12 DEG C, 15 DEG C, 17 DEG C, 19 DEG C, 21 DEG C, 23 DEG C or 24 DEG C of soaking temperatures, and the scope between any two in these concrete point values; Soak time for example 0.6 hour, 0.8 hour, 1.2 hours, 1.8 hours, 2.5 hours, 4 hours, 8 hours, 12 hours, 15 hours, 18 hours, 20 hours, 22 hours, 23 hours or 23.5 hours, and the scope between any two in these concrete point values; Be preferably at 12-20 DEG C and soak 1-12 hour.
In the third aspect, the invention provides the application in food, weaving, paper pulp papermaking or sewage disposal of immobilization laccase described in a kind of first aspect, the application in preferred coloring agent decolouring.
Beneficial effect of the present invention is: Laccase Immobilization of the present invention carries out under near reaction conditions very gentle normal temperature, normal pressure, water, pH neutrality etc., has greatly protected the activity of laccase; Described method adopts the method fixing laccase in the mesopore silicon oxide embedding of carrier fibre surface, has solved the low problem of traditional absorption method immobilization efficiency, and the low problem of enzymatic activity recovery of entrapping method and traditional fiber surface covalence graft laccase method.And, the immobilization laccase compared with micro-nano granules as carrier, immobilization laccase of the present invention has stronger reusability and operability.
Brief description of the drawings
Fig. 1 is reaction principle schematic diagram and the structural representation of immobilization laccase of the present invention, and wherein 1 represents carrier fibre, and 2 represent bioactive molecule, and 3 represent mesopore silicon oxide layer, and 4 represent laccase.
Fig. 2 be the surface sweeping Electronic Speculum picture of ultra-fine fibre before fixing laccase in the embodiment of the present invention 1 (a and scanning electron microscope picture c) and after fixing laccase (and b and d), show fixing laccase after ultra-fine fibre surface have coating.
Fig. 3 is prepared in the embodiment of the present invention 1 in the process of immobilization laccase, along with the variation of immobilization time, and the variation of the laccase content in laccase and silicane hydrolysate mixing solutions, when visible immobilization 120min, obtaining Laccase Immobilization efficiency is 98%.
Fig. 4 be in the embodiment of the present invention 1 preparation immobilization laccase and free laccase under high temperature (60 DEG C) condition, enzyme activity is over time.
Fig. 5 is immobilization laccase and the free laccase of preparation in the embodiment of the present invention 1 hatch altogether respectively (37 DEG C, Proteinase K concentration is 100 μ g/mL, 50mM Tris-HCl, pH7.5) during with Proteinase K, and enzyme activity over time.
Fig. 6 be the immobilization laccase of preparation in the embodiment of the present invention 1 while reusing enzyme activity with the variation of access times.
Fig. 7 be in the embodiment of the present invention 1 preparation immobilization laccase and free laccase to the percent of decolourization of Reactive Brilliant Blue KN-R (a) over time, and when reusing immobilization laccase Reactive Brilliant Blue KN-R being decoloured, every day to the decolorizing efficiency of new Reactive Brilliant Blue KN-R with the variation (b) of reusing number of times (reuse once every day).
Embodiment
Describe the present invention in detail below in conjunction with accompanying drawing and by embodiment.
As shown in Figure 1, immobilization laccase based on fibrous carrier provided by the invention, comprising: as the mesopore silicon oxide layer 3 of the fiber 1 of carrier, the bioactive molecule 2 that is distributed in described fiber 1 surface, coated described fiber 1 and bioactive molecule 2 and be embedded in the laccase 4 in described mesopore silicon oxide layer 3; Wherein said mesopore silicon oxide layer 3 is that described bioactive molecule 2 catalysis organosilanes form at described fiber 1 surface deposition.
Deposition reaction of the present invention, under near physiological condition (for example, normal temperature, normal pressure, pH value are neutral and water condition), form mesopore silicon oxide layer 3 by bioactive molecule 2 catalysis organosilanes at fiber 1 surface deposition, laccase 4 is embedded in mesopore silicon oxide layer 3 and forms immobilization laccase simultaneously.Deposition reaction of the present invention does not need severe condition, has protected the activity of laccase.
Below in conjunction with embodiment, embodiment of the present invention are described in detail.It will be understood to those of skill in the art that following examples are only the preferred embodiments of the present invention, so that understand better the present invention, thereby should not be considered as limiting scope of the present invention.
Experimental technique in following embodiment, if no special instructions, is ordinary method; Experiment material used, if no special instructions, is and is purchased available from routine biochemistry chemical reagent work.
In this example, adopt polystyrene (PS) the electrostatic spinning ultra-fine fibre of the most difficult modification as the carrier of immobilization laccase, the mean diameter of this fiber is 700nm.
First, get in the vitriol oil that 10mg fiber is immersed in 9.8M, after 5min, pull out, with deionized water rinsing repeatedly, until before and after rinsing, rinsing liquid pH value no longer changes.Thereafter, fiber is immersed in to the aqueous solution of the diallyl dimethyl ammoniumchloride (PDADMAC) of 1mg/mL, after 10min, fiber is pulled out, with deionized water wash 3 times, then steep in polyacrylic acid grafted alkyl bromide (PAA-g-Alkyl Bromide) aqueous solution at 1mg/mL, after 10min, pull fiber out, use deionized water wash 3 times.The operation that repeats above-mentioned PDADMAC and PAA-g-Alkyl Bromide aqueous solution soaking and wash 2 times.,, 10mg fiber dispersion, in 16mL water, and is added to 2mL methacrylic acid N therein thereafter; N-dimethylamino ethyl ester (DMAEMA) and 8mg2,2-dipyridyl, freeze-take out-melt operation 3 times after; under gas shield, add 5mg cuprous bromide (CuBr), stirred overnight under normal temperature.So far, on polystyrene (PS) fiber, modified the PDMAEMA of rich amido.
The 3-mercaptopropyltriethoxysilane that compound concentration is 1mM in 1mM hydrochloric acid, leaves standstill until observation solution is as clear as crystal.Take laccase 20mg, be dispersed in 18mL deionized water, concussion repeatedly, until solution is as clear as crystal.Laccase solution is mixed with 2mL solution of silane, then the fiber that 10mg is modified is immersed in mixing solutions, under normal temperature, 2h or 4 DEG C spend the night, can being fixed laccase.Before and after fixing laccase, the morphology change of fiber as shown in Figure 2.Before comparing fixing laccase, the fiber surface after fixing laccase becomes coarse, and it is large that Fibre diameter becomes, and fiber surface can be observed silicon shell.
Embodiment 2: at the surface immobilized laccase of polystyrene (PS) ultra-fine fibre
Get 10mg fiber (with embodiment 1) and be immersed in the vitriol oil of 9.8M, after 5min, pull out, with deionized water rinsing repeatedly, until before and after rinsing, rinsing liquid pH value no longer changes., fiber be immersed in the aqueous solution of polyetherimide (PEI) of 1mg/mL, after 10min, fiber pulled out thereafter, with deionized water wash 3 times, then steep in polyacrylic acid (PAA) aqueous solution at 1mg/mL, after 10min, pull fiber out, use deionized water wash 3 times.The operation that repeats above-mentioned PEI and PAA aqueous solution soaking and wash 2 times, finally repeats immersion and a rinsing in the PEI aqueous solution again.Because PEI molecule itself is rich in amido, so far, polystyrene (PS) fiber surface has been introduced the molecule that is rich in amido.
The 3-mercaptopropyltriethoxysilane that compound concentration is 1mM in 1mM hydrochloric acid, leaves standstill until observation solution is as clear as crystal.Take laccase 20mg, be dispersed in 18mL deionized water, concussion repeatedly, until solution is as clear as crystal.Laccase solution is mixed with 2mL solution of silane, then the fiber that 10mg is modified is immersed in mixing solutions, under normal temperature, 12h or 4 DEG C leave standstill two days, can being fixed laccase.
Embodiment 3: at the surface immobilized laccase of polyethylene terephthalate (PET) ultra-fine fibre
In the present embodiment, adopting PET yarn is carrier, and this carrier fibre mean diameter is 50 μ m.
First, get in the vitriol oil that 10mg fiber is immersed in 9.8M, after 5min, pull out, with deionized water rinsing repeatedly, until before and after rinsing, rinsing liquid pH value no longer changes.Thereafter, fiber is immersed in to the aqueous solution of the diallyl dimethyl ammoniumchloride (PDADMAC) of 1mg/mL, after 10min, fiber is pulled out, with deionized water wash 3 times, then steep in polyacrylic acid grafted alkyl bromide (PAA-g-AlkylBromide) aqueous solution of 1mg/mL, after 10min, pull fiber out, use deionized water wash 3 times.The operation that repeats above-mentioned PDADMAC and PAA-g-Alkyl Bromide aqueous solution soaking and wash 2 times.Thereafter, 10mg fiber dispersion, in 16mL water, and is added to 2mL DMAEMA and 8mg2 therein, 2-dipyridyl, freezes-takes out-melt after operation 3 times, under gas shield, adds 5mg CuBr, stirred overnight under normal temperature.So far, on PET fiber, modified the PDMAEMA of rich amido.
The 3-mercaptopropyltriethoxysilane that compound concentration is 1mM in 1mM hydrochloric acid, leaves standstill until observation solution is as clear as crystal.Take laccase 20mg, be dispersed in 18mL deionized water, concussion repeatedly, until solution is as clear as crystal.Laccase solution is mixed with 2mL solution of silane, then the fiber that 10mg is modified is immersed in mixing solutions, under normal temperature, 2h or 4 DEG C spend the night, can being fixed laccase.
The difference of the present embodiment and embodiment 1 is, laccase fixation procedure is as follows: the chloropropyl triethoxysilane that compound concentration is 0.2mM in 0.1mM sulfuric acid, leaves standstill until to observe solution as clear as crystal.Take laccase 4mg, be dispersed in 18mL deionized water, concussion repeatedly, until solution is as clear as crystal.Laccase solution is mixed with 2mL solution of silane, then the fiber that 10mg is modified is immersed in mixing solutions, 12h under normal temperature, can being fixed laccase.
Embodiment 5
The difference of the present embodiment and embodiment 1 is, laccase fixation procedure is as follows: the tetraethoxysilane that compound concentration is 5mM in 2mM hydrochloric acid, leaves standstill until to observe solution as clear as crystal.Take laccase 50mg, be dispersed in 38mL deionized water, concussion repeatedly, until solution is as clear as crystal.Laccase solution is mixed with 2mL solution of silane, then the fiber that 40mg is modified is immersed in mixing solutions, 6h at 8 DEG C, can being fixed laccase.
The difference of the present embodiment and embodiment 1 is, laccase fixation procedure is as follows: the tetramethoxy-silicane that compound concentration is 10mM in 5mM hydrochloric acid, leaves standstill until to observe solution as clear as crystal.Take laccase 80mg, be dispersed in 98mL deionized water, concussion repeatedly, until solution is as clear as crystal.Laccase solution is mixed with 2mL solution of silane, then the fiber that 100mg is modified is immersed in mixing solutions, 0.5h at 30 DEG C, can being fixed laccase.
Embodiment 7
The difference of the present embodiment and embodiment 2 is, laccase fixation procedure is as follows: 3-(2,3-epoxy the third oxygen) propyl trimethoxy silicane that compound concentration is 10mM in 3mM hydrochloric acid, leaves standstill until to observe solution as clear as crystal.Take laccase 50mg, be dispersed in 58mL deionized water, concussion repeatedly, until solution is as clear as crystal.Laccase solution is mixed with 2mL solution of silane, then the fiber that 50mg is modified is immersed in mixing solutions, 2h at 15 DEG C, can being fixed laccase.
Test example 1: the test of immobilization laccase and free laccase activity
Preparation 0.5mM2,2-connection nitrogen-bis-(3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts (ABTS) aqueous solution.Get the about 1mg of immobilization laccase (embodiment 1), the free laccase aqueous solution 0.1mL of 1mg/mL adds respectively 3mL and the 2.9mL ABTS aqueous solution, concussion 1min.Survey the absorbance that it goes out at 420nm.
Test example 2: immobilization efficiency and enzymatic activity recovery test
In the immobilization process of embodiment 1, constantly detect the content of laccase in laccase silicane hydrolysate mixed solution with the variation of immobilization time, result is as shown in Figure 3.When fixing beginning (0min), in the laccase content in laccase and silicane hydrolysate mixed solution and certain moment mixed solution, the difference of laccase content, is the amount that this moment is fixed on the laccase on ultra-fine fibre.As seen from Figure 3, after the fixing 120min of normal temperature, immobilization efficiency has reached 98%, and enzyme activity recovery experiment shows, enzymatic activity recovery has reached 94%.
Test example 3: immobilization laccase stability at high temperature
The free laccase of immobilization laccase (embodiment 1) and 1mg/mL is placed in 60 DEG C of water-baths, takes out laccase every 30min, measure its energy value.During with water-bath 0min, the energy value of laccase is 100%, and energy value, to the mapping of water-bath time, is obtained to the result shown in Fig. 4.As shown in Figure 4, the vigor of immobilization laccase is at high temperature stable a lot of than the vigor of free laccase.
Test example 4: the stability of immobilization laccase in the time that Proteinase K exists
The free laccase of immobilization laccase (embodiment 1) and 1mg/mL is added respectively to damping fluid (the 100 μ g/mL that contain Proteinase K, 50mM Tris-HCl, pH7.5), and be placed in 37 DEG C of water-baths, take out at set intervals laccase, measure its energy value.During with water-bath 0min, the energy value of laccase is 100%, and energy value, to the mapping of water-bath time, is obtained to the result shown in Fig. 5.As shown in Figure 5, the vigor of immobilization laccase is stable more a lot of than the vigor of free laccase in the time that Proteinase K exists.
Test example 5: immobilization laccase reuse stability
Be fixed on the laccase on fiber, can realize easily separating of immobilization laccase and reaction system by pulling the operations such as fiber out.In the time that needs reuse laccase, then the fiber of having fixed laccase is soaked into reaction system and can realizes the recycling of immobilization laccase.In order to measure the repeat performance of immobilization laccase, continuous ten same immobilization laccases of use (embodiment 1) react with ABTS, and survey its vigor, concrete operations are with test example 1, repeating ten test examples 1 operates, and the ABTS solution more renewing each time, uses washed with de-ionized water immobilization laccase three times after each operation.Result as shown in Figure 6.As shown in Figure 6, this immobilization laccase is enzyme activity fall maximum in using for the first time, but also less than 5%, from for the second time, the vigor of immobilization laccase is almost constant.Illustrate that this immobilization laccase has good reusable performance, and laccase after immobilization is not easy to depart from carrier.
Test example 6: the research of immobilization laccase to Reactive Brilliant Blue KN-R decolouring
Get 2mg immobilization laccase (embodiment 1), and the 0.1mL concentration free laccase aqueous solution that is 1mg/mL, adding respectively 100mL concentration is the Reactive Brilliant Blue KN-R of 1mg/mL, water-bath at 37 DEG C.At set intervals, sample thief is a small amount of, tests its absorbance at 565nm place, converses decolorizing efficiency, and result as shown in Figure 7a, can be found out by Fig. 7 a, and the final decolorizing efficiency of immobilization laccase will be higher than free laccase.
Within continuous ten days, process ten batches of new Reactive Brilliant Blue KN-Rs with same immobilization laccase, change once new reactive brilliant bule dye solution every day.Result as shown in Figure 7b, this immobilization laccase energy steady operation 6 days, but since the 5th day, the obviously fiber deliquescing of sensation immobilization laccase, start to occur the chip of immobilization laccase at reactor bottom, thereby cause the loss of immobilization laccase enzyme activity, this phenomenon is aggravation afterwards at the 6th day, the fiber fragment of immobilization laccase increases, and the loss of immobilization laccase vigor is serious.Can expect: change more firm fibrous carrier, or select diameter thicker fibrous carrier, this phenomenon can be improved significantly.
Applicant's statement, the present invention illustrates detailed features of the present invention and method detailed by above-described embodiment, but the present invention is not limited to above-mentioned detailed features and method detailed, do not mean that the present invention must rely on above-mentioned detailed features and method detailed could be implemented.Person of ordinary skill in the field should understand, any improvement in the present invention is selected the selection of the equivalence replacement of component and the interpolation of ancillary component, concrete mode etc., within all dropping on protection scope of the present invention and open scope to the present invention.
Claims (10)
1. the immobilization laccase based on fibrous carrier, comprising: as the mesopore silicon oxide layer of the fiber of carrier, the bioactive molecule that is distributed in described fiber surface, coated described fiber and bioactive molecule and be embedded in the laccase in described mesopore silicon oxide layer; Wherein said mesopore silicon oxide layer is that described bioactive molecule catalysis organosilane is at described fiber surface formation of deposits.
2. immobilization laccase according to claim 1, is characterized in that, described bioactive molecule has amido;
Preferably, described bioactive molecule is selected from polymethyl acrylic acid N, N-dimethylamino ethyl ester, polyetherimide or their multipolymer.
3. immobilization laccase according to claim 1 and 2, is characterized in that, by self-assembly layer by layer, surface grafting and/or surperficial initiated polymerization, described bioactive molecule is modified to described fiber surface.
4. according to the immobilization laccase described in claim 1-3 any one, it is characterized in that, described fiber is selected from styroflex, pet fiber, polyethylene fibre, polypropylene fibre, polyether-imide fiber, nylon fiber, cellulosic fibre and/or fibroin fiber;
Preferably, the diameter of described fiber is 50nm～50 μ m;
Preferably, described fiber obtains by direct spinning, spinning-mechanically peel method, spinning-chemical stripping method or co-blended spinning-dissolving stripping method technique.
5. according to the immobilization laccase described in claim 1-4 any one, it is characterized in that, the thickness of described mesopore silicon oxide layer is 10nm～10 μ m.
6. according to the immobilization laccase described in claim 1-5 any one, it is characterized in that, described organosilane is can in water, be hydrolyzed and the silane of condensation;
Preferably, described organosilane is selected from tetramethoxy-silicane, tetraethoxysilane, Trimethoxy silane, 3-mercaptopropyltriethoxysilane, chloropropyl triethoxysilane, 3-(2,3-epoxy the third oxygen) propyl trimethoxy silicane, 3-aminopropyl triethoxysilane and/or vinyltrimethoxy silane.
7. prepare the method for the immobilization laccase described in claim 1-6 any one for one kind, comprise: fiber surface to described bioactive molecule is soaked in the solution that contains described organosilane and laccase, treat that described organosilane is at described fiber surface formation of deposits mesopore silicon oxide layer, and described laccase is embedded in described mesopore silicon oxide layer, obtain described immobilization laccase.
8. method according to claim 7, it is characterized in that, described method comprises: surface is had to the fiber of described bioactive molecule with the amount of 0.05-5mg fiber/mL solution, be soaked in and contain in 0.01-1mM organosilane, 0.1-10mg/mL laccase and the hydrionic solution of 0.001-1mM, at 4-30 DEG C, soak 0.1-48 hour, obtain described immobilization laccase.
9. according to the method described in claim 7 or 8, it is characterized in that, the amount of described fiber is 0.1-1mg fiber/mL solution, is preferably 0.2-0.8mg fiber/mL solution, more preferably 0.3-0.6mg fiber/mL solution;
Preferably, in described solution, the concentration of organosilane is 0.02-0.8mM, is preferably 0.05-0.5mM, more preferably 0.1-0.3mM;
Preferably, in described solution, the concentration of laccase is 0.2-8mg/mL, is preferably 0.5-5mg/mL, more preferably 0.8-2mg/mL;
Preferably, in described solution, hydrionic concentration is 0.001-0.8mM, is preferably 0.005-0.5mM, more preferably 0.05-0.1mM;
Preferably, in described solution, hydrionic source is hydrochloric acid, sulfuric acid and/or nitric acid;
Preferably, at 8-25 DEG C, soak 0.5-24 hour, be preferably at 12-20 DEG C and soak 1-12 hour.
10. the application of the immobilization laccase described in a claim 1-6 any one in food, weaving, paper pulp papermaking or sewage disposal.
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| CN105802950A (en) * | 2016-05-24 | 2016-07-27 | 天津工业大学 | Beaded electrospun nanofiber immobilized saccharomycete and preparation method thereof |
| CN105995037A (en) * | 2016-05-25 | 2016-10-12 | 昆明诺本科技有限公司 | Production process for feed lipase with high stability and high activity |
| CN106277349A (en) * | 2016-07-18 | 2017-01-04 | 天津科技大学 | A kind of preparation method of the immobilized enzyme for processing mthod of white water from paper making |
| CN112689671A (en) * | 2018-10-24 | 2021-04-20 | 国立大学法人东京工业大学 | Enzyme immobilization carrier and immobilized enzyme |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105802950A (en) * | 2016-05-24 | 2016-07-27 | 天津工业大学 | Beaded electrospun nanofiber immobilized saccharomycete and preparation method thereof |
| CN105995037A (en) * | 2016-05-25 | 2016-10-12 | 昆明诺本科技有限公司 | Production process for feed lipase with high stability and high activity |
| CN106277349A (en) * | 2016-07-18 | 2017-01-04 | 天津科技大学 | A kind of preparation method of the immobilized enzyme for processing mthod of white water from paper making |
| CN106277349B (en) * | 2016-07-18 | 2019-06-11 | 天津科技大学 | It is a kind of for handling the preparation method of the immobilised enzymes of mthod of white water from paper making |
| CN112689671A (en) * | 2018-10-24 | 2021-04-20 | 国立大学法人东京工业大学 | Enzyme immobilization carrier and immobilized enzyme |
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