CN101058824A - Immobilized enzyme biological catalyst, preparation method and application - Google Patents
Immobilized enzyme biological catalyst, preparation method and application Download PDFInfo
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Abstract
The invention relates to an immobilized enzyme biocatalyst and the manufacturing method and application. The immobilized enzyme biocatalyst regards tubular hollow silica dioxide dielectric hole material as a carrier external surface, inner surface and micropore in the wall of which are fixed biological enzyme molecule. The method includes physical adsorption cast investment or chemical coupling/ crosslinking method; the biocatalyst is provided with good enzyme dispersibility and high carrying quantity, high recovery ratio of enzymatic activity, low enzyme flow rate. The invention can fix penicillin acylating enzyme, glucose oxidase, peroxidase, cytase and so on, which also can be used for removing sugar in protein, removing sugar in total egg, removing oxygen in food , microbiological sensing device, antibiosis and disinfection reaction, wherein immobilized penicillin acylating enzyme biocatalyst can be used to hydrolyze penicillin potassium and manufacture 6-APA.
Description
Technical field
The present invention relates to a kind of immobilized enzyme biocatalyst, particularly a kind of is the immobilized enzyme biocatalyst and preparation method thereof of carrier with silicon-dioxide, and the application in catalytic hydrolysis benzylpenicillin potassium reactant salt.
Background technology
Enzyme catalysis technology successfully is applied to the production of food, pharmaceuticals, agricultural chemicals, and is applied to the synthetic field of organic chemistry more and more.After but enzyme catalysis method exists enzyme to break away from its physical environment in actual applications, problems such as structural instability, and most of enzyme is water miscible, with polluted product, also is difficult to recycle after enzyme is water-soluble.The immobilization of enzyme (Enzymatic Immobilization) is to overcome the successful method of above problem.As: immobilized penicillin acylated enzyme (Immobilized Penicillin Acylase), the technology that is used for cracking penicillin production 6-amino-penicillanic acid (6-APA) demonstrates very big superiority.It can not only overcome lyoenzyme or thalline in use poor stability, be difficult for the shortcoming that reclaims, can not use repeatedly, and, simplified purifying technique, improved product production and quality because this technology can not brought protein and other impurity into product.Immobilized penicillin acylated enzyme also has the ability of catalytic hydrolysis and syncillin and cephamycin simultaneously.Because different enzymes has different character and composition, the different in kind of substrate and product, and the purposes difference of product, therefore carrier and the process for fixation that various enzymes are adopted is not quite similar, wherein the selection of solid support material is the key of technology with preparation, excellent carrier can improve the catalytic performance of immobilized enzyme, reduces the cost of Production by Enzymes.
At present, the immobilized enzyme of industrialization many with organic polymer as carrier, these fixation supports seldom can reuse, and have the aftertreatment of material and the idle problem of useless carrier.[Curr Sci 1988 such as Singh; 57 (22): 1229-1231] be carrier with agar-polyacrylamide resin, adopt the fixing penicillin acylase that extracts from E.coli NCIM2563 of the attached technology of double suction.Alginic acid salt and polyacrylamide carrier have been obtained certain success in the application of others, but they and be not suitable for penicillin acylation enzyme-fixing because the use of phosphate buffered saline buffer makes this class carrier stability decreases.
Inorganic carrier exists bigger economic advantages than organic carrier, and its higher physical strength more helps carrying out industrial production in bio-reactor.Inorganic carrier has colloidal sol-gel, pottery, kaolin and the macropore glass of silicon-dioxide and functional silane, sees [Appl.Clay Sci.2005,29,111-126; J.Membrane Sci.2004,241,161-166; Current Appl.Physics 2003,3,129-133].But these carriers are subjected to the restriction of factors such as carrier material hole footpath, specific surface area, grain graininess, and the covering amount of enzyme on carrier is generally not high; The molecular diffusion resistance is bigger, influences substrate and the effective of enzyme contacts, and influences that product in time breaks away from reaction system and the catalytic activity that reduced enzyme; And some carrier and enzyme molecule bonding force a little less than, immobilized enzyme is in the repeated use process, enzyme is easy to from the carrier loss that comes off, thereby has also limited its industrial application.
Mesoporous material is the most popular research object as the fixation support of enzyme, wherein various mesoporous materials are owing to the larger aperture, high-specific surface area, the less diffusional resistance that itself have, and the surface, duct is rich in the hydroxyl functional group and is shown excellent immobilization characteristic.Wherein, silicon-dioxide series material causes the concern and the research of Chinese scholars especially because of it has strong physics, machinery, chemical property and stronger organism and biological tolerance.The controllable bore diameter of the ordered nano mesoporous silicon oxide of bibliographical information is 20-500 at present, and specific surface area is up to 500-1000m
2/ g, Metaporous silicon dioxide material MCM-41[J.Mol.Catal.B.2000,11,45-53], SBA[Micropor.Mecropor.Mater.2001,44/45,755-762], FSM[J.Am.Chem.Soc.2002,124,1142-1149] the immobilization research that is in the news and is used for enzyme, J.He[J.Mol.Catal.B.2000,11,45-53] etc. the people find that penicillin acylase has high charge capacity on carrier SiO 2 molecular sieve MCM-41, they have adopted two kinds of process for fixation: a kind of is that physisorphtion is also referred to as direct immobilization method; Another kind is covalent cross-linking method (glutaraldehyde as cross linker).Result of study shows that the activity by the immobilized enzyme of physisorphtion preparation is 5 times of immobilized enzyme of covalent cross-linking method preparation, but the enzyme that in use is adsorbed on the carrier comes off easily.Can strengthen reactive force between enzyme and the carrier by linking agent and coupling agent to the modification of carrier surface, guarantee the active adsorption of enzyme, can reduce coming off of enzyme molecule again, thereby prolong the work-ing life of immobilized enzyme.Studies show that pattern, surface property, aperture and the pore structure of solid support material influence the useful load of enzyme.It is template that the Chinese patent CN1445311A of the applicant (Beijing University of Chemical Technology) application and CN1511785A disclose with the nano-calcium carbonate, with the water glass is the silicon source, by being reflected at lime carbonate surface coated silica, and regulate and control according to the granular size and the shape of lime carbonate, prepare spherical, difform hollow silicon dioxide mesoporous material such as tubulose, this material not only has the advantage of mesopore molecular sieve, its exclusive hollow structure can improve carrying capacity greatly, can also be by selecting different support shapes, satisfy the requirement that different catalytic reaction activity components distribute, in catalyst field potentiality to be exploited is widely arranged, as: Chinese patent (application number 200510085389.5) is at the distribution requirement of hydrogenation catalyst chain carrier, proposition utilizes this hollow silicon dioxide mesoporous material to be carrier, loaded metal active ingredient on its wall, obtain active ingredient and be metal catalyst and the preparation method that eggshell type distributes, improved the utilization ratio of metal active.In addition, the applicant has carried out series of studies to the application of this material, is carrier with this hollow silicon dioxide mesoporous material, and preparing new immobilized enzyme biocatalyst is task of the present invention, has the wide development prospect.
Summary of the invention
The objective of the invention is to propose a kind of with tubular, hollow SiO
2Mesoporous material is immobilized enzyme biocatalyst of carrier and preparation method thereof, and this catalyzer has good enzyme active center dispersiveness, higher enzymatic activity and storage, operational stability; This preparation method effectively raise carrier to the immobilization characteristic of enzyme, make the enzyme molecular energy be dispersed in tubular, hollow SiO
2Outside surface, internal surface and wall in the micropore, improve the catalytic activity of enzyme;
Another object of the present invention is with tubular, hollow SiO
2Mesoporous material is carrier penicillin acylation enzyme-fixing and the hydrolysis reaction that is applied to potassium salt of penicillin.
Biological catalyst of the present invention: on silica supports, be fixed with biological enzyme, wherein said carrier is a tubular, hollow mesoporous SiO 2 particle, is fixed with the biological enzyme molecule in the micropore in tubular, hollow mesoporous SiO 2 particulate outside surface, internal surface and wall.With immobilized enzyme weight of formulation per-cent is benchmark, and it consists of 0.01%～10.00% biological enzyme, 80.00%～99.99% tubular, hollow structural silica dioxide carrier, and 0～10.00% coupling agent is or/and linking agent.
Described biological enzyme is known suitable silica supported various enzymes, as: one or more in proteolytic enzyme, amylase, glucose isomerase, cellulase, polygalacturonase, lipase, glucose oxidase, amino acylase, altheine enzyme, penicillin acylase, L-Aspartase, Polyribonucleotide phosphorylase, dextrorotation candy acid anhydride enzyme, Lysozyme, horseradish peroxidase or the acetylcholinesterase;
Described have a hollow structure SiO
2The mesoporous particles carrier is prepared from by disclosed method among the Chinese invention patent Shen Qing Publication specification sheets CN 1511785A.Catalyzer of the present invention is then selected wherein tubular structure for use, has bigger pore volume and specific surface area, and the SiO in the small duct of queueing discipline is arranged in wall
2Particle is a carrier, tubular, hollow SiO
2The particulate specific surface area is 200～1000m
2/ g is preferably 500～800m
2/ g; Pore volume is 0.1～1.0mL/g, is preferably 0.4～0.8mL/g; Mean diameter is 20～600nm, is preferably 80～250nm, and mean length is 1～10 μ m, is preferably 2～8 μ m.
The preparation method of biological catalyst of the present invention is: with tubular, hollow mesoporous SiO 2 particulate material is carrier, and the covalent linkage connection method after employing physisorphtion or the carrier surface modification prepares the immobilized enzyme preparation.
Physisorphtion, in turn include the following steps: tubular, hollow silica supports and at least a crystalline enzyme or solution enzyme are joined in deionized water, acetate solution, phosphate buffer soln or the halid solution, and thorough mixing with it, temperature is controlled at 0～50 ℃; Load is washed after finishing, and filters lyophilize 1～6h.The enzyme of this immobilized enzyme by the simple physical absorption method preparation rate of recovery of living is higher, and can keep the three-dimensional structure of enzyme molecule well.
The covalent linkage connection method in turn includes the following steps: with coupling agent or/and linking agent joins tubular, hollow SiO
2In the colloidal solution of carrier, and fully stir with it, stirring velocity is controlled at 400～600r/min, and the pH value of maintenance system is between 8～10, and churning time is 12～24h; Wash after the carrier surface modification, filter, be warmed up to 50～80 ℃ with 5～10 ℃/min then, drying; In the deionized water that powder after the modification and at least a crystalline enzyme or solution enzyme join, acetate solution, phosphate buffer soln or the halid solution, control pH value is between 5～8, and thorough mixing with it, and temperature is controlled at 0～50 ℃; Load is washed after finishing, and filters lyophilize 1～6h; Be kept in the refrigerator-freezer dried powder standby.
Described coupling agent is selected from one or more in the muriate of cyanogen bromide, equal three chlorotriazines, hydrazides, sulfur mustard and titanium, tin, zinc, vanadium and iron; The effect of coupling agent is to the surface treatment enhancing enzyme molecule of solid support material and the adhesive property between the carrier, thereby increases the adsorptive capacity of enzyme on carrier.
Described linking agent is selected from glutaraldehyde, two diazonium p-diaminodiphenyl-2,2 '-disulfonic acid, 4,4 '-two fluoro-3,3 '-diphenylsulfone dinitro, 1,5 '-two fluoro-2, one or more in 4 '-dinitrobenzene, the Toluene-2,4-diisocyanate-isocyanic acid-isothiocyanate.Linking agent is the reagent that contains bifunctional group, and its a kind of functional group can form with solid support material and combine than strong covalent bond, has reduced the enzyme molecule effectively and has come off from carrier.And another functional group can form firm covalent attachment with the amino or the carboxyl of enzyme molecule, and stronger reactive force may have influence on the three-dimensional structure of enzyme molecule, thereby makes losing activity of enzyme branch.Therefore the consumption of coupling agent and linking agent should be controlled at immobilized enzyme weight of formulation per-cent be benchmark 10.00% in.
The SiO of above-mentioned hollow tubular structures
2Carrier is according to disclosed nanometer CaCO among the CN 1511785A
3/ SiO
2Composite material and preparation method thereof obtains.With the needle-like calcium carbonate is template, is the silicon source with tetraethoxy/water glass, under alkaline condition, adds certain amount of surfactant, tetraethoxy generation hydrolysis reaction, and at the surface of lime carbonate coating one deck SiO
2Can also regulate and control simultaneously the size of micropore in granular size, wall thickness and the wall of tubulose hollow silicon dioxide according to the granular size of needle-like calcium carbonate, the amount that adds the amount of tetraethoxy and add tensio-active agent.The preferred mean diameter 20～400nm that adopts, length-to-diameter ratio is about 10～15, CaCO
3The aragonite phase content is 88.75% nanometer needle-like CaCO in the particle
3Particulate is made inorganic template, finally prepares tubular, hollow SiO
2The particulate specific surface area is 200～1000m
2/ g, pore volume are 0.1～1.0mL/g, and mean diameter is 20～600nm, and mean length is the solid support material of 1～10 μ m.
Is the hydrolysis process that the catalyzer of carrier penicillin acylation enzyme-fixing is applied to benzylpenicillin potassium with the present invention with the titanium dioxide/mesoporous particle of tubular, hollow; ordinary method operation according to the hydrolysis of benzylpenicillin potassium; catalyst levels is in parts by weight; be benzylpenicillin potassium 1-3 doubly; 4～70 ℃ of hydrolysising reacting temperatures; pH is controlled at 5.0～9.0, obtains pharmaceutical intermediate 6-APA.
The application of immobilized enzyme biocatalyst of the present invention is not limited to the said fixing penicillin acylase at hydrolysis process; also relate to and fix other various (exemplifying) biological enzymes as preceding institute; be used for different fields, as: the application in the deoxygenation of albumen desugar, shell egg desugar, glycosuria test paper, food, microbiosensor and in antibiotic and sterilization process.
Technique effect of the present invention:
1, immobilized enzyme biocatalyst of the present invention is to have the SiO of hollow tubular structures
2Be carrier, at hollow tubular structures SiO
2The inside and outside wall of the pipe of carrier, wall micropore internal burden have the enzyme molecule, therefore, be easy to realize the distribution of enzyme active center on carrier, and can give full play to the catalytic activity of enzyme active center, and can significantly reduce the consumption of enzyme, can be applied to liquid-solid heterogeneous enzyme catalysis reaction system targetedly;
2, preparation method of the present invention makes the enzyme molecular energy be dispersed in tubular, hollow SiO
2Outside surface, internal surface and wall in the micropore, improve the catalytic activity of enzyme; Effectively raise carrier to the immobilization characteristic of enzyme, realized the recycling behind the solution enzyme immobilization, reduce the use cost of zymin greatly;
3, the penicillin acylation enzyme-fixing catalyzer of Preparation of Catalyst of the present invention with free penicillin acylase relatively: the optimal pH of immobilized penicillin acylated enzyme (8.5) deflection is neutral, free penicillin acylase optimal pH (9.0), and under the same enzymic activity requirement condition, immobilized penicillin acylated enzyme has wideer pH scope; The optimum temperuture of immobilized penicillin acylated enzyme is 51 ℃, and the optimum temperuture of free penicillin acylase is 43 ℃, and the raising of optimum temperuture will help the industrial applications of this kind enzyme; Discover by free enzyme and immobilized enzyme stability in storage: immobilized enzyme was preserved 100 days at 4 ℃, and keeping enzymic activity is 86%, and under similarity condition, the reservation deficient enzyme activity 66% of free enzyme.Therefore penicillin acylation enzyme-fixing catalyzer of the present invention is used for the hydrolysis reaction of potassium salt of penicillin, can improve the ability of catalytic hydrolysis and syncillin and cephamycin.
Description of drawings
Fig. 1 is the TEM photo that is used to prepare the needle-like calcium carbonate template of support of the catalyst of the present invention.
Fig. 2 is the XRD figure spectrum of needle-like calcium carbonate template.
Fig. 3 is the TEM photo of tubular, hollow silica supports of the present invention.
Fig. 4 is the TEM photo of immobilized enzyme biocatalyst of the present invention.
Fig. 5 is that the FT-IR of tubular, hollow silicon-dioxide, penicillin acylase and immobilized enzyme biocatalyst compares collection of illustrative plates, and curve 1 is a tubular, hollow silicon-dioxide collection of illustrative plates among the figure, and curve 2 is the penicillin acylase collection of illustrative plates.Curve 3 is the immobilized penicillin acylated enzyme collection of illustrative plates.
Embodiment
Below preparation method of the present invention is described in detail:
1, physisorphtion prepares catalyzer of the present invention:
Needle-like CaCO with certainweight
3Particulate joins in 50～200mL deionized water, and Vltrasonic device disperses, and adds ethanol and the 0.01-3g tensio-active agent of 10～50mL again, fully stirs 15～30 minutes; Then a certain amount of tetraethoxy slowly is added drop-wise in the mixing solutions, ageing 2～3h filters, and with deionized water and washing with alcohol several, drying, calcining is removed template CaCO with dissolving with hydrochloric acid
3, finally make tubular, hollow SiO
2The mesoporous material carrier, standby; With the immobilized enzyme biocatalyst weight percent is benchmark, in deionized water, acetate solution, phosphate buffer soln or the halid solution that the solid support material of the tubular, hollow structural silica dioxide with 80.00%～99.99% and 0.01%～10.00% at least a crystalline enzyme or solution enzyme join, and thorough mixing with it, control pH value is between 5～8, and temperature is controlled at 0～50 ℃; Load is washed after finishing, and filters lyophilize; Be kept in the refrigerator-freezer dried powder standby.
2, the covalent linkage connection method after the carrier surface modification prepares catalyzer of the present invention
First kind: a certain amount of coupling agent is selected from the muriate of cyanogen bromide, equal three chlorotriazines, hydrazides, sulfur mustard and titanium, tin, zinc, vanadium and iron one or more and joins tubular, hollow SiO
2In the colloidal solution of carrier, and fully stir with it, stirring velocity is controlled at 400～600r/min, and the pH value of maintenance system is between 8～10, and churning time is 2～8h; Wash after the carrier surface modification, filter, be warmed up to 50～80 ℃ with 5～10 ℃/min then, drying; In deionized water, acetate solution, phosphate buffer soln or the halid solution that powder after the modification and at least a crystalline enzyme or solution enzyme are joined, control pH value is between 5～8, and thorough mixing with it, and temperature is controlled at 0～50 ℃; Load is washed after finishing, and filters lyophilize; Be kept in the refrigerator-freezer dried powder standby, be benchmark wherein with the immobilized enzyme biocatalyst weight percent, the solid support material of tubular, hollow structural silica dioxide is 80.00%～99.99%, and biological enzyme is 0.01%～10.00%, and coupling agent is in 10.00%.
Second kind: a certain amount of linking agent is selected from glutaraldehyde, two diazonium p-diaminodiphenyl-2,2 '-disulfonic acid, 4,4 '-two fluoro-3,3 '-diphenylsulfone dinitro, 1,5 '-two fluoro-2, one or more in 4 '-dinitrobenzene, the Toluene-2,4-diisocyanate-isocyanic acid-isothiocyanate join tubular, hollow SiO
2In the water/phosphate buffer solution of carrier, and fully stir with it, stirring velocity is controlled at 400～600r/min, and the pH value of maintenance system is between 8～10, and churning time is 12～24h; Wash after the carrier surface modification, filter, be warmed up to 50～80 ℃ with 5～10 ℃/min then, drying; In the deionized water that powder after the modification and at least a crystalline enzyme or solution enzyme join successively, acetate solution, phosphate buffer soln or the halid solution, control pH value is between 5～8, and thorough mixing with it, and temperature is controlled at 0～50 ℃; Load is washed after finishing, and filters lyophilize 1～6h; Be kept in the refrigerator-freezer dried powder standby, be benchmark wherein with the immobilized enzyme biocatalyst weight percent, the solid support material of tubular, hollow structural silica dioxide is 80.00%～99.99%, and biological enzyme is 0.01%～10.00%, and linking agent is in 10.00%.
The third: one or more coupling agents and linking agent described in above-mentioned second and third kind method are joined tubular, hollow SiO successively
2In the colloidal solution of carrier, and fully stir with it, stirring velocity is controlled at 400～600r/min, and the pH value of maintenance system is between 8～10, and churning time is 12～24h; Wash after the carrier surface modification, filter, be warmed up to 50～80 ℃ with 5～10 ℃/min then, drying; In the deionized water that powder after the modification and at least a crystalline enzyme or solution enzyme join, acetate solution, phosphate buffer soln or the halid solution, control pH value is between 5～8, and thorough mixing with it, and temperature is controlled at 0～50 ℃; Load is washed after finishing, and filters lyophilize 1～6h; Be kept in the refrigerator-freezer dried powder standby, be benchmark wherein with the immobilized enzyme biocatalyst weight percent, the solid support material of tubular, hollow structural silica dioxide is 80.00%～99.99%, and biological enzyme is 0.01%～10.00%, and linking agent and coupling agent are in 10.00%.
Method of the present invention prepares the biological catalyst of penicillin acylation enzyme-fixing, and this catalyzer is used for the hydrolysis process of potassium salt of penicillin, preparation pharmaceutical intermediate 6-APA:
With tubular, hollow mesoporous SiO 2 particle is carrier; according to above-mentioned method load penicillin acylase; make the biological catalyst that is fixed with penicillin acylase; this catalyzer is applied to the hydrolysis process of conventional potassium salt of penicillin; catalyst levels is 1-3 times of benzylpenicillin potassium in parts by weight, 40～60 ℃ of temperature of reaction; Ph5～8, the concentration of benzylpenicillin potassium salts solution are 4% (W/W).In order to the method test is active down: the benzylpenicillin potassium solution of getting 100mL4% (W/W) stirs in beaker, the 0.1mol/l phosphate buffer solution that adds 100mLpH=7.5 is in 30 ℃ of constant temperature, NaOH solution with 0.25mol/L, conditioned reaction liquid is to pH=8.00, add the immobilization phase enzyme sample that accurately takes by weighing, pick up counting, keep pH=8.00 ± 0.1 with the NaOH solution titration of 0.25mol/L simultaneously, the alkali consumption that the record reaction is preceding 5 minutes, the activity that disposal data calculates immobilized enzyme can reach 2000U/g.
The application of immobilized enzyme biocatalyst of the present invention is not limited to the said fixing penicillin acylase at hydrolysis process, also relate to and fix other various (exemplifying) biological enzymes as preceding institute, be used for different fields, as: the ankyrin enzyme is in the application of albumen desugar, shell egg desugar, glycosuria test paper, and fixing glucose oxidase is in the deoxygenation of food, the application in the microbiosensor; In Application for Field such as antibiotic and sterilizations etc.
By the following examples enforcement of the present invention is further specified, but the present invention should not be limited to these embodiment, also should comprise: not depart under the scope of the invention condition, disclosed method is carried out the apparent various changes of those skilled in the art.
Present embodiment is the needle-like calcium carbonate template of preparation carrier: the technical grade unslaked lime that takes by weighing certain mass, put into 7～8 times in unslaked lime quality, temperature are hot water about 70 ℃, digestion for some time, filter one time with 80 orders and 250 purpose standard sieves respectively after cooling, obtain purified Ca (OH)
2Suspension is then with the EDTA standard solution titration demarcated Ca (OH) wherein
2Concentration, then with the Ca (OH) for preparing
2Suspension carries out carburizing reagent, in this experiment, and gas phase (CO
2Gas) continue to feed, liquid phase circulates, and contacts in rotating packed bed with gas phase and reacts.The carburizing reagent concrete steps are as follows: (1) temperature adjustment: rotating packed bed and stirring tank are equipped with chuck, and the temperature that the temperature of reaction system is crossed the recirculated water of chuck by controlling flow is controlled.Before experiment, need circulating water temperature is controlled to default temperature of reaction; (2) reinforced: as pH electrode to be installed, to be opened rotating packed bed, measure the Ca (OH) after 4L makes with extra care
2Suspension joins in the stirring tank, opens feed pump and makes feed liquid circulate while and recirculated water heat exchange arrival temperature of reaction in pipeline; (3) reaction: after treating that system temperature reaches set(ting)value and tends towards stability, add additive (crystal formation control agent), circulate 5 minutes, begin air inlet, pick up counting simultaneously, when pH drops to when being lower than 7, reaction finishes, stop air inlet, circulation for some time is taken out CaCO to the basic no change of pH value
3Slurries; (4) cleaning of rotating bed: taking out CaCO
3After the slurries, use tap water, dilute nitric acid solution, tap water cleaning reaction device successively, close feed pump after cleaning finishes, close rotating packed bed and stirring tank motor, take off pH electrode.Then carry out the product aftertreatment; With gained CaCO
3Behind the slurries filtration in electric drying oven with forced convection dry 24 hours, temperature was set at 100 ℃, obtains needle-like CaCO
3Dry powder, wherein line stone phase content is 88.75%, with gained needle-like CaCO
3Dry powder is done the specific surface area test, and recording its specific surface area is 22.16m
2/ g.Fig. 1 is the TEM photo of needle-like calcium carbonate.Fig. 2 is the XRD figure spectrum of the aragonite phase crystal formation assay of needle-like calcium carbonate powder.
The needle-like calcium carbonate that mainly contains line stone phase crystal formation of embodiment 1 preparation is mixed with the suspension that concentration is 0.8mol/L, get 200mL nano-calcium carbonate suspension and place reactor, and beginning heat temperature raising and stirring, stirring velocity is controlled at 400～500r/min; Compound concentration is the sodium silicate solution 500mL of 0.68mol/L and the dilute hydrochloric acid solution of 10wt%.When temperature is raised to 80 ℃, begin to drip sodium silicate solution, add dilute hydrochloric acid simultaneously, the pH of regulation system generates CaCO between 8.5～9.5
3/ SiO
2Nuclear-shell structure material.After treating that sodium silicate solution all is added drop-wise in the system, stop to add acid, and stir ageing under this temperature of reaction, digestion time is controlled at 4h, so that make SiO
2At CaCO
3Surface deposition, curing.Slurry after the ageing is through filtering, washing, and dry 12h under 105 ℃ calcines 4 ℃/min of heat-up rate, 600～700 ℃ of calcining temperatures, calcination time 6h then in retort furnace.Through the powder after the calcining,, remove CaCO with the dilute hydrochloric acid 500mL dissolving of 20wt%
3Template, and dissolve 12h under less than 1 situation in the pH value; Process ethanol and deionized water wash several, filtration at last, dry under 105 ℃, promptly obtain the tubular, hollow silica dioxide granule.The specific surface area of tubular, hollow silica dioxide granule is 200～1000m
2/ g, mean diameter is 80-250nm, and pore volume is 0.3～1.0mL/g, and mean pore size is 1-10nm, and mean length is 1～10 μ m.
Needle-like calcium carbonate is mixed with the suspension that concentration is 0.8mol/L, gets the 200mL calcium carbonate suspension and place reactor, stir, stirring velocity is controlled at 400～500r/min; According to SiO
2/ CaCO
3Mass ratio is the tetraethoxy (containing the Si massfraction is 28%) that 0.2 ratio takes by weighing 5.35g, and use dissolve with ethanol, the ethanolic soln of tetraethoxy is joined in the calcium carbonate slurry, add the strong aqua of the 17wt% of 20mL again, at room temperature stir 3h, reacted slurry is through filtering, washing, dry 12h under 105 ℃ pulverizes the back and calcine 4 ℃/min of heat-up rate in retort furnace, 600～700 ℃ of calcining temperatures, calcination time 6h.Through the powder after the calcining,, remove CaCO with the dilute hydrochloric acid 500mL dissolving of 10wt%
3Template, and dissolve 12h under less than 1 situation in the pH value; Through washing, filtration, dry under 105 ℃ at last, promptly obtain the tubular, hollow silica dioxide granule.Tubular, hollow SiO
2The particulate specific surface area is 200～1000m
2/ g, pore volume are 0.1～1.0mL/g, and mean diameter is 20～600nm, and mean length is 1～10 μ m.
Embodiment 4
Needle-like calcium carbonate is mixed with the suspension that concentration is 0.8mol/L, gets the 200mL calcium carbonate suspension and place reactor, stir, stirring velocity is controlled at 400～500r/min; According to SiO
2/ CaCO
3Mass ratio is the tetraethoxy (containing the Si massfraction is 28%) that 0.2 ratio takes by weighing 5.35g, and use dissolve with ethanol, the ethanolic soln of tetraethoxy is joined in the calcium carbonate slurry, the strong aqua and the 0.5g cetyl trimethyl ammonia bromide tensio-active agent that add the 17wt% of 20mL again, at room temperature stir 3h, reacted slurry is through filtering, washing, dry 12h under 105 ℃, pulverizing the back calcines in retort furnace, 4 ℃/min of heat-up rate, 600～700 ℃ of calcining temperatures, calcination time 6h.Through the powder after the calcining,, remove CaCO with the dilute hydrochloric acid 500mL dissolving of 10wt%
3Template, and dissolve 12h under less than 1 situation in the pH value; Through washing, filtration, dry under 105 ℃ at last, promptly obtain the tubular, hollow silica dioxide granule.Tubular, hollow SiO
2The particulate specific surface area is 500～800m
2/ g, pore volume are 0.4～0.8mL/g, and mean diameter is 80～250nm, and mean length is 2～8 μ m.Fig. 3 is the TEM photo of this tubular, hollow silicon-dioxide.
Embodiment 5
Take by weighing the tubular, hollow silicon-dioxide 2g pressed powder of embodiment 4 preparations, join in the 100mL deionized water, obtain the tubular, hollow silica suspension behind the ultra-sonic dispersion, the 0.2g penicillin acylase is added in the above-mentioned suspension, begin simultaneously to stir, flood.The control load temperature is about 10 ℃, and churning time is 5h, carries out vacuum filtration then, and passes through deionized water wash for several times to remove impurity, filters postlyophilization, promptly gets the immobilized penicillin acylated enzyme preparation.Fig. 4 is the TEM photo of this catalyzer, and gained immobilization acylase is used for the hydrolysis of benzylpenicillin potassium, and its catalytic activity is 2000U/g, and optimum temperuture is 51 ℃, and optimal pH is 8.5.
Embodiment 6
Take by weighing the tubular, hollow silicon-dioxide 2g pressed powder of embodiment 4 preparations, join in the 100mL deionized water, obtain the tubular, hollow silica suspension behind the ultra-sonic dispersion, 0.15g glucose oxidase and 0.01g horseradish peroxidase are added in the above-mentioned suspension, begin simultaneously to stir, flood.The control load temperature is about 50 ℃, churning time is 0.5h, carry out vacuum filtration then, and the process deionized water wash is for several times to remove impurity, lyophilize after the filtration, promptly get glucose oxidase and horseradish peroxidase combination immobilization enzyme, can be used for preparing microbiosensor to measure the content of blood-sugar content and glucose in urine.
Embodiment 7
Take by weighing the tubular, hollow silicon-dioxide 2g pressed powder of embodiment 4 preparations, join in the 100mL deionized water, obtain the tubular, hollow silica suspension behind the ultra-sonic dispersion, the 0.1g glucose oxidase is added in the above-mentioned suspension, begin simultaneously to stir, flood.The control load temperature is about 4 ℃, and churning time is 24h, carries out vacuum filtration then, and the process deionized water wash is for several times to remove impurity, lyophilize after the filtration promptly gets immobilized glucose oxidase, and this immobilized enzyme can be with preparing glycosuria test paper and food deoxygenation.
Embodiment 8
Take by weighing the tubular, hollow silicon-dioxide 2g pressed powder of embodiment 3 preparations, join in the 100mL deionized water, obtain the tubular, hollow silica suspension behind the ultra-sonic dispersion, the 0.05g Lysozyme is added in the above-mentioned suspension, begin simultaneously to stir, flood.The control load temperature is about 10 ℃, and churning time is 5h, carries out vacuum filtration then, and the process deionized water wash is for several times to remove impurity, lyophilize after the filtration promptly gets fixation cell cell wall lytic enzyme, and this immobilized enzyme can be used in the antibiotic and sterilization process.
Embodiment 9
Take by weighing the tubular, hollow silicon-dioxide 2g pressed powder of embodiment 3 preparations, join in the 100mL deionized water, obtain the tubular, hollow silica suspension behind the ultra-sonic dispersion, the 0.1g cellulase is added in the above-mentioned suspension, begin simultaneously to stir, flood.The control load temperature is about 10 ℃, and churning time is 5h, carries out vacuum filtration then, and passes through deionized water wash for several times to remove impurity, and the lyophilize after the filtration promptly gets immobilized cellulase, can be used for cellulosic hydrolysis reaction.
Take by weighing the tubular, hollow silica 1 0g pressed powder of embodiment 4 preparations, join in the 200mL deionized water, obtain the tubular, hollow silica suspension behind the ultra-sonic dispersion, 5mL linking agent glutaraldehyde (15wt%) is added in the above-mentioned suspension, begin simultaneously to stir.Controlled temperature is about 30 ℃, and churning time is 12h, carries out vacuum filtration then, and through deionized water wash for several times removing impurity, after the filtration 100 ℃ of vacuum-dryings, promptly get the tubular, hollow silicon-dioxide of surface modification.
Embodiment 11
Take by weighing the tubular, hollow silica 1 0g pressed powder of the surface modification of embodiment 10 preparations, join in the 200mL deionized water, obtain the tubular, hollow silica suspension behind the ultra-sonic dispersion, with 8mL right-β-sulfuric acid ester ethyl sulfone aniline (5wt%) adds in the above-mentioned suspension, begins simultaneously to stir.Controlled temperature is about 30 ℃, and churning time is 24h, carries out vacuum filtration then, and through deionized water wash for several times removing impurity, after the filtration 100 ℃ of vacuum-dryings, promptly get the tubular, hollow silicon-dioxide that contains coupling agent active group surface modification.
Embodiment 12
Take by weighing the surface modification tubular, hollow silicon-dioxide 2g pressed powder of embodiment 10 preparations; join in the 100mL deionized water; obtain the tubular, hollow silica suspension behind the ultra-sonic dispersion, 0.05 penicillin acylase is joined in the above-mentioned suspension, begin simultaneously to stir, flood.The control load temperature is about 0 ℃; control load pH value is 5, and churning time is 1h, carries out vacuum filtration then; and the process deionized water wash is for several times to remove impurity; lyophilize after the filtration promptly gets immobilized penicillin acylated enzyme, and this immobilized penicillin acylated enzyme is used for the benzylpenicillin potassium hydrolysis; its catalytic activity is 1800U/g; optimum temperuture is 45 ℃, and optimal pH is 8.8, reuses 5 times and does not see that its catalytic activity reduces.
Embodiment 13
What take by weighing embodiment 11 preparation contains coupling agent active group surface modification tubular, hollow silicon-dioxide 2g pressed powder; join in the 100mL deionized water; obtain the tubular, hollow silica suspension behind the ultra-sonic dispersion; the 0.1g penicillin acylase is added in the above-mentioned suspension, begin simultaneously to stir, flood.The control load temperature is about 20 ℃, and control load pH value is 8, and churning time is 6h, carries out vacuum filtration then, and passes through deionized water wash for several times to remove impurity, and the lyophilize after the filtration promptly gets immobilized penicillin acylated enzyme.
Embodiment 14
What take by weighing embodiment 11 preparation contains coupling agent active group surface modification tubular, hollow silicon-dioxide 2g pressed powder, join in the 100mL deionized water, obtain the tubular, hollow silica suspension behind the ultra-sonic dispersion, the 0.15g Lysozyme is added in the above-mentioned suspension, begin simultaneously to stir, flood.The control load temperature is about 50 ℃, and churning time is 5h, carries out vacuum filtration then, and passes through deionized water wash for several times to remove impurity, and the lyophilize after the filtration promptly gets fixation cell cell wall lytic enzyme.
Embodiment 15
Take by weighing the surface modification tubular, hollow silicon-dioxide 2g pressed powder of embodiment 10 preparations, join in the 100mL deionized water, obtain the tubular, hollow silica suspension behind the ultra-sonic dispersion, just 0.05g glucose oxidase and 0.1g horseradish peroxidase add in the above-mentioned suspension, begin simultaneously to stir, flood.The control load temperature is about 50 ℃, churning time is 5h, carry out vacuum filtration then, and the process deionized water wash is for several times to remove impurity, lyophilize after the filtration, promptly get the complex solidifying enzyme of glucose oxidase and horseradish peroxidase, can be used for preparing microbiosensor to measure the content of blood-sugar content and glucose in urine.
Embodiment 16
What take by weighing embodiment 10 preparation contains coupling agent active group surface modification tubular, hollow silicon-dioxide 2g pressed powder, join in the 100mL deionized water, obtain the tubular, hollow silica suspension behind the ultra-sonic dispersion, 0.1g glucose oxidase and 0.05g horseradish peroxidase are added in the above-mentioned suspension, begin simultaneously to stir, flood.The control load temperature is about 20 ℃, and churning time is 5h, carries out vacuum filtration then, and through deionized water wash for several times removing impurity, the lyophilize after the filtration promptly gets the complex solidifying enzyme of glucose oxidase and horseradish peroxidase.
Claims (10)
1, a kind of immobilized enzyme biocatalyst, on silica supports, be fixed with biological catalytic enzyme, it is characterized in that described carrier is a tubular, hollow mesoporous SiO 2 particle, is fixed with the biological enzyme molecule in the micropore in outside surface, internal surface and the wall of tubular, hollow silica supports.
2, immobilized enzyme biocatalyst according to claim 1, it is characterized in that, with the immobilized enzyme biocatalyst weight percent is that the composition of benchmark comprises: 0.01%～10.00% biological enzyme, 80.00%～99.99% tubular, hollow silica supports, 0～10.00% coupling agent is or/and linking agent.
3, immobilized enzyme biocatalyst according to claim 1 is characterized in that, tubular, hollow mesoporous SiO 2 particulate specific surface area is 200～1000m
2/ g, pore volume are 0.1～1.0mL/g, and mean diameter is 40～600nm, and mean length is 1～10 μ m.
4, immobilized enzyme biocatalyst according to claim 3 is characterized in that, tubular, hollow mesoporous SiO 2 particulate specific surface area is 500～800m
2/ g, pore volume are 0.4～0.8mL/g, and mean diameter is 80～250nm, and mean length is 2～8 μ m.
5, immobilized enzyme biocatalyst according to claim 1; it is characterized in that biological enzyme is selected from one or more in proteolytic enzyme, amylase, glucose isomerase, cellulase, polygalacturonase, lipase, glucose oxidase, amino acylase, altheine enzyme, penicillin acylase, L-Aspartase, Polyribonucleotide phosphorylase, dextrorotation candy acid anhydride enzyme, Lysozyme, horseradish peroxidase, the acetylcholinesterase.
6, the preparation method of the described immobilized enzyme biocatalyst of a kind of claim 1, it is characterized in that, this method in turn includes the following steps: the hollow tubular silica supports is joined in the aqueous solution of at least a biological enzyme, and thorough mixing 0.5～24h with it, temperature is controlled at 4～50 ℃, soak and filter lyophilize then after burying end.
7, the preparation method of the described immobilized enzyme biocatalyst of a kind of claim 1 is characterized in that, this method in turn includes the following steps: with coupling agent or/and linking agent joins tubular, hollow SiO
2In the colloidal solution of carrier, and fully stir with it, stirring velocity is controlled at 400～600r/min, and the pH value of maintenance system is between 8～10, and churning time is 12～24h; Wash after the carrier surface modification, filter, be warmed up to 50～80 ℃ with 5～10 ℃/min then, drying; In the deionized water that powder after the modification and at least a crystalline enzyme or solution enzyme join, acetate solution, phosphate buffer soln or the halid solution, and thorough mixing carries out the load of enzyme with it, control pH value is between 5～8, and temperature is controlled at 0～50 ℃; Load is washed after finishing, and filters lyophilize 1～6h.
8, according to the preparation method of claim 6,7 described immobilized enzyme biocatalysts, it is characterized in that one or more in the muriate of coupling agent is selected from right-β-sulfuric acid ester ethyl sulfone aniline, cyanogen bromide, equal three chlorotriazines, hydrazides, sulfur mustard and titanium, tin, zinc, vanadium and iron.
9, according to the preparation method of claim 6,7 described immobilized enzyme biocatalysts, it is characterized in that, linking agent is selected from glutaraldehyde, two diazonium p-diaminodiphenyl-2,2 '-disulfonic acid, 4,4 '-two fluoro-3,3 '-diphenylsulfone dinitro, 1,5 '-two fluoro-2, one or more in 4 '-dinitrobenzene, the Toluene-2,4-diisocyanate-isocyanic acid-isothiocyanate.
10, a kind of immobilized penicillin acylated enzyme biological catalysts according to claim 6,7 preparations prepare application among the pharmaceutical intermediate 6-APA in the hydrolysis of potassium salt of penicillin.
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