CN101531732A - Immobilized porcine pancreatic lipase carrier, preparation method and application thereof - Google Patents
Immobilized porcine pancreatic lipase carrier, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an immobilized porcine pancreatic lipase carrier, which is a macroporous bead crosslinked polymer containing succinimidyl ester groups and has a structure represented by the formula I. The carrier forms a high polymer with acrylic acid as a reactive monomer and N,N'-methylenebisacrylamide as a crosslinking agent, and in the meantime, incorporates the succinimidyl ester active group so that the time required for immobilizing the porcine pancreatic lipase is short, namely only 1 hour is required, and the immobilizing efficient is high and stability of activity retention of the immobilized enzyme is excellent; the apparent activity of the immobilized enzyme is high and can reach 750U/g and above. The invention also discloses a preparation method and application of the immobilized porcine pancreatic lipase carrier.
Description
Technical field
The invention belongs to the preparing technical field of fixed enzyme vector, particularly a kind of immobilized porcine pancreatic lipase carrier and preparation method thereof.
Background technology
Enzyme is a biological catalyst, has that specificity is strong, catalytic efficiency is high and distinguishing feature such as action condition gentleness.Thereby its application is very extensive, can be used on a plurality of fields such as light industry, food, feed, medicine, chemical industry, environmental protection and the energy, analyzing and testing.But because the existence of free enzyme separates with the product difficulty and the shortcoming of easy inactivation, people have attempted by the whole bag of tricks the enzyme molecule being modified, wherein one of method is exactly the research of immobilized enzyme, thereby changes some characteristic and the function of enzyme, makes it overcome the shortcoming of free enzyme.The research of immobilized enzyme starts from the fifties in last century, through 50 years of researches and development, aspect the immobilization of enzyme, successively developed various solid support material and the process for fixation of performance, yet the immobilized enzyme that really drops into industrial applications is few, and its reason is that the reagent that uses of enzyme immobilization and carrier cost height, immobilization efficiency are low, poor stability etc.Vigorously advocate the today of setting up resource-conserving and environment-friendly society in China, the further excellent more and inexpensive solid support material of exploitation performance, make the immobilization of enzyme easier, more practical, allow more immobilized enzyme obtain plant-scale application, be still now the target of pursuing.
Aspect the enzyme immobilization carrier material, mainly comprise inorganic carrier material, organic support material and polymer carrier etc., wherein polymer carrier is the solid support material of outbalance.Polymer carrier is divided into natural polymer solid support material and synthetic macromolecule solid support material again.The synthetic macromolecule solid support material is the most active Carrier Materials of Immobilized Enzyme of research at present, also being a class Carrier Materials of Immobilized Enzyme of using at present and reporting at most, mainly is by the chemical stability height of synthetic macromolecule solid support material, good mechanical property, with the big decision of immobilization loading of enzyme.
The process for fixation of enzyme mainly comprises three kinds of entrapping method, crosslinking and carrier combined techniqueses; Carrier combined techniques wherein comprises ionic bond method, physisorphtion and covalent attachment method again.For the immobilization of synthetic macromolecule solid support material and enzyme, mainly be the covalent attachment method.The covalent attachment method is to form chemical covalent linkage between functional group on the zymoprotein molecule and the lip-deep reactive group of solid support to connect (the nonessential group of zymoprotein forms irreversible the connection by covalent linkage and carrier), thus the method for immobilized enzyme.Owing to be connected firmly between enzyme and carrier, be difficult for taking place enzyme and come off, satisfactory stability and reusability are arranged, be the most active class of enzymes process for fixation of research at present.
Covalent attachment method research for enzyme, require during carrier immobilized enzyme to contain the reactive group that can react in the carrier with a certain group (as wherein a kind of group of amino, carboxyl, sulfydryl, hydroxyl, phenolic group, imidazolyl etc.) in the enzyme molecule, and the active centre of destructive enzyme molecule not after reactive group and the enzyme molecular reaction.These reactive groups generally include diazonium groups, nitrogen groups, imido-carbonic acid group, two chloro-cyanuro groups, epoxide group etc. change.
At present, aspect the synthetic macromolecule solid support material, abroad the most representative comprising: Kotha etc. are that pore-creating agent, divinylbenzene are that linking agent makes GM-three vinylformic acid three methanol-based propane ester copolymers with the hexalin, the preparation of research such as Ponrathnam macro porous crosslinking glycidyl methacrylate (GMA) copolymerization carrier, the production EupergitC of ROM company of Germany etc. contains the fixed enzyme vector of epoxide group etc.And at home, be engaged in mainly comprising of this respect research: the synthetic fixed enzyme vector that contains epoxide group of people such as the Lu Guanzhong of East China University of Science and disciple's black clouds Gao Wa, Xue Ping, black clouds Gao Wa etc. selects reactive monomer glycidyl methacrylate (GMA) for use, with N, N '-methylene-bis (acrylamide) is a pore-creating agent for linking agent, methane amide (MBAA), makes a series of bead-like copolymer carriers with inverse suspension polymerization method; Xue Ping etc. select glycidyl methacrylate (GMA) and the two kinds of monomers of hydrophilic N-V-Pyrol RC (NVP) that contain active epoxy group for use, with N, N '-methylene-bisacrylamide (MBAA) is a linking agent, methanol aqueous solution is made pore-creating agent, whiteruss is main medium, successfully synthesized wetting ability macropore GMA-NVP-MBAA terpolymer carrier (GNM) by the inverse suspension polymerization technology, other designs the inverse suspension polymerization system, prepared poly (glycidyl methacrylate) (GMA)-hydroxyethyl methylacrylate (HEMA)-N, N '-methylene-bisacrylamide (MBAA) hydrophilic magnetic polymkeric substance GHM microballoon, and prepared the carrier that the mesopore molecular sieve that contains cobalt MCM-48 and MCM-41 etc. is used as enzyme immobilization; The synthetic fixed enzyme vector that contains the cyclic carbonate ester group of people such as the Huang Jiaxian of Nankai University, this synthetic vectors is to be reactive monomer with vinylene carbonic ether (VCA), wetting ability N, N '-methylene-bisacrylamide (MBAA) is a linking agent, selecting for use N-vinyl pyrrolidone (NVP) and senecioate-two kinds of wetting abilities of hydroxyl ethyl ester (HEMA) to be total to monomer respectively is the composition of synthetic enzyme carrier, be composite pore-forming with dimethyl formamide and linear macromolecule poly(oxyethylene glycol) 400 first, also pass through inverse suspension polymerization simultaneously, with the whiteruss is medium, VCA is a reactive monomer, methacrylic acid-beta-hydroxy ethyl ester (HEMA) and Propylene glycol monoacrylate (HPA) are hydrophilic polymerized monomer, synthesize a series of cross-linked resin polymkeric substance as fixed enzyme vector; The Li Yuan of University Of Hebei merits etc. are that linking agent, sherwood oil are pore-creating agent with vinylformic acid reactive monomer and Vinylstyrene, by the carrier of suspension polymerization immobilized enzyme; In addition, also have other investigators all to do the research of different aspects for fixed enzyme vector.
From above these synthetic macromolecule solid support materials, reactive group wherein mainly is reactive groups such as epoxide group, cyclic carbonate ester group.
(lipase E.C.3.1.1.3) also may be defined as Procaine esterase to lipase, and its catalysis long-chain acyl glycerine is hydrolyzed to glycerine, free fatty acids and single, double glyceryl ester.Can be used for the applicable every field of enzyme, as be used for food-processing and quality-improving, make the bread improving agent, be used for cheese flavouring etc.; In weaving processing, be used for skimming treatment, can reach better decontamination purpose as the additive of washing composition; The prior production biofuel that also can be used for, biofuel is a kind of form of biomass energy, has green, renewable, low power consumption and other advantages, compare with ordinary diesel oil, have eco-friendly characteristics, the toxic organic compound quantity discharged only is 10% in its exhaust gas from diesel vehicle, particulate matter is 20%, CO
2With the CO quantity discharged only be 10%, according to one's analysis, to whole atmospheric influence, the CO of biofuel discharging
2Lack approximately 50% than mineral diesel, as seen, it will be a up-and-coming cause that immobilized lipase is used for the production biofuel, can alleviate oil and be about to crisis exhausted and that environmental pollution causes the mankind.
Research report about lipase immobilization and application etc., mainly comprise: the carrier immobilized lipase research of Kanwar synthesizing propylene acids of India Himachal Pradesh university is abroad arranged, the Yunas of Universiti Putra Malaysia is with gathering (methacrylic ester, methyl methacrylate, Vinylstyrene) microsphere immobilized lipase, Mahiran Basri is fixed to lipase on porous polyethylene alcohol pearl, the polyacrolein pearl and studies with hydrophobic organic carrier fixed fat enzyme, the ToshioHayashi of Kyoto Univ Japan; The domestic Xin Jiaying that Lanzhou Inst. of Chemical Physics, Chinese Academy of Sciences is arranged etc., the Kou Xiufen of Institute of Microorganism, Academia Sinica, the Xu Huixian of the Nankai University carrier immobilized lipase of acrylic acid or the like, the Sheng plum of Jiangsu Petrochemical Engineering College, big and disciple Peng Lifeng of the Tan Tian of Beijing University of Chemical Technology etc. is to lipase immobilization and applied research thereof.
A kind of as in the lipase of porcine pancreatic lipase, the constructional feature that has lipase on the structure too, the one, all comprise homology segment: His-X-Y-Gly-Z-Ser-W-Gly or Y-Gly-His-Ser-W-Gly (X, Y, W, Z are variable amino-acid residues); The 2nd, the active centre is a serine residue, protected by an a-screw-cap.From having the relative merits of enzyme in nature simultaneously, a lot of concrete application are also arranged, as can catalysis with hexanodioic acid divinyl ester and 1, the 4-butyleneglycol is the achirality carboxylic acid derivative polyreaction of representative, polymerization that also can catalysis chiral carboxylic acids derivative, Dafha Knani etc. makes catalyzer with porcine pancreatic lipase (PPL), carries out the polymerization of photolytic activity side-draw for hydroxy ester, and the result obtains having optically active oligomer.
At present, for the synthetic macromolecule carrier also and few to the research of the immobilization of porcine pancreatic lipase and application facet, the Arieva V Paula of St. Paul,Brazil university is fixed in porcine pancreatic lipase and prepares biofuel on polysiloxane-vinyl alcohol, and the He Feng of Wuhan University, the Xie Zhidong of Nankai University etc. also carried out research to the immobilization of porcine pancreatic lipase.
But up to the present, also do not have and to realize the listing of commercial immobilized porcine pancreatic lipase.Major cause is spoken of as the front, because shortcomings such as preparation method's complexity, severe reaction conditions, cost height cause it to be difficult to reach industrialization demands, the efficient of the product enzyme immobilization that makes is low, and the poor stability of activity of the immobilized enzyme maintenance.
Summary of the invention
The purpose of this invention is to provide a kind of new immobilized porcine pancreatic lipase carrier, the efficient height of its immobilized porcine pancreatic lipase, and the good stability of activity of the immobilized enzyme maintenance.
Another object of the present invention provides a kind of preparation method of said fixing porcine pancreatic lipase carrier, can overcome shortcomings such as the preparing carriers complexity that exists in the prior art, severe reaction conditions, cost height, difficult industrialization.
The technical solution adopted for the present invention to solve the technical problems is:
A kind of immobilized porcine pancreatic lipase carrier, it is a kind of macropore dropwise cross-linked polymer that contains succinimide ester group group, has the represented structure of following general formula I:
Wherein,
Be to be reactive monomer, N with vinylformic acid (AA), N '-methylene-bisacrylamide (MBAA) is the part except that carboxyl in the superpolymer that forms of linking agent.
The said fixing porcine pancreatic lipase carrier, can prepare by following method: with tensio-active agent Span (sapn) 40 and Span80 is that composite dispersing agent, kerosene are that disperse phase, vinylformic acid (AA) are reactive monomer, N, N '-methylene-bisacrylamide (MBAA) is that linking agent, ethylene glycol are pore-creating agent, adopt anti-phase outstanding polymerization technique to make the macropore dropwise cross-linked polymer, by N-hydroxy-succinamide (NHS) activation, promptly make described immobilized porcine pancreatic lipase carrier again.
The HLB value of Span40 and Span80 (Hydrophily and Lipophilyty Balance, hydrophile-lipophile balance value) is respectively 6.7 and 4.3, in the preparation method of said fixing porcine pancreatic lipase carrier of the present invention during as composite dispersing agent, the two mixed HLB value is adjusted to 4.5～6 gets final product, control the two usage ratio of Span40 and Span80 with this; Total consumption of composite dispersing agent can calculate by 10%～30% of disperse phase kerosene quality.
Linking agent N, the quality of N '-methylene-bisacrylamide (MBAA) can be preferably 40%～60% of monomer vinylformic acid (AA) quality;
The consumption of pore-creating agent ethylene glycol can be 70%～100% of monomer vinylformic acid (AA) quality;
In addition, among the preparation method of said fixing porcine pancreatic lipase carrier of the present invention, also can add the catalyzer of ammonium persulphate (AP) as free radical reaction, the add-on of catalyst A P can be 1.5%～4% of monomer vinylformic acid (AA) quality; The accelerator that also can add Tetramethyl Ethylene Diamine (TMEDA) conduct acceleration AP generation free radical simultaneously (thereby reacts owing to TMEDA itself is easy to generate free radical acceleration AP generation free radical, so the add-on of TMEDA is relevant with actual reaction, as long as can make reaction take place just can, consider in conjunction with cost etc., can be by 7～10 times of calculating of catalyst A P quality).
Above-mentioned preparation method can preferably include following key step:
(1), preparation oil phase:
The composite dispersing agent of Span40 and Span80 composition is dissolved in organic disperse phase kerosene mixing; Add Tetramethyl Ethylene Diamine (TMEDA) again, mixing constitutes oil phase;
(2), preparation water:
With vinylformic acid (AA), N, N '-methylene-bisacrylamide (MBAA), ethylene glycol are dissolved in the water (calculate by volume, the consumption of water can be 1～4 times of vinylformic acid volume), add ammonium persulphate (AP), and mixing constitutes water;
(3), preparation macropore dropwise cross-linked polymer:
Speed with 0.5～2d/s goes on foot (2) water dropping (available dropping funnel or the alternate manner dropping that makes, similar substantially guarantee to drip the water drop size that enters oil phase) disperse that (wherein the volume ratio of oil phase and water is controlled at 〉=4 gets final product in the oil phase that makes in (1) step, from saving cost consideration, can 4:1～10:1 be good), the control stirring velocity is 100～350r/min; 10～30 ℃ room temperature is reacted under nitrogen protection, 1～2 hour reaction times;
After reaction finishes, reclaim upper solution (can reclaim) by toppling over modes such as supernatant liquid, the ethanol (its consumption gets final product with the submergence product) that in reaction product, adds volumetric concentration 〉=65%, leave standstill behind the mixing more than 5 hours and (consider the problem of production cycle, can leave standstill 5～24 hours), remove the upper strata ethanolic soln; Wash with water repeatedly again to no kerosene flavor and alcohol flavor, suction filtration, the macropore dropwise cross-linked polymer, in vacuum drying oven, dry to constant weight for 35～60 ℃, must activated carrier be not standby;
(4), preparation activated carrier:
Get the not activated carrier that (3) step is dried to constant weight, do under the condition of solvent at tetrahydrofuran (THF) (THF), dicyclohexylcarbodiimide (DCC) is made dewatering agent, reacted 2～4 hours at 10～30 ℃ with N-hydroxy-succinamide (NHS), constantly stir (because this is a thermopositive reaction, the purpose of stirring is heat radiation) in the reaction process; Reaction finishes the back and reclaims solvent; Remove by product dicyclohexylurea (DCU) with anhydrous alcohol solution again, suction filtration, in vacuum drying oven, dry to constant weight for 35～60 ℃, make activated carrier, be said fixing porcine pancreatic lipase carrier of the present invention (a kind of macropore dropwise cross-linked polymer that contains succinimide ester group group).
In above-mentioned steps (4) the preparation activated carrier process, reactant (not activated carrier, NHS), with the consumption of dewatering agent DCC can be by reactive monomer in the activated carrier not: N-hydroxy-succinamide: dicyclohexylcarbodiimide (mol ratio)=1:(1～1.2): (1～1.2) calculates (the molar weight of reactive monomer can not calculated according to reactivity in the activated carrier); NHS and DCC can be excessive slightly, fully react to guarantee the carboxyl in the activated carrier not; The consumption of solvent THF with can dissolve DCC, NHS gets final product.
In above-mentioned preparation method's the reaction process, the main chemical reactions formula that is taken place is as follows:
The process for fixation of synthetic macropore dropwise cross-linked polymer and porcine pancreatic lipase is the covalent attachment method among the above-mentioned preparation method of the present invention.
Constructional feature according to porcine pancreatic lipase, the present invention is reactive monomer, N with vinylformic acid (AA), N '-methylene-bisacrylamide (MBAA) is that linking agent, ethylene glycol are pore-creating agent, with kerosene is organic disperse phase, adopt the insoluble upholder cross-linked acrylic acid of inverse suspension polymerization technology synthetic water porous microsphere, activate this polymkeric substance and introduce the succinimide ester active group with N-hydroxy-succinamide (NHS), this active group at room temperature just can not destroy the purpose that the active centre reaches enzyme immobilization with the reaction of amino-acid residue in the porcine pancreatic lipase.
Immobilized porcine pancreatic lipase carrier of the present invention is when immobilized porcine pancreatic lipase, and above-mentioned activated carrier is by the H in succinimide ester active group and the porcine pancreatic lipase
2The N-radical reaction reaches the effect of immobilized porcine pancreatic lipase.
The said fixing porcine pancreatic lipase carrier except that the fixation support that can be used as porcine pancreatic lipase, also can be used as the fixation support of other lipase, neutral protease, α-Dian Fenmei etc.
In the process of immobilized porcine pancreatic lipase etc., its principal reaction formula can be expressed as follows:
Compared with prior art, the invention has the beneficial effects as follows:
Immobilized porcine pancreatic lipase carrier of the present invention, by being reactive monomer, N with vinylformic acid, N '-methylene-bisacrylamide is that linking agent forms superpolymer, introduced the succinimide ester active group simultaneously, make the required time such as its immobilized porcine pancreatic lipase shorter, only need 1 hour, and the immobilization efficiency height, the good stability that activity of the immobilized enzyme keeps; Its immobilized enzyme apparent activity height, can reach 750U/g and more than.And, except can be used for immobilized porcine pancreatic lipase, also can be used for other lipase of immobilization, neutral protease, α-Dian Fenmei etc.
In addition, the preparation method of said fixing porcine pancreatic lipase carrier provided by the invention can overcome shortcomings such as the preparing carriers complexity that exists in the prior art, severe reaction conditions, cost height, difficult industrialization, and its major advantage is as follows:
The first, raw material is cheap and easy to get, and all is low toxicity or nontoxic reagent, and cost is lower;
The second, the reaction conditions gentleness must not heat in China south throughout the year, and is simple to operate;
The 3rd, required device is simple, can further reduce cost;
The 4th, the reaction times is short, can shorten the production cycle, guarantees the annual production height, increases economic efficiency.
Comprehensive above-mentioned advantage, this preparation method is simple to operate, reaction conditions is gentle and cost is low, annual production is high, is easy to realize suitability for industrialized production, and is good in economic efficiency.
Description of drawings
Fig. 1 is the infared spectrum of the obtained not activated carrier of the embodiment of the invention 1 step (3).
Fig. 2 is the infared spectrum of the obtained activated carrier of the embodiment of the invention 1 step (4).
Embodiment
The present invention is described in further detail below in conjunction with embodiment.
But this should be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following embodiment.
The immobilized porcine pancreatic lipase carrier of present embodiment for making by following method:
With tensio-active agent Span (sapn) 40 and Span80 is that composite dispersing agent, kerosene are that disperse phase, vinylformic acid (AA) are reactive monomer, N, N '-methylene-bisacrylamide (MBAA) is that linking agent, ethylene glycol are pore-creating agent, adopt anti-phase outstanding polymerization technique to make the macropore dropwise cross-linked polymer, by N-hydroxy-succinamide (NHS) activation, make described immobilized porcine pancreatic lipase carrier again.
Used raw material comprises:
Reactive monomer vinylformic acid (AA) 5mL (about 5.3g),
Linking agent N, N '-methylene-bisacrylamide (MBAA) 2.1g (be AA quality about 40%),
Pore-creating agent ethylene glycol 4.0mL (about 4.5g, for the AA quality about 85%),
The usage ratio of dispersion agent Span40 and Span80 is controlled so that the two mixed HLB value is adjusted to 5, and its total consumption is 12g,
Disperse phase kerosene 100mL,
Catalyzer ammonium persulphate (AP) 0.1g (be AA quality about 1.9%),
Accelerator Tetramethyl Ethylene Diamine (TMEDA) 1mL (about 0.77g is about 7.7 times of AP quality);
In addition, in the preparation activated carrier process, reactant (not activated carrier, N-hydroxy-succinamide) is respectively with the consumption of dewatering agent dicyclohexylcarbodiimide:
Activated carrier 2g (wherein reactive monomer is about 0.02 mole) not,
N-hydroxy-succinamide (NHS) 2.2g (0.02 mole),
Dewatering agent dicyclohexylcarbodiimide (DCC) 3.9g (0.02 mole).
Concrete preparation method comprises following key step:
(1), preparation oil phase:
Use the composite dispersing agent of small beaker weighing Span40 and Span80 composition earlier, in beaker, add a small amount of organic dispersion kerosene, because Span40 is a solid, needing a little, heating makes it dissolving, change in the four-hole bottle then, be transferred to four-hole bottle 2 times with kerosene washing beaker again, stirring and logical nitrogen mix; Add Tetramethyl Ethylene Diamine (TMEDA) again, mixing constitutes oil phase;
(2), preparation water:
With vinylformic acid (AA), N, N '-methylene-bisacrylamide (MBAA), ethylene glycol are dissolved in the 10ml water, add ammonium persulphate (AP), and mixing constitutes water;
(3), preparation macropore dropwise cross-linked polymer:
Adopt dropping funnel, will (2) go on foot the water that makes with the speed of 1d/s and drip in the oil phase that (1) makes and disperse, the control stirring velocity is 250r/min; About 18 ℃ are reacted under nitrogen protection, 1 hour reaction times;
After reaction finished, employing was toppled over mode and is reclaimed upper solution, added 95% ethanol 100ml in reaction product, and standing over night behind the mixing is removed the upper strata ethanolic soln; Wash with water repeatedly again to no kerosene flavor and alcohol flavor, suction filtration, the macropore dropwise cross-linked polymer, in vacuum drying oven, dry to constant weight for 40～45 ℃, activated carrier 5.92g altogether not, the calculating reactivity is about 80%, and is standby;
The contriver adopt the KBr tabletting method (with reference to Wang Kunhua, Luo Chuanqiu, week makes a whistling sound. polymkeric substance instrumental analysis in modern age [M]. Beijing: press of Tsing-Hua University, 1991:20～44) activated carrier does not detect to this, has obtained infrared (IR) collection of illustrative plates as shown in Figure 1; As can be seen from Figure 1,3431.9 places are a broad peak, have illustrated-COOH exists, simultaneously at 1172.7 and 1115.1 characteristic peaks that vinylformic acid generation crosslinking polymerization occurs, 1721.5 be-COOH in-peak of C=O; 1650.2 and 1452 be the characteristic peak of amido linkage among the MBAA, 1650.2 is-peak of C=O, 1452 is the vibration peak of C-N key; Remaining is the skeletal vibration peak, can draw AA and MBAA and crosslinking polymerization has taken place really obtain the PAA-MBAA polymkeric substance from IR analyzes collection of illustrative plates.
(4), preparation activated carrier:
Get tetrahydrofuran (THF) (THF) and dissolve dicyclohexylcarbodiimide (DCC) (3.9g, 0.02 mole) and N-hydroxy-succinamide (NHS) (2.2g, 0.02 mole) respectively, (the THF consumption is respectively 40mL, 20mL) obtains DCC solution and NHS solution;
Other gets the not activated carrier (2g, wherein reactive monomer is about 0.02mol) that (3) step is dried to constant weight, adds above-mentioned NHS solution, drips above-mentioned DCC solution again; About 18 ℃ of reactions are 2.5 hours in airtight round-bottomed flask, constantly stir (reaction process is carried out on magnetic stirring apparatus, adopts magnetic stir bar to stir) in the reaction process; Reaction finishes the back and reclaims solvent; Remove by product dicyclohexylurea (DCU) with anhydrous alcohol solution again, suction filtration is dried to constant weight for 40～45 ℃ in vacuum drying oven, makes activated carrier, is described immobilized porcine pancreatic lipase carrier.
The contriver adopts aforementioned KBr tabletting method that this activated carrier is detected, and has obtained infrared (IR) collection of illustrative plates as shown in Figure 2; As can be seen from Figure 2, be spike more than 3000 in the collection of illustrative plates, illustrating does not have-existence of COOH; And, prove the generation of active ester at 1738.2,1208.8,1075.9 characteristic peaks that ester group occurred; Newly occurred on the NHS simultaneously-peak of C=O, illustrate it is the ester that NHS participates in the reaction generation really 1820.7,1782.3.
In addition, the contriver also adopt sweet oil emulsion method measured this activated carrier immobilized porcine pancreatic lipase to the hydrolysis of sweet oil emulsion apparent activity, measurement result is 850U/g (representing with the carrier dry weight).
Embodiment 2
The immobilized porcine pancreatic lipase carrier of present embodiment for making by following method:
With tensio-active agent Span (sapn) 40 and Span80 is that composite dispersing agent, kerosene are that disperse phase, vinylformic acid (AA) are reactive monomer, N, N '-methylene-bisacrylamide (MBAA) is that linking agent, ethylene glycol are pore-creating agent, adopt anti-phase outstanding polymerization technique to make the macropore dropwise cross-linked polymer, by N-hydroxy-succinamide (NHS) activation, make described immobilized porcine pancreatic lipase carrier again.
Used raw material comprises:
Reactive monomer vinylformic acid (AA) 10mL (about 10.5g),
Linking agent N, N '-methylene-bisacrylamide (MBAA) 6.2g (be AA quality about 59%),
Pore-creating agent ethylene glycol 6.7mL (about 7.5g, for the AA quality about 71%),
The usage ratio of dispersion agent Span40 and Span80 is controlled so that the two mixed HLB value is adjusted to 4.5, and its total consumption is 8g,
Disperse phase kerosene 100mL,
Catalyzer ammonium persulphate (AP) 0.16g (be AA quality about 1.5%),
Accelerator Tetramethyl Ethylene Diamine (TMEDA) 2mL (about 1.5g is about 9.4 times of AP quality);
In addition, in the preparation activated carrier process, reactant (not activated carrier, N-hydroxy-succinamide) is respectively with the consumption of dewatering agent dicyclohexylcarbodiimide:
Activated carrier 5g (wherein reactive monomer is about 0.047 mole) not,
N-hydroxy-succinamide (NHS) 5.5g (0.048 mole),
Dewatering agent dicyclohexylcarbodiimide (DCC) 9.9g (0.048 mole).
Concrete preparation method comprises following key step:
(1), preparation oil phase:
The composite dispersing agent of Span40 and Span80 composition is added in organic disperse phase kerosene, and suitably heating for dissolving adds and is equipped with in the four-hole bottle of airway and agitator, and stirring and logical nitrogen mix; Add Tetramethyl Ethylene Diamine (TMEDA) again, mixing constitutes oil phase;
(2), preparation water:
With vinylformic acid (AA), N, N '-methylene-bisacrylamide (MBAA), ethylene glycol are dissolved in the 25ml water, add ammonium persulphate (AP), and mixing constitutes water;
(3), preparation macropore dropwise cross-linked polymer:
Adopt dropping funnel, will (2) go on foot the water that makes with the speed of 1d/s and drip in the oil phase that (1) makes and disperse, the control stirring velocity is 350r/min; About 30 ℃ are reacted under nitrogen protection, 2 hours reaction times;
After reaction finished, employing was toppled over mode and is reclaimed upper solution, added 65% ethanol 220ml in reaction product, and standing over night behind the mixing is removed the upper strata ethanolic soln; Wash with water repeatedly again to no kerosene flavor and alcohol flavor, suction filtration, the macropore dropwise cross-linked polymer, in vacuum drying oven, dry to constant weight for 55～60 ℃, activated carrier 12.86g altogether not, the calculating reactivity is about 77%, and is standby;
(4), preparation activated carrier:
Get tetrahydrofuran (THF) (THF) and dissolve dicyclohexylcarbodiimide (DCC) (9.9g, 0.048 mole) and N-hydroxy-succinamide (NHS) (5.5g, 0.048 mole) respectively, (the THF consumption is respectively 70ml, 30ml) obtains DCC solution and NHS solution;
Other gets the not activated carrier (5g, wherein reactive monomer is about 0.047 mole) that (3) step is dried to constant weight, adds above-mentioned NHS solution, drips above-mentioned DCC solution again; About 30 ℃ of reactions are 4 hours in airtight round-bottomed flask, constantly stir (reaction process is carried out on magnetic stirring apparatus, adopts magnetic stir bar to stir) in the reaction process; Reaction finishes the back and reclaims solvent; Remove the by product dicyclohexylurea with anhydrous alcohol solution again
(DCU), suction filtration is dried to constant weight for 55～60 ℃ in vacuum drying oven, makes activated carrier, is described immobilized porcine pancreatic lipase carrier.
Equally, the contriver adopt sweet oil emulsion method measured this activated carrier immobilized porcine pancreatic lipase to the hydrolysis of sweet oil emulsion apparent activity, measurement result is 750U/g (representing with the carrier dry weight).
The immobilized porcine pancreatic lipase carrier of present embodiment for making by following method:
With tensio-active agent Span (sapn) 40 and Span80 is that composite dispersing agent, kerosene are that disperse phase, vinylformic acid (AA) are reactive monomer, N, N '-methylene-bisacrylamide (MBAA) is that linking agent, ethylene glycol are pore-creating agent, adopt anti-phase outstanding polymerization technique to make the macropore dropwise cross-linked polymer, by N-hydroxy-succinamide (NHS) activation, make described immobilized porcine pancreatic lipase carrier again.
Used raw material comprises:
Reactive monomer vinylformic acid (AA) 2.5mL (about 2.6g),
Linking agent N, N '-methylene-bisacrylamide MBAA) 1.3g (be AA quality about 50%),
Pore-creating agent ethylene glycol 2.3mL (about 2.6g, for the AA quality about 100%),
The usage ratio of dispersion agent Span40 and Span80 is controlled so that the two mixed HLB value is adjusted to 6, and its total consumption is 24g,
Disperse phase kerosene 100mL,
Catalyzer ammonium persulphate (AP) 0.1g (be AA quality about 3.8%),
Accelerator Tetramethyl Ethylene Diamine (TMEDA) 1mL (about 0.77g is about 7.7 times of AP quality);
In addition, in the preparation activated carrier process, reactant (not activated carrier, N-hydroxy-succinamide) is respectively with the consumption of dewatering agent dicyclohexylcarbodiimide:
Activated carrier 1g (wherein reactive monomer is about 0.01 mole) not,
N-hydroxy-succinamide (NHS) 1.3g (0.011 mole),
Dewatering agent dicyclohexylcarbodiimide (DCC) 2.3g (0.011 mole).
Concrete preparation method comprises following key step:
(1), preparation oil phase:
Composite dispersing agent Span40 and Span80 are added in organic disperse phase kerosene, and suitably heating for dissolving adds and is equipped with in the four-hole bottle of airway and agitator, and stirring and logical nitrogen mix; Add Tetramethyl Ethylene Diamine (TMEDA) again, mixing constitutes oil phase;
(2), preparation water:
With vinylformic acid (AA), N, N '-methylene-bisacrylamide (MBAA), ethylene glycol are dissolved in the 10ml water, add ammonium persulphate (AP), and mixing constitutes water;
(3), preparation macropore dropwise cross-linked polymer:
Adopt dropping funnel, will (2) go on foot the water that makes with the speed of 2d/s and drip in the oil phase that (1) makes and disperse, the control stirring velocity is 100r/min; About 10 ℃ are reacted under nitrogen protection, 2 hours reaction times;
After reaction finished, employing was toppled over mode and is reclaimed upper solution, added 70% ethanol 80ml in reaction product, and standing over night behind the mixing is removed the upper strata ethanolic soln; Wash with water repeatedly again to no kerosene flavor and alcohol flavor, suction filtration, the macropore dropwise cross-linked polymer, in vacuum drying oven, dry to constant weight for 35～40 ℃, activated carrier 3.16g altogether not, the calculating reactivity is about 80.9%, and is standby;
(4), preparation activated carrier:
Get tetrahydrofuran (THF) (THF) and dissolve dicyclohexylcarbodiimide (DCC) (2.3g, 0.011 mole) and N-hydroxy-succinamide (NHS) (1.3g, 0.011 mole) respectively, (the THF consumption is respectively 40mL, 20mL) obtains DCC solution and NHS solution;
Other gets the not activated carrier (1g, wherein reactive monomer is about 0.01 mole) that (3) step is dried to constant weight, adds above-mentioned NHS solution, drips above-mentioned DCC solution again; About 10 ℃ of reactions are 2 hours in airtight round-bottomed flask, constantly stir (reaction process is carried out on magnetic stirring apparatus, adopts magnetic stir bar to stir) in the reaction process; Reaction finishes the back and reclaims solvent, removes by product dicyclohexylurea (DCU) with anhydrous alcohol solution again, and suction filtration is dried to constant weight for 35～40 ℃ in vacuum drying oven, makes activated carrier, is described immobilized porcine pancreatic lipase carrier.
Equally, the contriver adopt sweet oil emulsion method measured this activated carrier immobilized porcine pancreatic lipase to the hydrolysis of sweet oil emulsion apparent activity, measurement result is 830U/g (representing with the carrier dry weight).
The immobilized porcine pancreatic lipase carrier of present embodiment for making by following method:
With tensio-active agent Span (sapn) 40 and Span80 is that composite dispersing agent, kerosene are that disperse phase, vinylformic acid (AA) are reactive monomer, N, N '-methylene-bisacrylamide (MBAA) is that linking agent, ethylene glycol are pore-creating agent, adopt anti-phase outstanding polymerization technique to make the macropore dropwise cross-linked polymer, by N-hydroxy-succinamide (NHS) activation, make described immobilized porcine pancreatic lipase carrier again.
Used raw material comprises:
Reactive monomer vinylformic acid (AA) 5mL (about 5.3g),
Linking agent N, N '-methylene-bisacrylamide MBAA) 2.1g (be AA quality about 40%),
Pore-creating agent ethylene glycol 4.6mL (about 5.2g, for the AA quality about 100%),
The usage ratio of dispersion agent Span40 and Span80 is controlled so that the two mixed HLB value is adjusted to 5, and its total consumption is 5.4g,
Disperse phase kerosene 45mL,
Catalyzer ammonium persulphate (AP) 0.11g (be AA quality about 2%),
Accelerator Tetramethyl Ethylene Diamine (TMEDA) 1mL (about 0.77g is 7 times of AP quality);
In addition, in the preparation activated carrier process, reactant (not activated carrier, N-hydroxy-succinamide) is respectively with the consumption of dewatering agent dicyclohexylcarbodiimide:
Activated carrier 2g (wherein reactive monomer is about 0.02 mole) not,
N-hydroxy-succinamide (NHS) 2.8g (0.024 mole),
Dewatering agent dicyclohexylcarbodiimide (DCC) 5.0g (0.024 mole).
Concrete preparation method comprises following key step:
(1), preparation oil phase:
Composite dispersing agent Span40 and Span80 are added in organic disperse phase kerosene, and suitably heating for dissolving adds and is equipped with in the four-hole bottle of airway and agitator, and stirring and logical nitrogen mix; Add Tetramethyl Ethylene Diamine (TMEDA) again, mixing constitutes oil phase;
(2), preparation water:
With vinylformic acid (AA), N, N '-methylene-bisacrylamide (MBAA), ethylene glycol are dissolved in the 5ml water, add ammonium persulphate (AP), and mixing constitutes water;
(3), preparation macropore dropwise cross-linked polymer:
Adopt dropping funnel, will (2) go on foot the water that makes with the speed of 0.5d/s and drip in the oil phase that (1) makes and disperse, the control stirring velocity is 250r/min; About 10 ℃ are reacted under nitrogen protection, 2 hours reaction times;
After reaction finished, employing was toppled over mode and is reclaimed upper solution, added 95% ethanol 80ml in reaction product, and standing over night behind the mixing is removed the upper strata ethanolic soln; Wash with water repeatedly again to no kerosene flavor and alcohol flavor, suction filtration, the macropore dropwise cross-linked polymer, in vacuum drying oven, dry to constant weight for 35～40 ℃, activated carrier 5.1g altogether not, the calculating reactivity is about 68.7%, and is standby;
(4), preparation activated carrier:
Get tetrahydrofuran (THF) (THF) and dissolve dicyclohexylcarbodiimide (DCC) (5.0g, 0.024 mole) and N-hydroxy-succinamide (NHS) (2.8g, 0.024 mole) respectively, (the THF consumption is respectively 60mL, 20mL) obtains DCC solution and NHS solution;
Other gets the not activated carrier (2g, wherein reactive monomer is about 0.02 mole) that (3) step is dried to constant weight, adds above-mentioned NHS solution, drips above-mentioned DCC solution again; About 10 ℃ of reactions are 2 hours in airtight round-bottomed flask, constantly stir (reaction process is carried out on magnetic stirring apparatus, adopts magnetic stir bar to stir) in the reaction process; Reaction finishes the back and reclaims solvent, removes by product dicyclohexylurea (DCU) with anhydrous alcohol solution again, and suction filtration is dried to constant weight for 35～40 ℃ in vacuum drying oven, makes activated carrier, is described immobilized porcine pancreatic lipase carrier.
Equally, the contriver adopt sweet oil emulsion method measured this activated carrier immobilized porcine pancreatic lipase to the hydrolysis of sweet oil emulsion apparent activity, measurement result is 800U/g (representing with the carrier dry weight).
In the various embodiments described above, measure the sweet oil emulsion method of immobilized porcine pancreatic lipase carrier apparent activity, all be adopt document (the Zhao Yahua chief editor. Biochemistry Experiment technical tutorial [M]. Guangzhou: press of South China Science ﹠ Engineering University, 2000:157-159) Ji Zai classical sweet oil emulsion method.According to the said determination result as can be known, its apparent activity all can reach 750U/g or 750U/g above (representing with the carrier dry weight).
Claims (10)
1. the preparation method of an immobilized porcine pancreatic lipase carrier is characterized in that:
It is a kind of macropore dropwise cross-linked polymer that contains succinimide ester group group, has the represented structure of following general formula I:
General formula I,
Wherein
Be to be reactive monomer, N with vinylformic acid, N '-methylene-bisacrylamide is the part except that carboxyl in the superpolymer that forms of linking agent.
2. the preparation method of the described immobilized porcine pancreatic lipase carrier of claim 1 is characterized in that:
This preparation method is that composite dispersing agent, kerosene are that disperse phase, vinylformic acid are reactive monomer, N with tensio-active agent Span40 and Span80, N '-methylene-bisacrylamide is that linking agent, ethylene glycol are pore-creating agent, adopt anti-phase outstanding polymerization technique to make the macropore dropwise cross-linked polymer, by the N-hydroxy-succinamide activation, make described immobilized porcine pancreatic lipase carrier again; Specifically comprise following key step:
(1), preparation oil phase:
The composite dispersing agent of Span40 and Span80 composition is dissolved in organic disperse phase kerosene mixing; Add Tetramethyl Ethylene Diamine again, mixing constitutes oil phase;
(2), preparation water:
With vinylformic acid, N, N '-methylene-bisacrylamide, ethylene glycol are dissolved in the water, and add ammonium persulphate, and mixing constitutes water;
(3), preparation macropore dropwise cross-linked polymer:
The water that (2) step is made with the speed of 0.5～2d/s drips in the oil phase that makes in (1) step and disperses, and the control stirring velocity is 100～350r/min; 10～30 ℃ are reacted under nitrogen protection, 1～2 hour reaction times;
After reaction finishes, reclaim upper solution, in reaction product, add the ethanol of volumetric concentration 〉=65%, left standstill behind the mixing 5～24 hours, remove the upper strata ethanolic soln; Wash with water repeatedly again to no kerosene flavor and alcohol flavor, suction filtration, the macropore dropwise cross-linked polymer, in vacuum drying oven, dry to constant weight for 35～60 ℃, must activated carrier be not standby;
(4), preparation activated carrier:
Get the not activated carrier that (3) step is dried to constant weight, do at tetrahydrofuran (THF) under the condition of solvent, dicyclohexylcarbodiimide is made dewatering agent, reacts 2～4 hours at 10～30 ℃ with N-hydroxy-succinamide, constantly stirs in the reaction process; Reaction finishes the back and reclaims solvent; Remove the by product dicyclohexylurea with anhydrous alcohol solution again, suction filtration is dried to constant weight for 35～60 ℃ in vacuum drying oven, makes activated carrier, is said fixing porcine pancreatic lipase carrier of the present invention.
3. preparation method according to claim 2 is characterized in that: in the described composite dispersing agent, the ratio of Span40 and Span80 is 4.5～6 to regulate according to the two mixed HLB value; Total consumption of composite dispersing agent is 10%～30% of a disperse phase kerosene quality.
4. preparation method according to claim 2 is characterized in that: described linking agent N, the quality of N '-methylene-bisacrylamide is 40%～60% of a monomer vinylformic acid quality.
5. preparation method according to claim 2 is characterized in that: the consumption of described pore-creating agent ethylene glycol is 70%～100% of a monomer vinylformic acid quality.
6. preparation method according to claim 2 is characterized in that: the add-on of described ammonium persulphate is 1.5%～4% of a monomer vinylformic acid quality; The add-on of described Tetramethyl Ethylene Diamine is 7～10 times of ammonium persulphate quality.
7. preparation method according to claim 2 is characterized in that: in the described step (2), the consumption of water is 1～4 times of vinylformic acid volume.
8. preparation method according to claim 2 is characterized in that: in the described step (3), the volume ratio of oil phase and water is 4:1～10:1.
9. preparation method according to claim 2, it is characterized in that: in the described step (4), not activated carrier, N-hydroxy-succinamide, with the consumption of dicyclohexylcarbodiimide by reactive monomer in the activated carrier not: N-hydroxy-succinamide: calculate dicyclohexylcarbodiimide mol ratio=1:1～1.2:1～1.2; The consumption of solvent THF with can dissolve DCC, NHS gets final product.
10. the application of the described immobilized porcine pancreatic lipase carrier of claim 1 is with its fixation support as porcine pancreatic lipase, other lipase, neutral protease, α-Dian Fenmei.
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Cited By (6)
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| CN101899130A (en) * | 2010-08-02 | 2010-12-01 | 陕西师范大学 | Preparation method and application of macroporous polyacryamide resin |
| CN103275814A (en) * | 2013-06-07 | 2013-09-04 | 华东理工大学 | Method for preparing biodiesel without by-product of glycerin by utilization of high acid value waste oil |
| CN104357434A (en) * | 2014-12-15 | 2015-02-18 | 广西民族大学 | Amino modified rosin based macroporous adsorption resin immobilized lipase and preparation method thereof |
| CN110760495A (en) * | 2019-05-07 | 2020-02-07 | 宁波大学 | Co-crosslinking immobilization method of porcine pancreatic lipase |
| CN110777141A (en) * | 2019-05-07 | 2020-02-11 | 宁波大学 | Co-crosslinking immobilization method of acid urease |
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- 2009-02-19 CN CN2009100583771A patent/CN101531732B/en not_active Expired - Fee Related
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| CN101899130A (en) * | 2010-08-02 | 2010-12-01 | 陕西师范大学 | Preparation method and application of macroporous polyacryamide resin |
| CN103275814A (en) * | 2013-06-07 | 2013-09-04 | 华东理工大学 | Method for preparing biodiesel without by-product of glycerin by utilization of high acid value waste oil |
| CN103275814B (en) * | 2013-06-07 | 2015-02-18 | 华东理工大学 | Method for preparing biodiesel without by-product of glycerin by utilization of high acid value waste oil |
| CN104357434A (en) * | 2014-12-15 | 2015-02-18 | 广西民族大学 | Amino modified rosin based macroporous adsorption resin immobilized lipase and preparation method thereof |
| CN104357434B (en) * | 2014-12-15 | 2017-12-19 | 广西民族大学 | A kind of abietyl macroporous absorbent resin immobilized lipase of amido modification and preparation method thereof |
| CN110760495A (en) * | 2019-05-07 | 2020-02-07 | 宁波大学 | Co-crosslinking immobilization method of porcine pancreatic lipase |
| CN110777141A (en) * | 2019-05-07 | 2020-02-11 | 宁波大学 | Co-crosslinking immobilization method of acid urease |
| CN110760495B (en) * | 2019-05-07 | 2023-03-17 | 宁波大学 | Co-crosslinking immobilization method of porcine pancreatic lipase |
| CN110777141B (en) * | 2019-05-07 | 2023-03-17 | 宁波大学 | Co-crosslinking immobilization method of acid urease |
| CN117229529A (en) * | 2023-09-05 | 2023-12-15 | 江苏好多收农业科技有限公司 | Biological hydrogel as immobilized active enzyme carrier and preparation method thereof |
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