CN108486095A - A kind of preparation method and its device of Cephalosporin Acylases immobilized enzyme - Google Patents
A kind of preparation method and its device of Cephalosporin Acylases immobilized enzyme Download PDFInfo
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Abstract
The invention discloses a kind of preparation methods and its device of Cephalosporin Acylases immobilized enzyme, belong to biotechnology, and the method includes (1) to prepare three dimensional elasticity carrier by composite oil phase solution and compound aqueous phase solution emulsification；(2) three dimensional elasticity carrier is activated using 3 dimethylaminopropylamines, increases the adhesion strength to enzyme；(3) crosslinking modification is carried out using two acetaldehyde enemy's spore rhzomorph acylase of glucan, three dimensional elasticity carrier；(4) gradient increased temperature post-processing is carried out to crosslinking modification enzyme.In short, the Cephalosporin Acylases immobilized enzyme prepared by the present invention has many advantages, such as that enzyme binding force is strong and stability is good, mechanical performance is strong, enzymatic activity is high, long half time.
Description
Technical field
The invention belongs to biotechnologies, and in particular to a kind of preparation method of Cephalosporin Acylases immobilized enzyme and its
Device.
Background technology
Cephalosporin Acylases, which are one kind, can be catalyzed cephalosporin (CPC) and Glularyl-7-amino-cephalo-alkanoic acid (GL-
7ACA) generate the antibiotic industrial enzymes of 7-amino-cephalosporanic acid (7-ACA).But it is quick to environment due to existing as resolvase
The shortcomings of sense, property are unstable, and inactivation and product are difficult to separating-purifying easily in enzymatic reaction.Therefore the prior art mainly adopts
Enzyme immobilization technology solves this defect.
Enzyme immobilization technology is generated from the sixties in 20th century, is widely used to the industry of biocatalysis field at present
In production.Enzyme immobilizatio method can be divided into four major class：Absorption method, investment, covalent coupling method, cross-linking method.Absorption method is
The enzyme immobilization method occurred earliest, including physical absorption and ion exchange adsorption.The method mild condition, the conformation change of enzyme compared with
Small or be basically unchanged, therefore influenced on the catalytic activity of enzyme small, but binding force is weak between enzyme and carrier, in uncomfortable pH, with high salt dense
Under the conditions of degree, high temperature etc., enzyme easily falls off from carrier and pollutes catalytic reaction products etc..The basic principle of investment is carrier and enzyme
After solution mixing, polymerisation is carried out by initiator, enzyme is limited in the grid of carrier by physical action, to realize
The method of enzyme immobilization.The method is not related to the conformation of enzyme and the chemical change of enzyme molecule, and reaction condition is mild, thus enzyme activity returns
Yield is higher.Investment immobilised enzymes is easily missed, and the problems such as diffusion limits is commonly present, and catalysis reaction is influenced by resistance to mass tranfer,
The reaction of macromolecule substrate should not be catalyzed.Covalent coupling method refers to the nonessential group of enzyme molecule and the active function of carrier surface
Group connect jail with carrier by forming the strong enzyme fixing means for realizing Irreversible binding of chemical covalent, the immobilised enzymes of gained
Gu having good stability and reusability, but the more other fixing means reactions of the method are violent, activity of the immobilized enzyme loses more
Add serious.Cross-linking method is to form covalent bond between enzyme molecule and cross-linking reagent using difunctional or multi-functional cross-linking reagent, adopt
Different materials is added with different crosslinking conditions and in cross-linking system, can generate the different immobilised enzymes of physical property.
Supplementary means of the cross-linking method generally as other process for fixation.Advantage is available that cross-linking reagent is more, and technology is simple；The knot of enzyme
Resultant force is strong, highly stable.It is poor but there are still mechanical performances, the shortcomings of enzyme is easy to inactivate in immobilization process, immobilized enzyme
Successful key is to select a good carrier.
Invention content
For the technical problem present on, the present invention provide a kind of Cephalosporin Acylases immobilized enzyme preparation method and
Its device.
The technical scheme is that：A kind of preparation method of Cephalosporin Acylases immobilized enzyme, includes the following steps：
(1) preparation of three dimensional elasticity carrier：By composite oil phase solution and compound aqueous phase solution with volume ratio for 1:1 independent dress
Entering in multifunctional temperature control water bath device, carries out gradient input stirring, speed of agitator 400-3000r/min, temperature is 2-10 DEG C,
Compared to conventional high-temperature, emulsion reaction is carried out at a lower temperature, can further improve the machine of three dimensional elasticity carrier obtained
Tool intensity and resilience make reduction and alleviate the impact force in immobilization process suffered by enzyme molecule, maintain the normal of zymoprotein
Conformation, to reduce the loss of enzyme activity；Mixing time is 4-24h, meanwhile, it is passed through nitrogen and carries out the micro- aeration of ultrasonic wave nanometer, obtain
To microbead latex, compared with being passed through air, the generation of oxidation reaction can be prevented by being passed through nitrogen, by the way of the micro- aeration of nanometer,
Nanometer nitrogen microbubble is passed through in blended liquid phase, assists spreading with ultrasonic wave, in the emulsion process of lower temperature, to utilize
The effect of nanometer nitrogen microbubble occupy-place to form a large amount of intercommunicating pore in bead particles.Be conducive to obtain porosity it is flourishing and
The uniform three dimensional elasticity carrier in aperture；The microbead latex is filtered using filter membrane, takes microballon, the microballon is utilized
The deionized water of 5 times of volume ratios is rinsed 3-5 times repeatedly, obtains wet microballon；The wet microballon is subjected to absorbent drying again, freezing is deposited
Storage is to get three dimensional elasticity carrier；
(2) activation of three dimensional elasticity carrier：Before use, it is 1-2 that volume ratio is first added in the three dimensional elasticity carrier
Times, the deionized water that temperature is 30-40 DEG C keeps the temperature 3-10min, is soaked and thawed；Then it adds as dosage 10-
The 3- dimethylaminopropylamines of 30ml/kg, at a temperature of 30-40 DEG C, with rotating speed for 60-80r/min mechanical agitation 60-90min, mistake
Filter obtains activation three dimensional elasticity carrier；Three dimensional elasticity carrier after overactivation can increase the adhesion strength to enzyme.
(3) enzyme immobilizatio：By the activation three dimensional elasticity carrier, the cephalo of two acetaldehyde of glucan and a concentration of 250g/L
It is 60-80 that rhzomorph, which is acylated enzyme solutions with mass ratio,:3-4:20 are fitted into the multifunctional temperature control water bath device, adjust pH to 7.5-
8.0, reaction temperature is 45-50 DEG C, and 12-14h is stirred by ultrasonic, and obtains crosslinking modification enzyme；Cephalosporin acyl after crosslinked modification
Change enzyme than unmodified 4-6 times preceding of Increased Plasma Half-life；
(4) it is crosslinked the post-processing of modification enzyme：Modification enzyme three is crosslinked described in EMSA wash buffers with pH for 7.5-8.0
Secondary, each 1min obtains amixia connection modification enzyme after membrane filtration；By amixia connection modification enzyme and a concentration of 50-
The tea polyphenols of 250mg/L are 1 according to mass ratio:After the ratio mixing of 2-3, it is transferred in multifunctional temperature control water bath device, with 2-4
DEG C/heating rate of h stirs gradually from room temperature to 55 DEG C with the rotating speed assisting ultrasonic of 30-60r/min, it filters, collects
Solid is to get Cephalosporin Acylases immobilized enzyme；Tea polyphenols can protect crosslinking modification enzyme to exist as a kind of natural
It is not interfered and is destroyed by oxygen radical in gradient increased temperature heat treatment process, keep the activity of Cephalosporin Acylases immobilized enzyme,
And after gradient increased temperature is heat-treated, it is also greatly improved the thermal stability of Cephalosporin Acylases immobilized enzyme.
Further, the composite oil phase solution includes according to weight percent：7-10% carbon tetrachloride, 14-16% are different
Propyl alcohol, 15-17% silicone oils, 9-11% ether, 1-2% isoflavones, 15-17% turpentine oil, 1-3% fluoro-carbon surface actives
Agent, surplus are wood wax oil.Isoflavones can prevent the autoxidation of oil-phase component, fluoro-carbon surface active as antioxidant
Agent can significantly improve dispersibility of the composite oil phase solution in compound aqueous phase solution, to improve the three dimensional elasticity carrier of preparation
Homogeneity.
Further, the compound aqueous phase solution includes according to weight percent：The poly- second of 8-10% polyaminoacid, 9-13%
Glycol-polycarboxylic acid polymer, 14-16% polyvinylpyrrolidones, 10-13% polylactic acid, 3-5% gelatin, 6-9% colloid eggs
In vain, 1-3% guar gums, 1-3% furcellarans, 0.5-1% lattice Tegosept Ps, surplus is pure water.Lattice Tegosept P can be carried as curing agent
The stability of high emulsion system.
Further, the gradient input method is：The composite oil phase solution and compound aqueous phase solution are according to 1:1 matter
Ratio is measured, in 0min, 10min, 20min, 30min, feeds composite oil phase solution and compound aqueous phase solution gross mass respectively
25%.Input in batches, it is ensured that composite oil phase solution and compound aqueous phase solution emulsifying power
Further, the aeration quantity of the nitrogen is 0.1-0.5L/min, and aeration quantity is too small, then it is impossible to ensure that in microballon
Microbubble uniform amount, aeration quantity is excessive, then the intercommunicating pore as caused by microbubble occupy-place is excessive, can reduce the hole of microballon instead
Gap rate.
Further, the multifunctional temperature control water bath device includes water bath, liner slot, pedestal, supersonic generator, adds
Hot device, General controller, L-shaped support arm, stirring rod, motor, aeration tube, Gas Connector, the water bath are fixedly connected on described
The top opening of the upper surface of pedestal, the liner slot is equipped with outer layer, and the outer layer is connected at the top of water bath, keeps liner slot outstanding
For sky in the inner hub location of water bath, the inner hub location in pedestal, the supersonic generator is arranged in the General controller
It is electrically connected at the top of General controller, the transmitting terminal of supersonic generator is connected with water-bath trench bottom, and the heater uniformly divides
Cloth is electrically connected in water-bath trench bottom, and with General controller, and the perpendicular section of the L-shaped support arm is fixedly connected on the rear portion of pedestal, L-shaped
The crosspiece of support arm is located at the surface of liner slot, and the motor is fixed on above the crosspiece of L-shaped support arm, the gas connection
Device is fixedly connected below the crosspiece of L-shaped support arm, and Gas Connector is connected with external air source, and the aeration tube is connected to gas
The upper end of the lower section of connector, the stirring rod is connected through aeration tube with the output shaft of motor, and the lower end of stirring rod, which is equipped with, to be stirred
Mix impeller.
Further, the water bath includes liquid storage box one, liquid storage box two, condensing agent box, and the liquid storage box one is arranged
On the left of water bath outer wall, liquid storage box two is arranged on the right side of water bath outer wall, and the condensing agent box is arranged before water bath outer wall
Side, liquid storage box one, liquid storage box two top respectively by rotate inlet tube one extend at the madial wall of the liner slot, condense
Agent box is extended to by rotating inlet tube two at the madial wall of water bath, is divided on the rotation inlet tube one, rotation inlet tube two
It She You not miniature metering pump, the miniature metering pump and General controller electric connection.
Further, the perpendicular section of the L-shaped support arm is retractable structure, is facilitated stirring and exposure attached below
Device of air proposes liner slot, and then is separately separated out liner slot and internal objects.
Compared with prior art, beneficial effects of the present invention are：
(1) present invention is mixed to prepare three-dimensional under 2-10 DEG C of lower temperature through composite oil phase solution and compound aqueous phase solution
Resilient support carries out emulsion reaction at a lower temperature compared to conventional high-temperature, can further improve three dimensional elasticity obtained
The mechanical strength and resilience of carrier make reduction and alleviate the impact force in immobilization process suffered by enzyme molecule, maintain enzyme egg
White normal conformation, to reduce the loss of enzyme activity.
(2) by the way of the micro- aeration of nanometer, nanometer nitrogen microbubble is led to while emulsion reaction occurs by the present invention
Enter in blended liquid phase, assist spreading with ultrasonic wave, in the emulsion process of lower temperature, to utilize nanometer nitrogen microbubble occupy-place
Effect to form a large amount of intercommunicating pore in bead particles, be conducive to obtain the three dimensional elasticity that porosity is flourishing and aperture is uniform
Carrier；Huge space can be provided for crosslinking modification enzyme.
(3) Cephalosporin Acylases after the modification of the method for the present invention are than unmodified 4-6 times preceding of Increased Plasma Half-life.
In short, being had by Cephalosporin Acylases immobilized enzyme prepared by the present invention, enzyme binding force is strong and stability is good, mechanical
The advantages that performance is strong, enzymatic activity is high, long half time.
Description of the drawings
Fig. 1 is the main sectional view of the multifunctional temperature control water bath device of the present invention；
Fig. 2 is the right view of the multifunctional temperature control water bath device of the present invention.
Wherein, 1- water baths, 2- liners slot, 3- pedestals, 4- supersonic generators, 5- heaters, 6- General controllers, 7-L shapes
Support arm, 8- stirring rods, 9- motors, 10- aeration tubes, 11- Gas Connectors, the outers 12- layer, 13- agitator arms, 14- liquid storages
Box one, 15- liquid storage boxes two, 16- condensing agents box, 17- rotations inlet tube one, 18- rotations inlet tube two, the miniature metering pumps of 19-.
Specific implementation mode
In order to more fully explain the present invention, the present invention is described further below by attached drawing 1-2.
Embodiment 1
A kind of preparation method of Cephalosporin Acylases immobilized enzyme, includes the following steps：
(1) preparation of three dimensional elasticity carrier：By composite oil phase solution and compound aqueous phase solution with volume ratio for 1:1 independent dress
Enter in multifunctional temperature control water bath device, wherein the composite oil phase solution includes according to weight percent：7% carbon tetrachloride,
14% isopropanol, 15% silicone oil, 9% ether, 1% isoflavones, 15% turpentine oil, 1% fluorocarbon surfactant, surplus
For wood wax oil.Isoflavones can prevent the autoxidation of oil-phase component, fluorocarbon surfactant from can show as antioxidant
Write and improve dispersibility of the composite oil phase solution in compound aqueous phase solution, to improve preparation three dimensional elasticity carrier it is uniform
Property.The compound aqueous phase solution includes according to weight percent：8% polyaminoacid, 9% polyethylene glycol carboxylic acid polyalcohol,
14% polyvinylpyrrolidone, 10% polylactic acid, 3% gelatin, 6% colloid albumen, 1% guar gum, 1% furcellaran, 0.5% lattice
Tegosept P, surplus are pure water.The stability of emulsion system can be improved as curing agent for lattice Tegosept P.Gradient input stirring is carried out,
The gradient input method is：The composite oil phase solution and compound aqueous phase solution are according to 1:1 mass ratio, 0min,
When 10min, 20min, 30min, the 25% of composite oil phase solution and compound aqueous phase solution gross mass is fed respectively.In batches into
Amount, it is ensured that composite oil phase solution and compound aqueous phase solution emulsifying power.Speed of agitator is 400r/min, and temperature is 2 DEG C, is compared
In conventional high-temperature, emulsion reaction is carried out at a lower temperature, and the machinery that can further improve three dimensional elasticity carrier obtained is strong
Degree and resilience, make reduction and alleviate the impact force in immobilization process suffered by enzyme molecule, maintain the normal conformation of zymoprotein,
To reduce the loss of enzyme activity；Mixing time is 4h, meanwhile, it is passed through nitrogen and carries out the micro- aeration of ultrasonic wave nanometer, obtain microballon breast
The aeration quantity of liquid, the nitrogen is 0.1L/min, and aeration quantity is too small, then cannot be guaranteed the microbubble uniform amount in microballon,
Aeration quantity is excessive, then the intercommunicating pore as caused by microbubble occupy-place is excessive, can reduce the porosity of microballon instead.Be passed through air phase
Than the generation of oxidation reaction can be prevented by being passed through nitrogen, and by the way of the micro- aeration of nanometer, nanometer nitrogen microbubble is passed through mixing
It in liquid phase, assists spreading with ultrasonic wave, in the emulsion process of lower temperature, to utilize the effect of nanometer nitrogen microbubble occupy-place
So that forming a large amount of intercommunicating pore in bead particles.Be conducive to obtain the three dimensional elasticity carrier that porosity is flourishing and aperture is uniform；
The microbead latex is filtered using filter membrane, takes microballon, the microballon is utilized into the deionized water of 5 times of volume ratios repeatedly
It rinses 3 times, obtains wet microballon；The wet microballon is subjected to absorbent drying again, freezer storage is to get three dimensional elasticity carrier；
(2) activation of three dimensional elasticity carrier：Before use, first in the three dimensional elasticity carrier be added volume ratio be 1 times,
The deionized water that temperature is 30 DEG C keeps the temperature 3min, is soaked and thawed；Then the 3- diformazans for dosage 10ml/kg are added
Amino propylamine, at a temperature of 30 DEG C, with rotating speed for 60r/min mechanical agitation 60min, filtering obtains activation three dimensional elasticity carrier；
Three dimensional elasticity carrier after overactivation can increase the adhesion strength to enzyme.
(3) enzyme immobilizatio：By the activation three dimensional elasticity carrier, the cephalo of two acetaldehyde of glucan and a concentration of 250g/L
Rhzomorph is acylated enzyme solutions with mass ratio as 60:3:20 are fitted into the multifunctional temperature control water bath device, adjust pH to 7.5, reaction
Temperature is 45 DEG C, and 12h is stirred by ultrasonic, and obtains crosslinking modification enzyme；Cephalosporin Acylases after crosslinked modification are than before unmodified
4 times of Increased Plasma Half-life；
(4) it is crosslinked the post-processing of modification enzyme：It is crosslinked modification enzyme three times described in the EMSA wash buffers for being 7.5 with pH, often
Secondary 1min obtains amixia connection modification enzyme after membrane filtration；The amixia is joined to the tea of modification enzyme and a concentration of 50mg/L
Polyphenol is 1 according to mass ratio:After 2 ratio mixing, it is transferred in multifunctional temperature control water bath device, with the heating rate of 2 DEG C/h
It gradually from room temperature to 55 DEG C, and is stirred, is filtered with the rotating speed assisting ultrasonic of 30r/min, collect solid to get cephalosporin
Acylase immobilized enzyme；Tea polyphenols can protect crosslinking modification enzyme heat treated in gradient increased temperature as a kind of natural
It is not interfered and is destroyed by oxygen radical in journey, keep the activity of Cephalosporin Acylases immobilized enzyme, and through gradient increased temperature heat
After processing, it is also greatly improved the thermal stability of Cephalosporin Acylases immobilized enzyme.
As shown in Figs. 1-2, multifunctional temperature control water bath device includes water bath 1, liner slot 2, pedestal 3, supersonic generator
4, heater 5, General controller 6, L-shaped support arm 7, stirring rod 8, motor 9, aeration tube 10, Gas Connector 11, water bath 1 are fixed
It is connected to the upper surface of pedestal 3, water bath 1 includes liquid storage box 1, liquid storage box 2 15, condensing agent box 16, and liquid storage box 1 is set
It sets on the left of 1 outer wall of water bath, liquid storage box 2 15 is arranged on the right side of 1 outer wall of water bath, and condensing agent box 16 is arranged outside water bath 1
On front side of wall, liquid storage box 1, liquid storage box 2 15 top the madial wall of liner slot 2 is extended to by rotating inlet tube 1 respectively
Place, condensing agent box 16 are extended to by rotating inlet tube 2 18 at the madial wall of water bath 1, and rotation inlet tube 1 is rotated into
Miniature metering pump 19 is respectively equipped in liquid pipe 2 18, miniature metering pump 19 is electrically connected with General controller 6.The top opening of liner slot 2 is set
There are outer layer 12, outer layer 12 to be connected to 1 top of water bath, make inner hub location of the liner slot 2 vacantly in water bath 1, always
The inner hub location in pedestal 3 is arranged in control device 6, and supersonic generator 4 is electrically connected at the top of General controller 6, ultrasonic wave hair
The transmitting terminal of raw device 4 is connected with 1 bottom of water bath, and heater 5 is evenly distributed on 1 bottom of water bath, and electrically connects with General controller 6
Connect, the perpendicular section of L-shaped support arm 7 is fixedly connected on the rear portion of pedestal 3, and the perpendicular section of L-shaped support arm 7 is retractable structure, facilitate by
Stirring attached below and aerator propose liner slot 2, and then are separately separated out liner slot 2 and internal objects.L-shaped supports
The crosspiece of arm 7 is located at the surface of liner slot 2, and motor 9 is fixed on above the crosspiece of L-shaped support arm 7, and Gas Connector 11 is fixed
It is connected to below the crosspiece of L-shaped support arm 7, Gas Connector 11 is connected with external air source, and aeration tube 10 is connected to gas connection
The upper end of the lower section of device 11, stirring rod 8 is connected through aeration tube 10 with the output shaft of motor 9, and the lower end of stirring rod 8 is equipped with stirring
Impeller 13.
Interpretation of result：By treated, Cephalosporin Acylases immobilized enzyme carries out enzyme activity determination, enzyme activity 98U/g, in pH
=9.0, under conditions of temperature is 45 DEG C, it is 80min to measure half-life.
Embodiment 2
A kind of preparation method of Cephalosporin Acylases immobilized enzyme, includes the following steps：
(1) preparation of three dimensional elasticity carrier：By composite oil phase solution and compound aqueous phase solution with volume ratio for 1:1 independent dress
Enter in multifunctional temperature control water bath device, wherein the composite oil phase solution includes according to weight percent：8.5% carbon tetrachloride,
15% isopropanol, 16% silicone oil, 10% ether, 1.5% isoflavones, 16% turpentine oil, 2% fluorocarbon surfactant,
Surplus is wood wax oil.Isoflavones can prevent the autoxidation of oil-phase component, fluorocarbon surfactant can as antioxidant
To significantly improve dispersibility of the composite oil phase solution in compound aqueous phase solution, to improve preparation three dimensional elasticity carrier it is equal
One property.The compound aqueous phase solution includes according to weight percent：9% polyaminoacid, 10.5% polyethylene glycol carboxylic acid are poly-
Close object, 15% polyvinylpyrrolidone, 11.5% polylactic acid, 4% gelatin, 7.5% colloid albumen, 2% guar gum, 2% red algae
Glue, 0.75% lattice Tegosept P, surplus is pure water.The stability of emulsion system can be improved as curing agent for lattice Tegosept P.Carry out ladder
Input stirring is spent, the gradient input method is：The composite oil phase solution and compound aqueous phase solution are according to 1:1 mass ratio,
In 0min, 10min, 20min, 30min, the 25% of composite oil phase solution and compound aqueous phase solution gross mass is fed respectively.
Input in batches, it is ensured that composite oil phase solution and compound aqueous phase solution emulsifying power.Speed of agitator is 1700r/min, temperature
It is 4 DEG C, compared to conventional high-temperature, carries out emulsion reaction at a lower temperature, can further improves three dimensional elasticity obtained and carry
The mechanical strength and resilience of body make reduction and alleviate the impact force in immobilization process suffered by enzyme molecule, maintain zymoprotein
Normal conformation, to reduce the loss of enzyme activity；Mixing time is 14h, meanwhile, it is passed through nitrogen and carries out the micro- exposure of ultrasonic wave nanometer
Gas obtains microbead latex, and the aeration quantity of the nitrogen is 0.3L/min, and aeration quantity is too small, then it is impossible to ensure that micro- in microballon
Number of bubbles is uniform, and aeration quantity is excessive, then the intercommunicating pore as caused by microbubble occupy-place is excessive, can reduce the hole of microballon instead
Rate.Compared with being passed through air, the generation of oxidation reaction can be prevented by being passed through nitrogen, by the way of the micro- aeration of nanometer, by nanometer nitrogen
Gas microbubble is passed through in blended liquid phase, assists spreading with ultrasonic wave, in the emulsion process of lower temperature, to utilize nanometer nitrogen
The effect of microbubble occupy-place to form a large amount of intercommunicating pore in bead particles.Be conducive to obtain porosity prosperity and aperture is uniform
Three dimensional elasticity carrier；The microbead latex is filtered using filter membrane, takes microballon, the microballon is utilized into 5 times of volumes
The deionized water of ratio is rinsed 4 times repeatedly, obtains wet microballon；The wet microballon is subjected to absorbent drying again, freezer storage is to get three
Tie up resilient support；
(2) activation of three dimensional elasticity carrier：Before use, first in the three dimensional elasticity carrier be added volume ratio be 2 times,
The deionized water that temperature is 35 DEG C keeps the temperature 6min, is soaked and thawed；Then the 3- diformazans for dosage 20ml/kg are added
Amino propylamine, at a temperature of 35 DEG C, with rotating speed for 70r/min mechanical agitation 75min, filtering obtains activation three dimensional elasticity carrier；
Three dimensional elasticity carrier after overactivation can increase the adhesion strength to enzyme.
(3) enzyme immobilizatio：By the activation three dimensional elasticity carrier, the cephalo of two acetaldehyde of glucan and a concentration of 250g/L
Rhzomorph is acylated enzyme solutions with mass ratio as 70:3:20 are fitted into the multifunctional temperature control water bath device, adjust pH to 7.9, reaction
Temperature is 47 DEG C, and 13h is stirred by ultrasonic, and obtains crosslinking modification enzyme；Cephalosporin Acylases after crosslinked modification are than before unmodified
6 times of Increased Plasma Half-life；
(4) it is crosslinked the post-processing of modification enzyme：It is crosslinked modification enzyme three times described in the EMSA wash buffers for being 7.9 with pH, often
Secondary 1min obtains amixia connection modification enzyme after membrane filtration；By amixia connection modification enzyme with a concentration of 150mg/L's
Tea polyphenols are 1 according to mass ratio:After 2.5 ratio mixing, it is transferred in multifunctional temperature control water bath device, with the heating of 3 DEG C/h
Speed is stirred gradually from room temperature to 55 DEG C with the rotating speed assisting ultrasonic of 45r/min, is filtered, and collects solid to get cephalo
Rhzomorph acylase immobilized enzyme；Tea polyphenols can protect crosslinking modification enzyme at gradient increased temperature heat as a kind of natural
It is not interfered and is destroyed by oxygen radical during reason, keep the activity of Cephalosporin Acylases immobilized enzyme, and through gradient liter
After warm processing, it is also greatly improved the thermal stability of Cephalosporin Acylases immobilized enzyme.
As shown in Figs. 1-2, multifunctional temperature control water bath device includes water bath 1, liner slot 2, pedestal 3, supersonic generator
4, heater 5, General controller 6, L-shaped support arm 7, stirring rod 8, motor 9, aeration tube 10, Gas Connector 11, water bath 1 are fixed
It is connected to the upper surface of pedestal 3, water bath 1 includes liquid storage box 1, liquid storage box 2 15, condensing agent box 16, and liquid storage box 1 is set
It sets on the left of 1 outer wall of water bath, liquid storage box 2 15 is arranged on the right side of 1 outer wall of water bath, and condensing agent box 16 is arranged outside water bath 1
On front side of wall, liquid storage box 1, liquid storage box 2 15 top the madial wall of liner slot 2 is extended to by rotating inlet tube 1 respectively
Place, condensing agent box 16 are extended to by rotating inlet tube 2 18 at the madial wall of water bath 1, and rotation inlet tube 1 is rotated into
Miniature metering pump 19 is respectively equipped in liquid pipe 2 18, miniature metering pump 19 is electrically connected with General controller 6.The top opening of liner slot 2 is set
There are outer layer 12, outer layer 12 to be connected to 1 top of water bath, make inner hub location of the liner slot 2 vacantly in water bath 1, always
The inner hub location in pedestal 3 is arranged in control device 6, and supersonic generator 4 is electrically connected at the top of General controller 6, ultrasonic wave hair
The transmitting terminal of raw device 4 is connected with 1 bottom of water bath, and heater 5 is evenly distributed on 1 bottom of water bath, and electrically connects with General controller 6
Connect, the perpendicular section of L-shaped support arm 7 is fixedly connected on the rear portion of pedestal 3, and the perpendicular section of L-shaped support arm 7 is retractable structure, facilitate by
Stirring attached below and aerator propose liner slot 2, and then are separately separated out liner slot 2 and internal objects.L-shaped supports
The crosspiece of arm 7 is located at the surface of liner slot 2, and motor 9 is fixed on above the crosspiece of L-shaped support arm 7, and Gas Connector 11 is fixed
It is connected to below the crosspiece of L-shaped support arm 7, Gas Connector 11 is connected with external air source, and aeration tube 10 is connected to gas connection
The upper end of the lower section of device 11, stirring rod 8 is connected through aeration tube 10 with the output shaft of motor 9, and the lower end of stirring rod 8 is equipped with stirring
Impeller 13.
Interpretation of result：By treated, Cephalosporin Acylases immobilized enzyme carries out enzyme activity determination, enzyme activity 121U/g, in pH
=9.0, under conditions of temperature is 45 DEG C, it is 120min to measure half-life.
Embodiment 3
A kind of preparation method of Cephalosporin Acylases immobilized enzyme, includes the following steps：
(1) preparation of three dimensional elasticity carrier：By composite oil phase solution and compound aqueous phase solution with volume ratio for 1:1 independent dress
Enter in multifunctional temperature control water bath device, wherein the composite oil phase solution includes according to weight percent：10% carbon tetrachloride,
16% isopropanol, 17% silicone oil, 11% ether, 2% isoflavones, 17% turpentine oil, 3% fluorocarbon surfactant, it is remaining
Amount is wood wax oil.Isoflavones can prevent the autoxidation of oil-phase component as antioxidant, and fluorocarbon surfactant can be with
Significantly improve dispersibility of the composite oil phase solution in compound aqueous phase solution, to improve preparation three dimensional elasticity carrier it is uniform
Property.The compound aqueous phase solution includes according to weight percent：10% polyaminoacid, the polymerization of 13% polyethylene glycol carboxylic acid
Object, 16% polyvinylpyrrolidone, 13% polylactic acid, 5% gelatin, 9% colloid albumen, 3% guar gum, 3% furcellaran, 1%
Lattice Tegosept P, surplus are pure water.The stability of emulsion system can be improved as curing agent for lattice Tegosept P.Gradient input is carried out to stir
It mixes, the gradient input method is：The composite oil phase solution and compound aqueous phase solution are according to 1:1 mass ratio, 0min,
When 10min, 20min, 30min, the 25% of composite oil phase solution and compound aqueous phase solution gross mass is fed respectively.In batches into
Amount, it is ensured that composite oil phase solution and compound aqueous phase solution emulsifying power.Speed of agitator is 3000r/min, and temperature is 10 DEG C, phase
Compared with conventional high-temperature, emulsion reaction is carried out at a lower temperature, can further improve the machinery of three dimensional elasticity carrier obtained
Intensity and resilience make reduction and alleviate the impact force in immobilization process suffered by enzyme molecule, maintain the normal structure of zymoprotein
As to reduce the loss of enzyme activity；Mixing time be for 24 hours, meanwhile, be passed through nitrogen carry out the micro- aeration of ultrasonic wave nanometer, obtain micro-
The aeration quantity of pearl lotion, the nitrogen is 0.5L/min, and aeration quantity is too small, then it is impossible to ensure that the microbubble quantity in microballon is equal
Even, aeration quantity is excessive, then the intercommunicating pore as caused by microbubble occupy-place is excessive, can reduce the porosity of microballon instead.Be passed through sky
Gas is compared, and the generation of oxidation reaction can be prevented by being passed through nitrogen, and by the way of the micro- aeration of nanometer, nanometer nitrogen microbubble is passed through
It in blended liquid phase, assists spreading with ultrasonic wave, in the emulsion process of lower temperature, to utilize nanometer nitrogen microbubble occupy-place
Effect in bead particles so that form a large amount of intercommunicating pore.Be conducive to obtain the three dimensional elasticity load that porosity is flourishing and aperture is uniform
Body；The microbead latex is filtered using filter membrane, takes microballon, the microballon is utilized to the deionized water of 5 times of volume ratios
It rinses 5 times repeatedly, obtains wet microballon；The wet microballon is subjected to absorbent drying again, freezer storage is to get three dimensional elasticity carrier；
(2) activation of three dimensional elasticity carrier：Before use, first in the three dimensional elasticity carrier be added volume ratio be 2 times,
The deionized water that temperature is 40 DEG C keeps the temperature 10min, is soaked and thawed；Then the 3- diformazans for dosage 30ml/kg are added
Amino propylamine, at a temperature of 40 DEG C, with rotating speed for 80r/min mechanical agitation 90min, filtering obtains activation three dimensional elasticity carrier；
Three dimensional elasticity carrier after overactivation can increase the adhesion strength to enzyme.
(3) enzyme immobilizatio：By the activation three dimensional elasticity carrier, the cephalo of two acetaldehyde of glucan and a concentration of 250g/L
Rhzomorph is acylated enzyme solutions with mass ratio as 80:4:20 are fitted into the multifunctional temperature control water bath device, adjust pH to 8.0, reaction
Temperature is 50 DEG C, and 14h is stirred by ultrasonic, and obtains crosslinking modification enzyme；Cephalosporin Acylases after crosslinked modification are than before unmodified
5 times of Increased Plasma Half-life；
(4) it is crosslinked the post-processing of modification enzyme：It is crosslinked modification enzyme three times described in the EMSA wash buffers for being 8.0 with pH, often
Secondary 1min obtains amixia connection modification enzyme after membrane filtration；By amixia connection modification enzyme with a concentration of 250mg/L's
Tea polyphenols are 1 according to mass ratio:It after 3 ratio mixing, is transferred in multifunctional temperature control water bath device, with the heating speed of 4 DEG C/h
Degree is stirred gradually from room temperature to 55 DEG C with the rotating speed assisting ultrasonic of 60r/min, is filtered, and collects solid to get cephalo bacterium
Plain acylase immobilized enzyme；Tea polyphenols can protect crosslinking modification enzyme to be heat-treated in gradient increased temperature as a kind of natural
It is not interfered and is destroyed by oxygen radical in the process, keep the activity of Cephalosporin Acylases immobilized enzyme, and through gradient increased temperature
After heat treatment, it is also greatly improved the thermal stability of Cephalosporin Acylases immobilized enzyme.
As shown in Figs. 1-2, multifunctional temperature control water bath device includes water bath 1, liner slot 2, pedestal 3, supersonic generator
4, heater 5, General controller 6, L-shaped support arm 7, stirring rod 8, motor 9, aeration tube 10, Gas Connector 11, water bath 1 are fixed
It is connected to the upper surface of pedestal 3, water bath 1 includes liquid storage box 1, liquid storage box 2 15, condensing agent box 16, and liquid storage box 1 is set
It sets on the left of 1 outer wall of water bath, liquid storage box 2 15 is arranged on the right side of 1 outer wall of water bath, and condensing agent box 16 is arranged outside water bath 1
On front side of wall, liquid storage box 1, liquid storage box 2 15 top the madial wall of liner slot 2 is extended to by rotating inlet tube 1 respectively
Place, condensing agent box 16 are extended to by rotating inlet tube 2 18 at the madial wall of water bath 1, and rotation inlet tube 1 is rotated into
Miniature metering pump 19 is respectively equipped in liquid pipe 2 18, miniature metering pump 19 is electrically connected with General controller 6.The top opening of liner slot 2 is set
There are outer layer 12, outer layer 12 to be connected to 1 top of water bath, make inner hub location of the liner slot 2 vacantly in water bath 1, always
The inner hub location in pedestal 3 is arranged in control device 6, and supersonic generator 4 is electrically connected at the top of General controller 6, ultrasonic wave hair
The transmitting terminal of raw device 4 is connected with 1 bottom of water bath, and heater 5 is evenly distributed on 1 bottom of water bath, and electrically connects with General controller 6
Connect, the perpendicular section of L-shaped support arm 7 is fixedly connected on the rear portion of pedestal 3, and the perpendicular section of L-shaped support arm 7 is retractable structure, facilitate by
Stirring attached below and aerator propose liner slot 2, and then are separately separated out liner slot 2 and internal objects.L-shaped supports
The crosspiece of arm 7 is located at the surface of liner slot 2, and motor 9 is fixed on above the crosspiece of L-shaped support arm 7, and Gas Connector 11 is fixed
It is connected to below the crosspiece of L-shaped support arm 7, Gas Connector 11 is connected with external air source, and aeration tube 10 is connected to gas connection
The upper end of the lower section of device 11, stirring rod 8 is connected through aeration tube 10 with the output shaft of motor 9, and the lower end of stirring rod 8 is equipped with stirring
Impeller 13.
Interpretation of result：By treated, Cephalosporin Acylases immobilized enzyme carries out enzyme activity determination, enzyme activity 105U/g, in pH
=9.0, under conditions of temperature is 45 DEG C, it is 100min to measure half-life.
Finally it should be noted that：The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although
Present invention has been described in detail with reference to the aforementioned embodiments, it will be understood by those of ordinary skill in the art that：It still may be used
To modify to the technical solution recorded in previous embodiment or equivalent replacement of some of the technical features；And
These modifications or replacements, the spirit and model of technical solution of the embodiment of the present invention that it does not separate the essence of the corresponding technical solution
It encloses.
Claims (8)
1. a kind of preparation method of Cephalosporin Acylases immobilized enzyme, which is characterized in that include the following steps：
(1) preparation of three dimensional elasticity carrier：By composite oil phase solution and compound aqueous phase solution with volume ratio for 1:1 independent loading is more
In function temperature controlled water bath device, gradient input stirring, speed of agitator 400-3000r/min are carried out, temperature is 2-10 DEG C, stirring
Time is 4-24h, meanwhile, it is passed through nitrogen and carries out the micro- aeration of ultrasonic wave nanometer, obtain microbead latex；The microbead latex is utilized
Filter membrane is filtered, and takes microballon, and the microballon is rinsed 3-5 times repeatedly using the deionized water of 5 times of volume ratios, is obtained wet micro-
Pearl；The wet microballon is subjected to absorbent drying again, freezer storage is to get three dimensional elasticity carrier；
(2) activation of three dimensional elasticity carrier：It is warm before use, it is 1-2 times that volume ratio is first added in the three dimensional elasticity carrier
Degree is 30-40 DEG C of deionized water, keeps the temperature 3-10min, is soaked and thawed；Then it adds as dosage 10-30ml/kg's
3- dimethylaminopropylamines, at a temperature of 30-40 DEG C, with rotating speed for 60-80r/min mechanical agitation 60-90min, filtering is lived
Change three dimensional elasticity carrier；
(3) enzyme immobilizatio：By the activation three dimensional elasticity carrier, the cephalosporin of two acetaldehyde of glucan and a concentration of 250g/L
Acylated enzyme solutions are 60-80 with mass ratio:3-4:20 are fitted into the multifunctional temperature control water bath device, adjust pH to 7.5-8.0,
Reaction temperature is 45-50 DEG C, and 12-14h is stirred by ultrasonic, and obtains crosslinking modification enzyme；
(4) it is crosslinked the post-processing of modification enzyme：It is crosslinked modification enzyme three times described in EMSA wash buffers with pH for 7.5-8.0, often
Secondary 1min obtains amixia connection modification enzyme after membrane filtration；By amixia connection modification enzyme and a concentration of 50-250mg/L
Tea polyphenols according to mass ratio be 1:After the ratio mixing of 2-3, it is transferred in multifunctional temperature control water bath device, with 2-4 DEG C/h's
Heating rate is stirred gradually from room temperature to 55 DEG C with the rotating speed assisting ultrasonic of 30-60r/min, is filtered, is collected solid,
Up to Cephalosporin Acylases immobilized enzyme.
2. a kind of preparation method of Cephalosporin Acylases immobilized enzyme as described in claim 1, which is characterized in that described compound
Oil-phase solution includes according to weight percent：7-10% carbon tetrachloride, 14-16% isopropanols, 15-17% silicone oils, 9-11%
Ether, 1-2% isoflavones, 15-17% turpentine oil, 1-3% fluorocarbon surfactants, surplus are wood wax oil.
3. a kind of preparation method of Cephalosporin Acylases immobilized enzyme as described in claim 1, which is characterized in that described compound
Aqueous phase solution includes according to weight percent：8-10% polyaminoacid, 9-13% polyethylene glycols carboxylic acid polyalcohol, 14-
16% polyvinylpyrrolidone, 10-13% polylactic acid, 3-5% gelatin, 6-9% colloid albumens, 1-3% guar gums, 1-3% are red
Phycocolloid, 0.5-1% lattice Tegosept Ps, surplus is pure water.
4. a kind of preparation method of Cephalosporin Acylases immobilized enzyme as described in claim 1, which is characterized in that the gradient
Input method is：The composite oil phase solution and compound aqueous phase solution are according to 1:1 mass ratio, 0min, 10min,
When 20min, 30min, the 25% of composite oil phase solution and compound aqueous phase solution gross mass is fed respectively.
5. a kind of preparation method of Cephalosporin Acylases immobilized enzyme as described in claim 1, which is characterized in that the gradient
Input method is：The composite oil phase solution and compound aqueous phase solution are according to 1:1 mass ratio, in 0min, feeding is compound
The 25% of oil-phase solution and compound aqueous phase solution gross mass；When 10min, feeds composite oil phase solution and compound aqueous phase solution is total
The 25% of quality；When 20min, the 25% of composite oil phase solution and compound aqueous phase solution gross mass is fed；When 30min, into
To the 25% of composite oil phase solution and compound aqueous phase solution gross mass.
6. a kind of preparation method of Cephalosporin Acylases immobilized enzyme as described in claim 1, which is characterized in that more work(
Energy temperature controlled water bath device includes water bath (1), liner slot (2), pedestal (3), supersonic generator (4), heater (5), master control
Device (6), L-shaped support arm (7), stirring rod (8), motor (9), aeration tube (10), Gas Connector (11), the water bath (1)
It is fixedly connected on the upper surface of the pedestal (3), the top opening of the liner slot (2) is equipped with outer layer (12), the outer layer
(12) it is connected at the top of water bath (1), makes liner slot (2) vacantly in the inner hub location of water bath (1), the General controller
(6) inner hub location in pedestal (3) is set, the supersonic generator (4) is electrically connected at the top of General controller (6),
The transmitting terminal of supersonic generator (4) is connected with water bath (1) bottom, and the heater (5) is evenly distributed on water bath (1) bottom
Portion, and be electrically connected with General controller (6), the perpendicular section of the L-shaped support arm (7) is fixedly connected on the rear portion of pedestal (3), L-shaped branch
The crosspiece of brace (7) is located at the surface of liner slot (2), and the motor (9) is fixed on above the crosspiece of L-shaped support arm (7), institute
It states Gas Connector (11) to be fixedly connected below the crosspiece of L-shaped support arm (7), Gas Connector (11) and external air source phase
Even, the aeration tube (10) is connected to the lower section of Gas Connector (11), and aeration tube (10) is run through in the upper end of the stirring rod (8)
It is connected with the output shaft of motor (9), the lower end of stirring rod (8) is equipped with agitator arm (13).
7. a kind of preparation method of Cephalosporin Acylases immobilized enzyme as claimed in claim 6, which is characterized in that the water-bath
Slot (1) includes liquid storage box one (14), liquid storage box two (15), condensing agent box (16), and the liquid storage box one (14) is arranged in water bath
(1) on the left of outer wall, on the right side of water bath (1) outer wall, the condensing agent box (16) is arranged in water bath for liquid storage box two (15) setting
(1) on front side of outer wall, liquid storage box one (14), liquid storage box two (15) top respectively by rotate inlet tube one (17) extend to it is described
At the madial wall of liner slot (2), condensing agent box (16) extends to the madial wall of water bath (1) by rotating inlet tube two (18)
Locate, miniature metering pump (19), the miniature metering are respectively equipped on the rotation inlet tube one (17), rotation inlet tube two (18)
(19) are pumped to be electrically connected with the General controller (6).
8. a kind of preparation method of Cephalosporin Acylases immobilized enzyme as claimed in claim 6, which is characterized in that the L-shaped
The perpendicular section of support arm (7) is retractable structure.
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