CN108486095B - Preparation method and device of cephalosporin acylase immobilized enzyme - Google Patents
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Abstract
The invention discloses a preparation method and a device of cephalosporin acylase immobilized enzyme, belonging to the technical field of biology, wherein the method comprises the following steps of (1) emulsifying a composite oil phase solution and a composite water phase solution to prepare a three-dimensional elastic carrier; (2) 3-dimethylaminopropylamine is used for activating the three-dimensional elastic carrier, so that the adhesion to enzyme is increased; (3) performing cross-linking modification on the cephalosporin acylase and the three-dimensional elastic carrier by utilizing glucan diacetal; (4) and carrying out gradient temperature rise post-treatment on the crosslinking modification enzyme. In a word, the cephalosporin acylase immobilized enzyme prepared by the invention has the advantages of strong enzyme binding force, good stability, strong mechanical property, high enzyme activity, long half-life period and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a preparation method and a device of cephalosporin acylase immobilized enzyme.
Background
Cephalosporin acylases are antibiotic industrial enzymes which can catalyze cephalosporin C (CPC) and glutaryl-7-aminocephalosporanic acid (GL-7ACA) to generate 7-aminocephalosporanic acid (7-ACA). However, the free enzyme has the disadvantages of environmental sensitivity, unstable property, easy inactivation in enzymatic reaction, difficult separation and purification of products and the like. Therefore, the prior art mostly adopts the immobilized enzyme technology to solve the defect.
The immobilized enzyme technology is generated in the sixties of the 20 th century and is widely applied to industrial production in the field of biological catalysis at present. Immobilization methods of enzymes can be divided into four major categories: adsorption, embedding, covalent bonding, crosslinking. Adsorption methods are the earliest emerging methods of enzyme immobilization, including physical adsorption and ion exchange adsorption. The method has mild conditions, and the conformational change of the enzyme is small or basically unchanged, so the method has small influence on the catalytic activity of the enzyme, but the binding force between the enzyme and the carrier is weak, and the enzyme is easy to fall off from the carrier and pollute catalytic reaction products and the like under the conditions of uncomfortable pH, high salt concentration, high temperature and the like. The basic principle of the embedding method is that after a carrier is mixed with an enzyme solution, polymerization reaction is carried out by means of an initiator, and the enzyme is limited in a grid of the carrier through physical action, so that the enzyme immobilization method is realized. The method does not relate to the conformation of the enzyme and the chemical change of enzyme molecules, and has mild reaction conditions, so the recovery rate of the enzyme activity is higher. The immobilized enzyme by the embedding method is easy to leak, and has the problems of diffusion limitation and the like, and the catalytic reaction is influenced by mass transfer resistance and is not suitable for catalyzing the reaction of a macromolecular substrate. The covalent binding method is an enzyme immobilization method for realizing irreversible binding by forming a chemical covalent bond between an unnecessary group of an enzyme molecule and an active functional group on the surface of a carrier, and the obtained immobilized enzyme is firmly connected with the carrier and has good stability and reusability, but compared with other immobilization methods, the method has violent reaction and more serious activity loss of the immobilized enzyme. The cross-linking method is to form covalent bond between enzyme molecule and cross-linking reagent by using bifunctional or multifunctional cross-linking reagent, and to generate immobilized enzymes with different physical properties by adopting different cross-linking conditions and adding different materials into a cross-linking system. The cross-linking method is generally used as an auxiliary means for other immobilization methods. The method has the advantages of more available crosslinking reagents and simple technology; the enzyme has strong binding force and is very stable. But still has the disadvantages of poor mechanical properties, easy inactivation of enzyme in the immobilization process and the like, and the key of the successful enzyme immobilization lies in selecting a good carrier.
Disclosure of Invention
Aiming at the technical problems, the invention provides a preparation method and a device of cephalosporin acylase immobilized enzyme.
The technical scheme of the invention is as follows: a preparation method of cephalosporin acylase immobilized enzyme comprises the following steps:
(1) preparing a three-dimensional elastic carrier: independently filling the composite oil phase solution and the composite water phase solution into a multifunctional temperature-controlled water bath device according to the volume ratio of 1:1, performing gradient input stirring, wherein the stirring speed is 400-3000r/min, the temperature is 2-10 ℃, and compared with the traditional high temperature, the emulsion reaction is performed at a lower temperature, so that the mechanical strength and the resilience of the prepared three-dimensional elastic carrier can be further improved, the collision force applied to enzyme molecules in the immobilization process is reduced and relieved, the normal conformation of the enzyme protein is maintained, and the loss of enzyme activity is reduced; stirring time is 4-24h, simultaneously, nitrogen is introduced to carry out ultrasonic nano micro aeration to obtain a micro-bead emulsion, compared with air, the introduction of nitrogen can prevent oxidation reaction, nano micro aeration is adopted to introduce nano nitrogen micro-bubbles into a mixed liquid phase, ultrasonic auxiliary diffusion is adopted, and a large number of communicating holes are formed on micro-bead particles by utilizing the occupying effect of the nano nitrogen micro-bubbles in the emulsification process at a lower temperature. The three-dimensional elastic carrier with developed porosity and uniform pore diameter is obtained; filtering the microbead emulsion by using a filter membrane, taking microbeads, and repeatedly washing the microbeads for 3-5 times by using deionized water with the volume ratio of 5 times to obtain wet microbeads; then, carrying out water absorption drying and freezing storage on the wet micro-beads to obtain the three-dimensional elastic carrier;
(2) activation of the three-dimensional elastic carrier: before use, deionized water with the volume ratio of 1-2 times and the temperature of 30-40 ℃ is added into the three-dimensional elastic carrier, and the temperature is kept for 3-10min, and then the three-dimensional elastic carrier is moistened and unfrozen; then adding 10-30ml/kg of 3-dimethylaminopropylamine, mechanically stirring for 60-90min at the temperature of 30-40 ℃ and the rotating speed of 60-80r/min, and filtering to obtain an activated three-dimensional elastic carrier; the activated three-dimensional elastic carrier can increase the adhesion force to enzyme.
(3) Immobilization of the enzyme: loading the activated three-dimensional elastic carrier, glucan-acetaldehyde and a cephalosporin acylase solution with the concentration of 250g/L into the multifunctional temperature-controlled water bath device according to the mass ratio of 60-80:3-4:20, adjusting the pH to 7.5-8.0, controlling the reaction temperature to 45-50 ℃, and ultrasonically stirring for 12-14h to obtain a cross-linked modified enzyme; the half-life of the cephalosporin acylase after cross-linking modification is prolonged by 4-6 times compared with that before modification;
(4) post-treatment of the cross-linking modified enzyme: washing the cross-linked modified enzyme with EMSA buffer solution with the pH value of 7.5-8.0 for three times, wherein each time is 1min, and filtering with a filter membrane to obtain the cross-linked modified enzyme; mixing the non-cross-linked modified enzyme and tea polyphenol with the concentration of 50-250mg/L according to the mass ratio of 1:2-3, transferring the mixture into a multifunctional temperature-controlled water bath device, gradually heating the mixture from room temperature to 55 ℃ at the heating speed of 2-4 ℃/h, carrying out auxiliary ultrasonic stirring at the rotating speed of 30-60r/min, carrying out suction filtration, and collecting solids to obtain the cephalosporin acylase immobilized enzyme; the tea polyphenol as a natural antioxidant can protect the cross-linking modified enzyme from being interfered and damaged by oxygen free radicals in the gradient temperature-rising heat treatment process, keep the activity of the cephalosporin acylase immobilized enzyme, and greatly improve the heat stability of the cephalosporin acylase immobilized enzyme after the gradient temperature-rising heat treatment.
Further, the composite oil phase solution comprises the following components in percentage by weight: 7-10% of carbon tetrachloride, 14-16% of isopropanol, 15-17% of silicone oil, 9-11% of diethyl ether, 1-2% of soybean isoflavone, 15-17% of turpentine, 1-3% of fluorocarbon surfactant and the balance of wood wax oil. The soybean isoflavone is used as an antioxidant, so that the autoxidation of oil phase components can be prevented, and the fluorocarbon surfactant can remarkably improve the dispersibility of the composite oil phase solution in the composite water phase solution, so that the uniformity of the prepared three-dimensional elastic carrier is improved.
Further, the composite aqueous phase solution comprises the following components in percentage by weight: 8-10% of polyamino acid, 9-13% of polyethylene glycol-polycarboxylic acid polymer, 14-16% of polyvinylpyrrolidone, 10-13% of polylactic acid, 3-5% of gelatin, 6-9% of gliadin, 1-3% of guar gum, 1-3% of furcellaran, 0.5-1% of guriposide and the balance of pure water. The geniposide serving as a curing agent can improve the stability of an emulsion system.
Further, the gradient advance method comprises the following steps: the composite oil phase solution and the composite water phase solution are respectively fed by 25 percent of the total mass of the composite oil phase solution and the composite water phase solution according to the mass ratio of 1:1 at 0min, 10min, 20min and 30 min. The feed amount is fed in batches, so that the emulsification efficiency of the composite oil phase solution and the composite water phase solution can be ensured
Further, the aeration amount of the nitrogen is 0.1-0.5L/min, if the aeration amount is too small, the uniform amount of microbubbles in the microbeads cannot be ensured, and if the aeration amount is too large, the communication holes caused by the occupation of the microbubbles are too large, so that the porosity of the microbeads is reduced.
Further, the multifunctional temperature control water bath device comprises a water bath tank, a liner tank, a base, an ultrasonic generator, a heater, a master controller, an L-shaped supporting arm, a stirring rod, a motor, an aeration pipe and a gas connector, wherein the water bath tank is fixedly connected to the upper surface of the base, an outer edge layer is arranged at the top opening of the liner tank, the outer edge layer is clamped at the top of the water bath tank, the liner tank is suspended at the inner central position of the water bath tank, the master controller is arranged at the inner central position of the base, the ultrasonic generator is electrically connected above the master controller, the transmitting end of the ultrasonic generator is connected with the bottom of the water bath tank, the heater is uniformly distributed at the bottom of the water bath tank and is electrically connected with the master controller, the vertical section of the L-shaped supporting arm is fixedly connected to the rear part of the base, the transverse section of the L-shaped supporting arm is positioned right above the liner tank, and the motor, the gas connector is fixedly connected below the transverse section of the L-shaped supporting arm and connected with an external gas source, the aeration pipe is connected below the gas connector, the upper end of the stirring rod penetrates through the aeration pipe and is connected with an output shaft of the motor, and the lower end of the stirring rod is provided with a stirring impeller.
Furthermore, the water bath comprises a first liquid storage box, a second liquid storage box and a condensate box, the first liquid storage box is arranged on the left side of the outer wall of the water bath, the second liquid storage box is arranged on the right side of the outer wall of the water bath, the condensate box is arranged on the front side of the outer wall of the water bath, the tops of the first liquid storage box and the second liquid storage box extend to the inner side wall of the inner container groove through the first rotary liquid inlet pipe, the condensate box extends to the inner side wall of the water bath through the second rotary liquid inlet pipe, micro metering pumps are respectively arranged on the first rotary liquid inlet pipe and the second rotary liquid inlet pipe, and the micro metering pumps are electrically connected with the master controller.
Furthermore, the vertical section of the L-shaped supporting arm is of a telescopic structure, so that the stirring and aeration device connected below can be conveniently lifted out of the inner container groove, and the inner container groove and internal objects can be separated out independently.
Compared with the prior art, the invention has the beneficial effects that:
(1) the three-dimensional elastic carrier is prepared by mixing the composite oil phase solution and the composite water phase solution at a lower temperature of 2-10 ℃, and compared with the traditional high temperature, the three-dimensional elastic carrier is subjected to an emulsification reaction at a lower temperature, so that the mechanical strength and the resilience of the prepared three-dimensional elastic carrier can be further improved, the collision force of enzyme molecules in the immobilization process is reduced and relieved, the normal conformation of enzyme protein is maintained, and the loss of enzyme activity is reduced.
(2) When the emulsification reaction occurs, a nano micro-aeration mode is adopted, nano nitrogen microbubbles are introduced into a mixed liquid phase, ultrasonic wave is used for assisting diffusion, and in the emulsification process at a lower temperature, a large number of communicating holes are formed on the microsphere particles by utilizing the occupation effect of the nano nitrogen microbubbles, so that a three-dimensional elastic carrier with developed porosity and uniform aperture can be obtained; can provide huge space for cross-linking modified enzyme.
(3) The half-life of cephalosporin acylase modified by the method of the invention is prolonged by 4-6 times compared with the half-life before modification.
In a word, the cephalosporin acylase immobilized enzyme prepared by the invention has the advantages of strong enzyme binding force, good stability, strong mechanical property, high enzyme activity, long half-life period and the like.
Drawings
FIG. 1 is a front sectional view of the multifunctional temperature controlled water bath apparatus of the present invention;
FIG. 2 is a right side view of the multifunctional temperature controlled water bath apparatus of the present invention.
The system comprises a water bath tank 1, a liner tank 2, a base 3, an ultrasonic generator 4, a heater 5, a master controller 6, a supporting arm 7-L, a stirring rod 8, a motor 9, an aerator pipe 10, an air connector 11, an outer edge layer 12, a stirring impeller 13, a liquid storage box I14, a liquid storage box II 15, a condenser box 16, a rotary liquid inlet pipe I17, a rotary liquid inlet pipe II 18 and a micro metering pump 19.
Detailed Description
The present invention will now be further described with reference to FIGS. 1-2 for a more complete explanation of the invention.
Example 1
A preparation method of cephalosporin acylase immobilized enzyme comprises the following steps:
(1) preparing a three-dimensional elastic carrier: independently filling the composite oil phase solution and the composite water phase solution into a multifunctional temperature-controlled water bath device according to the volume ratio of 1:1, wherein the composite oil phase solution comprises the following components in percentage by weight: 7% of carbon tetrachloride, 14% of isopropanol, 15% of silicone oil, 9% of diethyl ether, 1% of soybean isoflavone, 15% of turpentine, 1% of fluorocarbon surfactant and the balance of wood wax oil. The soybean isoflavone is used as an antioxidant, so that the autoxidation of oil phase components can be prevented, and the fluorocarbon surfactant can remarkably improve the dispersibility of the composite oil phase solution in the composite water phase solution, so that the uniformity of the prepared three-dimensional elastic carrier is improved. The composite aqueous phase solution comprises the following components in percentage by weight: 8% of polyamino acid, 9% of polyethylene glycol-polycarboxylic acid polymer, 14% of polyvinylpyrrolidone, 10% of polylactic acid, 3% of gelatin, 6% of gliadin, 1% of guar gum, 1% of furcellaran, 0.5% of guripod and the balance of pure water. The geniposide serving as a curing agent can improve the stability of an emulsion system. Carrying out gradient input stirring, wherein the gradient input method comprises the following steps: the composite oil phase solution and the composite water phase solution are respectively fed by 25 percent of the total mass of the composite oil phase solution and the composite water phase solution according to the mass ratio of 1:1 at 0min, 10min, 20min and 30 min. The emulsification efficiency of the composite oil phase solution and the composite water phase solution can be ensured by feeding in batches. The stirring speed is 400r/min, the temperature is 2 ℃, and compared with the traditional high temperature, the emulsion reaction is carried out at a lower temperature, so that the mechanical strength and the rebound resilience of the prepared three-dimensional elastic carrier can be further improved, the collision force of enzyme molecules in the immobilization process is reduced and relieved, the normal conformation of the enzyme protein is maintained, and the loss of enzyme activity is reduced; the stirring time is 4h, and simultaneously, nitrogen is introduced to carry out ultrasonic nano micro aeration to obtain the micro-bead emulsion, wherein the aeration rate of the nitrogen is 0.1L/min, the aeration rate is too small, so that the uniformity of the quantity of micro bubbles in the micro beads cannot be ensured, and the aeration rate is too large, so that the communication holes caused by micro bubble occupation are too large, and the porosity of the micro beads is reduced. Compared with the air, the nitrogen is introduced to prevent oxidation reaction, and the nano nitrogen microbubbles are introduced into the mixed liquid phase in a nano micro aeration mode to be subjected to ultrasonic wave-assisted diffusion so as to form a large number of communicating holes on the microsphere particles by utilizing the occupied effect of the nano nitrogen microbubbles in the emulsification process at a lower temperature. The three-dimensional elastic carrier with developed porosity and uniform pore diameter is obtained; filtering the microbead emulsion by using a filter membrane, taking microbeads, and repeatedly washing the microbeads for 3 times by using deionized water with the volume ratio of 5 times to obtain wet microbeads; then, carrying out water absorption drying and freezing storage on the wet micro-beads to obtain the three-dimensional elastic carrier;
(2) activation of the three-dimensional elastic carrier: before use, deionized water with the volume ratio of 1 time and the temperature of 30 ℃ is added into the three-dimensional elastic carrier, the temperature is kept for 3min, and the three-dimensional elastic carrier is moistened and unfrozen; then adding 3-dimethylamino propylamine with the dose of 10ml/kg, mechanically stirring for 60min at the temperature of 30 ℃ and the rotating speed of 60r/min, and filtering to obtain an activated three-dimensional elastic carrier; the activated three-dimensional elastic carrier can increase the adhesion force to enzyme.
(3) Immobilization of the enzyme: loading the activated three-dimensional elastic carrier, glucan-acetaldehyde and a cephalosporin acylase solution with the concentration of 250g/L into the multifunctional temperature-controlled water bath device according to the mass ratio of 60:3:20, adjusting the pH to 7.5, controlling the reaction temperature to be 45 ℃, and ultrasonically stirring for 12 hours to obtain a cross-linked modified enzyme; the half-life of the cephalosporin acylase after cross-linking modification is prolonged by 4 times compared with that before the modification;
(4) post-treatment of the cross-linking modified enzyme: washing the cross-linked modified enzyme with EMSA buffer solution with the pH of 7.5 for three times, wherein each time is 1min, and filtering through a filter membrane to obtain the cross-linked modified enzyme; mixing the non-cross-linked modified enzyme and 50mg/L tea polyphenol according to the mass ratio of 1:2, transferring the mixture into a multifunctional temperature-controlled water bath device, gradually heating the mixture from room temperature to 55 ℃ at the heating speed of 2 ℃/h, carrying out ultrasonic stirring at the rotation speed of 30r/min, carrying out suction filtration, and collecting solids to obtain the cephalosporin acylase immobilized enzyme; the tea polyphenol as a natural antioxidant can protect the cross-linking modified enzyme from being interfered and damaged by oxygen free radicals in the gradient temperature-rising heat treatment process, keep the activity of the cephalosporin acylase immobilized enzyme, and greatly improve the heat stability of the cephalosporin acylase immobilized enzyme after the gradient temperature-rising heat treatment.
As shown in fig. 1-2, the multifunctional temperature-controlled water bath device comprises a water bath 1, an inner container tank 2, a base 3, an ultrasonic generator 4, a heater 5, a master controller 6, an L-shaped supporting arm 7, a stirring rod 8, a motor 9, an aeration pipe 10 and a gas connector 11, wherein the water bath 1 is fixedly connected to the upper surface of the base 3, the water bath 1 comprises a first liquid storage box 14, a second liquid storage box 15 and a condenser box 16, the first liquid storage box 14 is arranged on the left side of the outer wall of the water bath 1, the second liquid storage box 15 is arranged on the right side of the outer wall of the water bath 1, the condenser box 16 is arranged on the front side of the outer wall of the water bath 1, the tops of the first liquid storage box 14 and the second liquid storage box 15 respectively extend to the inner side wall of the inner container tank 2 through a rotary liquid inlet pipe 17, the condenser box 16 extends to the inner side wall of the water bath 1 through a rotary liquid, the micro metering pump 19 is electrically connected with the master controller 6. The apical foramen of inner bag groove 2 is equipped with outer edge layer 12, outer edge layer 12 joint is at water bath 1 top, make inner bag groove 2 unsettled in water bath 1's inside central point put, total controller 6 sets up the inside central point at base 3 and puts, supersonic generator 4 electric connection is in the top of total controller 6, supersonic generator 4's transmitting terminal links to each other with water bath 1 bottom, heater 5 evenly distributed is in water bath 1 bottom, and with total controller 6 electric connection, L shape support arm 7's vertical section fixed connection is at base 3's rear portion, L shape support arm 7's vertical section is extending structure, the stirring and the aeration equipment that conveniently will connect in the below propose inner bag groove 2, and then separate out inner bag groove 2 and inside object alone. The horizontal segment of L shape support arm 7 is located inner bag groove 2 directly over, and motor 9 is fixed in the horizontal segment top of L shape support arm 7, and gas connector 11 fixed connection is in the horizontal segment below of L shape support arm 7, and gas connector 11 links to each other with outside air supply, and aeration pipe 10 is connected in gas connector 11's below, and aeration pipe 10 is run through to the upper end of puddler 8 and motor 9's output shaft links to each other, and the lower extreme of puddler 8 is equipped with impeller 13.
And (4) analyzing results: and (3) carrying out enzyme activity determination on the treated cephalosporin acylase immobilized enzyme, wherein the enzyme activity is 98U/g, and the half-life time is 80min under the conditions that the pH is 9.0 and the temperature is 45 ℃.
Example 2
A preparation method of cephalosporin acylase immobilized enzyme comprises the following steps:
(1) preparing a three-dimensional elastic carrier: independently filling the composite oil phase solution and the composite water phase solution into a multifunctional temperature-controlled water bath device according to the volume ratio of 1:1, wherein the composite oil phase solution comprises the following components in percentage by weight: 8.5 percent of carbon tetrachloride, 15 percent of isopropanol, 16 percent of silicone oil, 10 percent of diethyl ether, 1.5 percent of soybean isoflavone, 16 percent of turpentine, 2 percent of fluorocarbon surfactant and the balance of wood wax oil. The soybean isoflavone is used as an antioxidant, so that the autoxidation of oil phase components can be prevented, and the fluorocarbon surfactant can remarkably improve the dispersibility of the composite oil phase solution in the composite water phase solution, so that the uniformity of the prepared three-dimensional elastic carrier is improved. The composite aqueous phase solution comprises the following components in percentage by weight: 9% of polyamino acid, 10.5% of polyethylene glycol-polycarboxylic acid polymer, 15% of polyvinylpyrrolidone, 11.5% of polylactic acid, 4% of gelatin, 7.5% of gliadin, 2% of guar gum, 2% of furcellaran, 0.75% of guripod and the balance of pure water. The geniposide serving as a curing agent can improve the stability of an emulsion system. Carrying out gradient input stirring, wherein the gradient input method comprises the following steps: the composite oil phase solution and the composite water phase solution are respectively fed by 25 percent of the total mass of the composite oil phase solution and the composite water phase solution according to the mass ratio of 1:1 at 0min, 10min, 20min and 30 min. The emulsification efficiency of the composite oil phase solution and the composite water phase solution can be ensured by feeding in batches. The stirring speed is 1700r/min, the temperature is 4 ℃, and compared with the traditional high temperature, the emulsion reaction is carried out at a lower temperature, so that the mechanical strength and the rebound resilience of the prepared three-dimensional elastic carrier can be further improved, the collision force of enzyme molecules in the immobilization process is reduced and relieved, the normal conformation of the enzyme protein is maintained, and the loss of enzyme activity is reduced; the stirring time is 14 hours, meanwhile, nitrogen is introduced to carry out ultrasonic nano micro aeration to obtain the micro bead emulsion, the aeration amount of the nitrogen is 0.3L/min, the micro bubbles in the micro beads cannot be ensured to be uniform in quantity if the aeration amount is too small, and the communication holes caused by micro bubble occupation are too large if the aeration amount is too large, so that the porosity of the micro beads is reduced. Compared with the air, the nitrogen is introduced to prevent oxidation reaction, and the nano nitrogen microbubbles are introduced into the mixed liquid phase in a nano micro aeration mode to be subjected to ultrasonic wave-assisted diffusion so as to form a large number of communicating holes on the microsphere particles by utilizing the occupied effect of the nano nitrogen microbubbles in the emulsification process at a lower temperature. The three-dimensional elastic carrier with developed porosity and uniform pore diameter is obtained; filtering the microbead emulsion by using a filter membrane, taking microbeads, and repeatedly washing the microbeads for 4 times by using deionized water with the volume ratio of 5 times to obtain wet microbeads; then, carrying out water absorption drying and freezing storage on the wet micro-beads to obtain the three-dimensional elastic carrier;
(2) activation of the three-dimensional elastic carrier: before use, deionized water with the volume ratio of 2 times and the temperature of 35 ℃ is added into the three-dimensional elastic carrier, the temperature is kept for 6min, and the three-dimensional elastic carrier is moistened and unfrozen; then adding 20ml/kg of 3-dimethylamino propylamine, mechanically stirring for 75min at the temperature of 35 ℃ and the rotating speed of 70r/min, and filtering to obtain an activated three-dimensional elastic carrier; the activated three-dimensional elastic carrier can increase the adhesion force to enzyme.
(3) Immobilization of the enzyme: loading the activated three-dimensional elastic carrier, glucan-acetaldehyde and a cephalosporin acylase solution with the concentration of 250g/L into the multifunctional temperature-controlled water bath device according to the mass ratio of 70:3:20, adjusting the pH to 7.9, controlling the reaction temperature to 47 ℃, and ultrasonically stirring for 13 hours to obtain a cross-linked modified enzyme; the half-life of the cephalosporin acylase after cross-linking modification is prolonged by 6 times compared with that before the modification;
(4) post-treatment of the cross-linking modified enzyme: washing the cross-linked modified enzyme with EMSA buffer solution with the pH of 7.9 for three times, wherein each time is 1min, and filtering through a filter membrane to obtain the cross-linked modified enzyme; mixing the non-cross-linked modified enzyme and 150mg/L tea polyphenol according to the mass ratio of 1:2.5, transferring the mixture into a multifunctional temperature-controlled water bath device, gradually heating the mixture from room temperature to 55 ℃ at the heating speed of 3 ℃/h, carrying out ultrasonic stirring at the rotation speed of 45r/min, carrying out suction filtration, and collecting solids to obtain the cephalosporin acylase immobilized enzyme; the tea polyphenol as a natural antioxidant can protect the cross-linking modified enzyme from being interfered and damaged by oxygen free radicals in the gradient temperature-rising heat treatment process, keep the activity of the cephalosporin acylase immobilized enzyme, and greatly improve the heat stability of the cephalosporin acylase immobilized enzyme after the gradient temperature-rising heat treatment.
As shown in fig. 1-2, the multifunctional temperature-controlled water bath device comprises a water bath 1, an inner container tank 2, a base 3, an ultrasonic generator 4, a heater 5, a master controller 6, an L-shaped supporting arm 7, a stirring rod 8, a motor 9, an aeration pipe 10 and a gas connector 11, wherein the water bath 1 is fixedly connected to the upper surface of the base 3, the water bath 1 comprises a first liquid storage box 14, a second liquid storage box 15 and a condenser box 16, the first liquid storage box 14 is arranged on the left side of the outer wall of the water bath 1, the second liquid storage box 15 is arranged on the right side of the outer wall of the water bath 1, the condenser box 16 is arranged on the front side of the outer wall of the water bath 1, the tops of the first liquid storage box 14 and the second liquid storage box 15 respectively extend to the inner side wall of the inner container tank 2 through a rotary liquid inlet pipe 17, the condenser box 16 extends to the inner side wall of the water bath 1 through a rotary liquid, the micro metering pump 19 is electrically connected with the master controller 6. The apical foramen of inner bag groove 2 is equipped with outer edge layer 12, outer edge layer 12 joint is at water bath 1 top, make inner bag groove 2 unsettled in water bath 1's inside central point put, total controller 6 sets up the inside central point at base 3 and puts, supersonic generator 4 electric connection is in the top of total controller 6, supersonic generator 4's transmitting terminal links to each other with water bath 1 bottom, heater 5 evenly distributed is in water bath 1 bottom, and with total controller 6 electric connection, L shape support arm 7's vertical section fixed connection is at base 3's rear portion, L shape support arm 7's vertical section is extending structure, the stirring and the aeration equipment that conveniently will connect in the below propose inner bag groove 2, and then separate out inner bag groove 2 and inside object alone. The horizontal segment of L shape support arm 7 is located inner bag groove 2 directly over, and motor 9 is fixed in the horizontal segment top of L shape support arm 7, and gas connector 11 fixed connection is in the horizontal segment below of L shape support arm 7, and gas connector 11 links to each other with outside air supply, and aeration pipe 10 is connected in gas connector 11's below, and aeration pipe 10 is run through to the upper end of puddler 8 and motor 9's output shaft links to each other, and the lower extreme of puddler 8 is equipped with impeller 13.
And (4) analyzing results: and (3) carrying out enzyme activity determination on the treated cephalosporin acylase immobilized enzyme, wherein the enzyme activity is 121U/g, and the half-life time is 120min under the conditions that the pH is 9.0 and the temperature is 45 ℃.
Example 3
A preparation method of cephalosporin acylase immobilized enzyme comprises the following steps:
(1) preparing a three-dimensional elastic carrier: independently filling the composite oil phase solution and the composite water phase solution into a multifunctional temperature-controlled water bath device according to the volume ratio of 1:1, wherein the composite oil phase solution comprises the following components in percentage by weight: 10% of carbon tetrachloride, 16% of isopropanol, 17% of silicone oil, 11% of diethyl ether, 2% of soybean isoflavone, 17% of turpentine, 3% of fluorocarbon surfactant and the balance of wood wax oil. The soybean isoflavone is used as an antioxidant, so that the autoxidation of oil phase components can be prevented, and the fluorocarbon surfactant can remarkably improve the dispersibility of the composite oil phase solution in the composite water phase solution, so that the uniformity of the prepared three-dimensional elastic carrier is improved. The composite aqueous phase solution comprises the following components in percentage by weight: 10% of polyamino acid, 13% of polyethylene glycol-polycarboxylic acid polymer, 16% of polyvinylpyrrolidone, 13% of polylactic acid, 5% of gelatin, 9% of gliadin, 3% of guar gum, 3% of furcellaran, 1% of guripod and the balance of pure water. The geniposide serving as a curing agent can improve the stability of an emulsion system. Carrying out gradient input stirring, wherein the gradient input method comprises the following steps: the composite oil phase solution and the composite water phase solution are respectively fed by 25 percent of the total mass of the composite oil phase solution and the composite water phase solution according to the mass ratio of 1:1 at 0min, 10min, 20min and 30 min. The emulsification efficiency of the composite oil phase solution and the composite water phase solution can be ensured by feeding in batches. The stirring speed is 3000r/min, the temperature is 10 ℃, and compared with the traditional high temperature, the emulsion reaction is carried out at a lower temperature, so that the mechanical strength and the rebound resilience of the prepared three-dimensional elastic carrier can be further improved, the collision force of enzyme molecules in the immobilization process is reduced and relieved, the normal conformation of the enzyme protein is maintained, and the loss of enzyme activity is reduced; the stirring time is 24 hours, meanwhile, nitrogen is introduced to carry out ultrasonic nano micro aeration to obtain the micro bead emulsion, the aeration amount of the nitrogen is 0.5L/min, the micro bubbles in the micro beads cannot be ensured to be uniform in quantity if the aeration amount is too small, and the communication holes caused by micro bubble occupation are too large if the aeration amount is too large, so that the porosity of the micro beads is reduced. Compared with the air, the nitrogen is introduced to prevent oxidation reaction, and the nano nitrogen microbubbles are introduced into the mixed liquid phase in a nano micro aeration mode to be subjected to ultrasonic wave-assisted diffusion so as to form a large number of communicating holes on the microsphere particles by utilizing the occupied effect of the nano nitrogen microbubbles in the emulsification process at a lower temperature. The three-dimensional elastic carrier with developed porosity and uniform pore diameter is obtained; filtering the microbead emulsion by using a filter membrane, taking microbeads, and repeatedly washing the microbeads for 5 times by using deionized water with the volume ratio of 5 times to obtain wet microbeads; then, carrying out water absorption drying and freezing storage on the wet micro-beads to obtain the three-dimensional elastic carrier;
(2) activation of the three-dimensional elastic carrier: before use, deionized water with the volume ratio of 2 times and the temperature of 40 ℃ is added into the three-dimensional elastic carrier, the temperature is kept for 10min, and the three-dimensional elastic carrier is moistened and unfrozen; then adding 3-dimethylamino propylamine with the dose of 30ml/kg, mechanically stirring for 90min at the temperature of 40 ℃ at the rotating speed of 80r/min, and filtering to obtain an activated three-dimensional elastic carrier; the activated three-dimensional elastic carrier can increase the adhesion force to enzyme.
(3) Immobilization of the enzyme: loading the activated three-dimensional elastic carrier, glucan-acetaldehyde and a cephalosporin acylase solution with the concentration of 250g/L into the multifunctional temperature-controlled water bath device according to the mass ratio of 80:4:20, adjusting the pH to 8.0, controlling the reaction temperature to be 50 ℃, and ultrasonically stirring for 14h to obtain a cross-linked modified enzyme; the half-life of the cephalosporin acylase after cross-linking modification is prolonged by 5 times compared with that before the modification;
(4) post-treatment of the cross-linking modified enzyme: washing the cross-linked modified enzyme with EMSA buffer solution with the pH value of 8.0 for three times, wherein each time is 1min, and filtering through a filter membrane to obtain the cross-linked modified enzyme; mixing the non-cross-linked modified enzyme and 250mg/L tea polyphenol according to the mass ratio of 1:3, transferring the mixture into a multifunctional temperature-controlled water bath device, gradually heating the mixture from room temperature to 55 ℃ at the heating speed of 4 ℃/h, carrying out ultrasonic stirring at the rotating speed of 60r/min, carrying out suction filtration, and collecting solids to obtain the cephalosporin acylase immobilized enzyme; the tea polyphenol as a natural antioxidant can protect the cross-linking modified enzyme from being interfered and damaged by oxygen free radicals in the gradient temperature-rising heat treatment process, keep the activity of the cephalosporin acylase immobilized enzyme, and greatly improve the heat stability of the cephalosporin acylase immobilized enzyme after the gradient temperature-rising heat treatment.
As shown in fig. 1-2, the multifunctional temperature-controlled water bath device comprises a water bath 1, an inner container tank 2, a base 3, an ultrasonic generator 4, a heater 5, a master controller 6, an L-shaped supporting arm 7, a stirring rod 8, a motor 9, an aeration pipe 10 and a gas connector 11, wherein the water bath 1 is fixedly connected to the upper surface of the base 3, the water bath 1 comprises a first liquid storage box 14, a second liquid storage box 15 and a condenser box 16, the first liquid storage box 14 is arranged on the left side of the outer wall of the water bath 1, the second liquid storage box 15 is arranged on the right side of the outer wall of the water bath 1, the condenser box 16 is arranged on the front side of the outer wall of the water bath 1, the tops of the first liquid storage box 14 and the second liquid storage box 15 respectively extend to the inner side wall of the inner container tank 2 through a rotary liquid inlet pipe 17, the condenser box 16 extends to the inner side wall of the water bath 1 through a rotary liquid, the micro metering pump 19 is electrically connected with the master controller 6. The apical foramen of inner bag groove 2 is equipped with outer edge layer 12, outer edge layer 12 joint is at water bath 1 top, make inner bag groove 2 unsettled in water bath 1's inside central point put, total controller 6 sets up the inside central point at base 3 and puts, supersonic generator 4 electric connection is in the top of total controller 6, supersonic generator 4's transmitting terminal links to each other with water bath 1 bottom, heater 5 evenly distributed is in water bath 1 bottom, and with total controller 6 electric connection, L shape support arm 7's vertical section fixed connection is at base 3's rear portion, L shape support arm 7's vertical section is extending structure, the stirring and the aeration equipment that conveniently will connect in the below propose inner bag groove 2, and then separate out inner bag groove 2 and inside object alone. The horizontal segment of L shape support arm 7 is located inner bag groove 2 directly over, and motor 9 is fixed in the horizontal segment top of L shape support arm 7, and gas connector 11 fixed connection is in the horizontal segment below of L shape support arm 7, and gas connector 11 links to each other with outside air supply, and aeration pipe 10 is connected in gas connector 11's below, and aeration pipe 10 is run through to the upper end of puddler 8 and motor 9's output shaft links to each other, and the lower extreme of puddler 8 is equipped with impeller 13.
And (4) analyzing results: and (3) carrying out enzyme activity determination on the treated cephalosporin acylase immobilized enzyme, wherein the enzyme activity is 105U/g, and the half-life time is 100min under the conditions that the pH value is 9.0 and the temperature is 45 ℃.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (4)
1. A preparation method of cephalosporin acylase immobilized enzyme is characterized by comprising the following steps:
(1) preparing a three-dimensional elastic carrier: independently filling the composite oil phase solution and the composite water phase solution into a multifunctional temperature-controlled water bath device according to the volume ratio of 1:1, performing gradient input stirring at the stirring speed of 400-3000r/min, the temperature of 2-10 ℃, the stirring time of 4-24h, and simultaneously introducing nitrogen gas to perform ultrasonic nano micro aeration to obtain a microbead emulsion; filtering the microbead emulsion by using a filter membrane, taking microbeads, and repeatedly washing the microbeads for 3-5 times by using deionized water with the volume ratio of 5 times to obtain wet microbeads; then, carrying out water absorption drying and freezing storage on the wet micro-beads to obtain the three-dimensional elastic carrier;
(2) activation of the three-dimensional elastic carrier: before use, deionized water with the volume ratio of 1-2 times and the temperature of 30-40 ℃ is added into the three-dimensional elastic carrier, and the temperature is kept for 3-10min, and then the three-dimensional elastic carrier is moistened and unfrozen; then adding 10-30ml/kg of 3-dimethylaminopropylamine, mechanically stirring for 60-90min at the temperature of 30-40 ℃ and the rotating speed of 60-80r/min, and filtering to obtain an activated three-dimensional elastic carrier;
(3) immobilization of the enzyme: loading the activated three-dimensional elastic carrier, glucan-acetaldehyde and a cephalosporin acylase solution with the concentration of 250g/L into the multifunctional temperature-controlled water bath device according to the mass ratio of 60-80:3-4:20, adjusting the pH to 7.5-8.0, controlling the reaction temperature to 45-50 ℃, and ultrasonically stirring for 12-14h to obtain a cross-linked modified enzyme;
(4) post-treatment of the cross-linking modified enzyme: washing the cross-linked modified enzyme with EMSA buffer solution with the pH value of 7.5-8.0 for three times, wherein each time is 1min, and filtering with a filter membrane to obtain the cross-linked modified enzyme; mixing the non-cross-linked modified enzyme and tea polyphenol with the concentration of 50-250mg/L according to the mass ratio of 1:2-3, transferring the mixture into a multifunctional temperature-controlled water bath device, gradually heating the mixture from room temperature to 55 ℃ at the heating speed of 2-4 ℃/h, carrying out auxiliary ultrasonic stirring at the rotating speed of 30-60r/min, carrying out suction filtration, and collecting solids to obtain the cephalosporin acylase immobilized enzyme;
the composite oil phase solution comprises the following components in percentage by weight: 7-10% of carbon tetrachloride, 14-16% of isopropanol, 15-17% of silicone oil, 9-11% of diethyl ether, 1-2% of soybean isoflavone, 15-17% of turpentine, 1-3% of fluorocarbon surfactant and the balance of wood wax oil;
the composite aqueous phase solution comprises the following components in percentage by weight: 8-10% of polyamino acid, 9-13% of polyethylene glycol-polycarboxylic acid polymer, 14-16% of polyvinylpyrrolidone, 10-13% of polylactic acid, 3-5% of gelatin, 6-9% of gliadin, 1-3% of guar gum, 1-3% of furcellaran, 0.5-1% of guriposide and the balance of pure water;
the gradient input method comprises the following steps: the composite oil phase solution and the composite water phase solution are respectively fed by 25 percent of the total mass of the composite oil phase solution and the composite water phase solution according to the mass ratio of 1:1 at 0min, 10min, 20min and 30 min;
the gradient input method comprises the following steps: feeding the composite oil phase solution and the composite water phase solution according to the mass ratio of 1:1, wherein 25% of the total mass of the composite oil phase solution and the composite water phase solution is fed at 0 min; feeding 25% of the total mass of the composite oil phase solution and the composite water phase solution in 10 min; at 20min, feeding 25% of the total mass of the composite oil phase solution and the composite water phase solution; at the time of 30min, 25% of the total mass of the complex oil phase solution and the complex water phase solution was fed.
2. The preparation method of cephalosporin acylase immobilized enzyme as claimed in claim 1, characterized in that the multifunctional temperature-controlled water bath device comprises a water bath tank (1), an inner container tank (2), a base (3), an ultrasonic generator (4), a heater (5), a master controller (6), an L-shaped supporting arm (7), a stirring rod (8), a motor (9), an aeration pipe (10) and a gas connector (11), wherein the water bath tank (1) is fixedly connected with the upper surface of the base (3), the top opening of the inner container tank (2) is provided with an outer edge layer (12), the outer edge layer (12) is clamped at the top of the water bath tank (1) to suspend the inner container tank (2) at the inner center of the water bath tank (1), the master controller (6) is arranged at the inner center of the base (3), the ultrasonic generator (4) is electrically connected above the master controller (6), the ultrasonic generator (4) is connected with the bottom of the water bath (1), the heaters (5) are uniformly distributed at the bottom of the water bath (1) and are electrically connected with the master controller (6), the vertical section of the L-shaped supporting arm (7) is fixedly connected to the rear part of the base (3), the transverse section of the L-shaped supporting arm (7) is positioned right above the inner liner (2), the motor (9) is fixed above the transverse section of the L-shaped supporting arm (7), the gas connector (11) is fixedly connected below the transverse section of the L-shaped supporting arm (7), the gas connector (11) is connected with an external gas source, the aeration pipe (10) is connected below the gas connector (11), the upper end of the stirring rod (8) penetrates through the output shaft of the aeration pipe (10) and is connected with the motor (9), and the lower end of the stirring rod (8) is provided with a stirring impeller (13).
3. The process for producing a cephalosporin acylase immobilized enzyme according to claim 2, the water bath (1) comprises a first liquid storage box (14), a second liquid storage box (15) and a condensate box (16), the first liquid storage box (14) is arranged on the left side of the outer wall of the water bath (1), the second liquid storage box (15) is arranged on the right side of the outer wall of the water bath (1), the condenser box (16) is arranged on the front side of the outer wall of the water bath (1), the tops of the first liquid storage box (14) and the second liquid storage box (15) respectively extend to the inner side wall of the inner container groove (2) through the first rotary liquid inlet pipe (17), the condenser box (16) extends to the inner side wall of the water bath (1) through the second rotary liquid inlet pipe (18), the first rotary liquid inlet pipe (17) and the second rotary liquid inlet pipe (18) are respectively provided with a micro metering pump (19), the miniature metering pump (19) is electrically connected with the master controller (6).
4. The process for preparing a cephalosporin acylase immobilized enzyme according to claim 2 characterized in that the vertical section of the L-shaped support arm (7) is a telescopic structure.
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