CN111304189A - Enzyme-loaded calcium alginate microsphere enhanced cascade enzymatic reaction method based on aqueous two-phase system - Google Patents
Enzyme-loaded calcium alginate microsphere enhanced cascade enzymatic reaction method based on aqueous two-phase system Download PDFInfo
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
- C12Y101/03004—Glucose oxidase (1.1.3.4)
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Abstract
The invention discloses an enzyme-loaded calcium alginate microsphere enhanced cascade enzymatic reaction method based on a two-aqueous-phase system, which comprises the following steps: step 1: respectively preparing a PEG aqueous solution with the mass concentration of 8% and a Dex aqueous solution with the mass concentration of 8%; fully mixing and then carrying out phase separation, wherein the upper phase is a PEG solution, and the lower phase is a Dex solution; step 2: adding sodium alginate and a target enzyme solution into the Dex solution to form a Dex-sodium alginate solution containing the target enzyme, wherein the mass concentration of the sodium alginate is 0.5%; and step 3: taking the PEG solution prepared in the step 1 as an external aqueous phase, taking the Dex-sodium alginate solution containing the target enzyme obtained in the step 2 as an internal aqueous phase, and forming a double aqueous phase sodium alginate liquid drop at the conical tip of the coaxial capillary; and 4, step 4: the double-aqueous-phase sodium alginate droplets are led into a PEG-calcium chloride aqueous solution to generate the enzyme-loaded calcium alginate microspheres, and the enzymatic reaction is carried out on the interface of the calcium alginate microspheres by spontaneous liquid discharge partition, so that the mass transfer distance of an intermediate product is shortened, and the cascade enzymatic reaction is strengthened. The invention constructs a cascade enzymatic reaction strengthening system with simple preparation method, high biocompatibility and high efficiency.
Description
Technical Field
The invention relates to the technical field of enhanced enzymatic reaction, in particular to an enzyme-loaded calcium alginate microsphere enhanced cascade enzymatic reaction method based on a two-aqueous-phase system.
Background
In recent years, with the increasing development of the enzyme industry, studies for enhancing enzymatic reactions have received increasing attention. Currently, immobilized enzyme technology has been available to enhance enzymatic reactions by enhancing enzyme stability and recycling efficiency. The calcium alginate microspheres are common immobilized enzyme carriers due to the characteristics of good biocompatibility, no toxicity and the like. However, the mass transfer rate in the ordinary enzyme-loaded calcium alginate microspheres is slow, the retention rate of the enzyme is low, and oil phase and surfactant are introduced in the preparation process of most calcium alginate microspheres, so that the biocompatibility of the microspheres can be reduced (Marcoux, Colloids and surfaces A: physical and Engineering industries, 2011,377,54), the technical cost of the immobilized enzyme is greatly lost, and the enzyme activity is lost in the immobilization process.
The double water phase catalysis technology is another novel intensified enzymatic reaction technology. It uses two aqueous solutions of different high polymers or one high polymer and one inorganic salt to form two immiscible liquid phase systems due to their different concentrations. The concentration is adjusted to distribute reactants and biocatalysts in the lower phase and products in the upper phase, so as to realize the coupling of biological reaction and product separation. The method is characterized in that cefalexin is synthesized in a double aqueous phase system consisting of PEG 400/magnesium sulfate by utilizing penicillin G acylase as a catalyst, and the enzymatic reaction efficiency can be improved (Wei Dong Zhi, Zhu Jiang, Cao Xue Jun. enzymatic synthesis cefalexin in aqueous two-phase systems [ J ]. Biochemical engineering-ing Journal,2002,11: 95-99.); but the reagents used are expensive. Zhuangyan et al synthesized cephalosporin IV by a two-aqueous phase biotransformation method to increase the yield of cephalosporin IV (Zhu Jian, Wei DongZhi, Ye Qi. partial dehavior of ceohalexin and 7-aminodeacetoxyphalosporanic acid in PEG/amo-nium sulfate aqueous phase of Chemical-pharmaceutical systems [ J ] journal of Chemical Technology Biotechnology, 2001,76(11): 1194-.
Both of the above techniques have achieved certain effects in enhancing enzymatic reactions, but due to their own property limitations, there are many disadvantages to their use alone. The laboratory preliminary study finds that the Double-water Phase system has stronger retention property to specific enzymes and proteins (Meng S.X., Xue L.H., Xie C.Y., Bai R.X., Yang X., Wang Y.L, Meng T. Enhanced enzyme Reaction by Y Aqueous Two-Phase Systems using paralleliaminar Flow in a Double Y-Branched Microfluidic device. chemical engineering Journal (SCI, IF 8.355),2018,335: 392-; meanwhile, the special water phase partition formed by the double-water phase system can enhance the mass transfer efficiency of the enzyme and the substrate at the double-water phase interface, thereby enhancing the enzymatic reaction and being convenient for recovery. Therefore, the development of the green and mild double-aqueous-phase enzymatic strengthening method with high catalytic efficiency based on the combination of the double-aqueous-phase system and the calcium alginate microspheres has important application value.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides an enzyme-loaded calcium alginate microsphere enhanced cascade enzymatic reaction method based on a two-aqueous-phase system, which has high flux, good biocompatibility and enhanced enzymatic reaction.
The technical scheme adopted by the invention is as follows:
a preparation method of enzyme-loaded calcium alginate microspheres based on an aqueous two-phase system comprises the following steps:
step 1: respectively preparing a PEG aqueous solution with the mass concentration of 8% and a Dex aqueous solution with the mass concentration of 8%; fully mixing and then carrying out phase separation, wherein the upper phase is a PEG solution, and the lower phase is a Dex solution;
step 2: adding sodium alginate and a target enzyme solution into the Dex solution to form a Dex-sodium alginate solution containing the target enzyme, wherein the mass concentration of the sodium alginate is 0.5%;
and step 3: taking the PEG solution prepared in the step 1 as an external aqueous phase, taking the Dex-sodium alginate solution containing the target enzyme obtained in the step 2 as an internal aqueous phase, and forming a double aqueous phase sodium alginate liquid drop at the conical tip of the coaxial capillary;
and 4, step 4: and (4) introducing the aqueous two-phase sodium alginate droplets obtained in the step (3) into a PEG-calcium chloride aqueous solution to generate the enzyme-loaded calcium alginate microspheres.
A double aqueous phase system-based enzyme-loaded calcium alginate microsphere enhanced cascade enzymatic reaction method is characterized in that enzyme-loaded calcium alginate microspheres obtained in step 4 are added into a PEG-glucose solution to generate a cascade enzymatic reaction.
Further, in the step 1, the PEG aqueous solution and the Dex aqueous solution are fully mixed through a rotary incubator, and phase separation is carried out after the mixture is kept still for 12 hours.
Further, the target enzymes in the step 2 are glucose oxidase and horseradish peroxidase; in the Dex-sodium alginate solution of the target enzyme, the concentration of glucose oxidase is 5 mug/mL, the concentration of horseradish peroxidase is 5 mug/mL, the concentrations of the glucose oxidase and the horseradish peroxidase are both 5 mug/mL or the concentrations of the glucose oxidase and the horseradish peroxidase are both 2.5 mug/mL.
Furthermore, in the step 3, the external water phase and the internal water phase are injected by an air pump, the injection pressure of the external water phase is 0.030MPa, and the injection pressure of the internal water phase is 0.015-0.030 MPa; and in the process of sampling the internal water phase, starting an air pump for 0.2s, and closing the air pump for 1.5s to serve as a sampling period.
Further, the diameter range of the enzyme-loaded calcium alginate microspheres in the step 4 is 200-400 microns.
Further, the PEG-calcium chloride aqueous solution in step 4 is formed by adding calcium chloride to the PEG solution in step 1, wherein the concentration of the calcium chloride is 10% by mass.
Further, the PEG-glucose solution is formed by adding glucose into the PEG solution in the step 1, wherein the concentration of the glucose is 23.25 mmol/L.
The invention has the beneficial effects that:
(1) the preparation method is simple, easy to adjust, low in material consumption and good in biocompatibility in the whole process;
(2) the calcium alginate microspheres obtained by the invention strengthen the cascade enzymatic reaction, and because the calcium alginate microspheres have large specific surface area and short mass transfer distance, the enzyme-loaded microspheres can resist mechanical shearing force with certain strength and can effectively strengthen the cascade enzymatic reaction;
(3) the method can be used in the fields of food industry, biochemical engineering and enzyme engineering; a system which has simple preparation method, high biocompatibility and short mass transfer distance and can efficiently strengthen the cascade enzymatic reaction is constructed.
Drawings
FIG. 1 is a schematic view showing the spontaneous "liquid discharge" phenomenon of the calcium alginate microspheres in example 1 of the present invention.
FIG. 2 is a statistical chart of "liquid discharge" at regular time in example 1 of the present invention.
FIG. 3 is a diagram of the experimental mechanism of the present invention.
FIG. 4 is a diagram of A, B, C showing three different encapsulation modes in example 2 of the present invention.
FIG. 5 shows O in example 2 of the present invention2Graph of concentration versus time.
FIG. 6 shows O in example 2 of the present invention2Graph of concentration versus time.
FIG. 7 shows A, B, C three groups of O in example 2 of the present invention2Concentration value comparison chart.
Detailed description of the invention
The invention is further described with reference to the following figures and specific embodiments.
Before preparing the enzyme-loaded calcium alginate microspheres, whether the enzyme-loaded calcium alginate microspheres are feasible or not is firstly verified, and the method is carried out according to the following steps:
step 1: preparation of aqueous two-phase system
Respectively preparing a polyethylene glycol aqueous solution with the mass fraction of 8% (w/w) and a glucan aqueous solution with the mass fraction of 8% (w/w); after fully mixing by a rotary incubatorStanding for 12 hours, and then splitting phases; wherein the upper phase is polyethylene glycol solution as external water phase, and the lower phase is dextran solution; adding sodium alginate into the lower phase, mixing to obtain dextran + sodium alginate aqueous solution with mass fraction of 0.5% as internal water phase, and receiving solution with mass fraction of 10% CaCl2-PEG solution.
Step 2: punching calcium alginate microspheres with different sizes in microfluidic device, and observing liquid discharge phenomenon
Connecting the aqueous phase prepared in the step 1 with a coaxial capillary device, and injecting samples of the internal aqueous phase and the external aqueous phase through an air pump; the external water phase sample injection pressure is 0.030 MPa; and in the process of sampling the internal water phase, an air pump is started for 0.2s, and is closed for 1.5s to serve as a sampling period, wherein the sampling pressure is 0.015-0.030 MPa. The reaction temperature is 25 ℃, the diameter range of the calcium alginate microspheres is 200-400 mu m by adjusting the air pressure of the internal water phase, and the spontaneous liquid discharge time is 4h through observation and test, as shown in figure 1.
Then preparing a double aqueous phase system containing the target enzyme
Preparing a Tris-HCl buffer solution with the pH value of 7.4 and the concentration of 1mg/mL, preparing glucose oxidase GOX and horseradish peroxidase HRP solutions with the pH value of 5 mug/mL and a Dex + sodium alginate solution with the concentrations of 5 mug/mL and 2.5 mug/mL by using the buffer solution respectively. The target enzyme solution was divided into three groups: group A is Dex + sodium alginate solution in which the two enzymes (GOX/HRP) are dissolved simultaneously and the concentration of the two enzymes is 5 mug/mL; the group B is Dex + sodium alginate solution dissolved with 5 mug/mL glucose oxidase GOX and horse radish peroxidase HRP respectively; group C had both enzymes GOX/HRP dissolved in a 2.5. mu.g/mL solution of Dex + sodium alginate.
And (3) preparing the three groups of target enzyme solutions into calcium alginate microspheres with uniform sizes under the same conditions, reacting the calcium alginate microspheres with a glucose substrate with the concentration of 23.25mmol/L according to a certain group, adding a color-developing agent (Amplex Red) with the concentration of 0.5mg/mL, and observing and analyzing the result.
Example 1
The observation of spontaneous liquid discharge phenomenon of calcium alginate microspheres prepared based on a double aqueous phase system comprises the following steps:
(1) Respectively weighing 1.6g of PEG8kDa and 1.6g of Dex 500kDa by using a precision electronic balance, and putting the weighed materials into the same 20mL screw-top glass bottle; then, 16.8mL of deionized water was added thereto using a pipette to prepare a mixed aqueous solution having mass fractions of both solutes (Dex 500kDa and PEG8kDa) of 8%. Placing on a rotary stirrer, slowly rotating, mixing and dissolving. When the solute is fully dissolved, forming 20mL aqueous two-phase solution with the mass fraction of 8%; standing for 24h to allow them to separate, and using a syringe to extract the thinner PEG phase at the upper layer and the thicker Dex phase at the lower layer. Stored in two new screw bottles for use.
(2) And weighing sodium alginate solid with the mass fraction of 0.5% by using a precision electronic balance, dissolving the sodium alginate solid in the prepared Dex solution, fully dissolving the sodium alginate solid on a rotary stirrer, standing and defoaming the sodium alginate solid for later use, and using the sodium alginate solid as an internal phase sample injection solution of the microchannel device. Weighing several anhydrous calcium chloride with a precision electronic balance, dissolving in the obtained PEG solution, and making into 20 mg/mL-1 CaCl2The solution is used as a collection of micro-droplets.
Step 2: punching calcium alginate microspheres with different sizes in microfluidic device, and observing liquid discharge phenomenon
(1) And (3) taking the PEG solution prepared in the step (1) and the Dex solution dissolved with the sodium alginate, regulating and controlling through a nitrogen pump, and injecting into the external phase sample injection capillary and the internal phase sample injection capillary of the device respectively.
(2) And (4) starting a nitrogen pump, setting the air pressure connected with the PEG phase to be 0.030MPa, and continuously feeding samples. The pressure intensity of the gas pressure connected with the Dex phase is 0.015-0.030 MPa, the intermittent sample introduction is carried out, and the gas pressure pump is closed for 1.5s every time when the gas pressure pump is started for 0.2 s. The PEG phase in the outer tube was first passed through, followed by the Dex phase in the inner tube. The generation process of the liquid drops in the micro-channel is observed in real time through an optical microscope, and a beaker filled with collecting liquid is placed to collect the liquid drops at the outlet of the capillary tube.
(3) After the liquid drops in the micro-channel are generated continuously and stably, shooting a video of the liquid drop generation process from an optical microscope; and gradually moving the glass slide along with the generation of the liquid drops, observing the change of the form in the liquid drop generation process, taking pictures, and arranging the pictures into a picture group for analysis.
(4) After the double-water-phase liquid drops are generated smoothly in the micro-liquid-drop channel device, a surface dish filled with a collected-phase solution is prevented from being arranged at the outlet of the device, and the output liquid is collected, so that the sodium alginate in the double-water-phase liquid drops and the Ca in the collected liquid2+The reaction produces calcium alginate. The liquid drops are stabilized into calcium alginate gel microspheres with uniform size. In the collecting process, the surface dish needs to be slightly shaken to disperse and settle the microspheres, so that the microspheres are prevented from being stacked and adhered. Standing for 5min after collection to settle and stabilize the microspheres, and observing the surface appearance of the microspheres under an optical microscope.
(5) After the droplet flow microchannel device is installed, the nitrogen pump is opened, the air pressure intensity of the PEG external phase is set to be 0.030MPa, and the air pressure intensity of the Dex internal phase is adjusted to be 0.015MPa, 0.018MPa, 0.021MPa, 0.024MPa, 0.0271MPa and 0.030MPa respectively. Sequentially preparing the calcium alginate gel microspheres under the air pressure condition, collecting the calcium alginate gel microspheres in a surface dish, and observing the calcium alginate gel microspheres under an optical microscope after the calcium alginate gel microspheres are settled and stabilized for 5 min. The diameter of the calcium alginate microspheres is within the range of 200-400 mu m by adjusting the air pressure of the internal water phase at the temperature of 25 ℃.
(6) And after the calcium alginate in the receiving liquid is stable, taking pictures every 10min in the first 30min to record the morphological change of the local specific microspheres, and then taking pictures every 30min to record the morphological change of the local specific microspheres. The spontaneous "drainage" time of the calcium alginate microspheres was determined to be stable after 4h, and the results are shown in fig. 1 and 2.
Example 2
An enzyme-loaded calcium alginate microsphere enhanced cascade enzymatic reaction method based on an aqueous two-phase system comprises the following steps:
step 1: preparation of aqueous two-phase system
PEG8kDa and Dex 500kDa of 1.6g each are weighed by a precision electronic balance and put into the same 20mL screw glass bottle. Then, 16.8mL of deionized water was added thereto using a pipette to prepare a mixed aqueous solution having 8% mass fractions of both solutes (Dex 500kDa and PEG8kDa), which was placed on a rotary stirrer and dissolved by slow rotary mixing. And (3) after the solute is fully dissolved to form 20mL of double aqueous phase solution with the mass fraction of 8%, standing for 24h to separate the two phases, and respectively extracting the thinner PEG phase at the upper layer and the thicker Dex phase at the lower layer by using a syringe. And stored in two new screw bottles for later use.
And weighing sodium alginate solid with the mass fraction of 0.5% by using a precision electronic balance. Dissolving in the Dex solution, dissolving on a rotary stirrer to obtain Dex-sodium alginate solution, standing, and defoaming to obtain internal phase sample solution of the microchannel device. Weighing several anhydrous calcium chloride with a precision electronic balance, dissolving in the obtained PEG solution, and making into 20 mg/mL-1In (C) is2The solution is used as a collection of micro-droplets.
Step 2: preparation of aqueous two-phase system containing target enzyme
(1) Taking a certain amount of Dex-sodium alginate solution and 1mg/mL Tris-enzyme solution, and respectively preparing 5 mu g/mL glucose oxidase GOX and horseradish peroxidase HRP solution and Dex-sodium alginate solution in which the two enzymes are dissolved and the concentrations of which are both 5 mu g/mL and 2.5 mu g/mL by using buffer solutions. The target enzyme solution was divided into three groups.
The group A is Dex-sodium alginate solution dissolved with the two enzymes GOX/HRP and horse radish peroxidase HRP.
The group B is Dex-sodium alginate solution dissolved with 5 mug/mL glucose oxidase GOX and horse radish peroxidase HRP respectively;
group C had both enzymes GOX/HRP dissolved in a 2.5. mu.g/mL solution of Dex-sodium alginate.
(2) After the droplet flow microchannel device is installed, the nitrogen pump is opened, and the pressure intensity of the PEG phase connected air pressure is set to be 0.030MPa, and the pressure intensity of the Dex phase connected air pressure is set to be 0.025 MPa. Under the atmospheric pressure external field conditions, the internal phase hoses were connected to the Dex phases in which the three groups of enzyme solutions were dissolved, which were disposed in (1). Calcium alginate gel microspheres, in which GOX and HRP were encapsulated respectively and enzymes were encapsulated at two concentrations of both GOX and HRP, were prepared, as shown in fig. 3.
(3) Because the internal phase nitrogen pump is intermittently started, every time the internal phase nitrogen pump is started for 1.5s, the internal phase nitrogen pump is closed for 0.2s, so that every 1.7s can collect one calcium alginate gel microsphere, 30 microspheres can be collected within 51s by calculation, and the microspheres are doubled in sequence, so that multiple microspheres of 30 can be collected, and the enzyme concentration difference in unit volume is formed by different numbers of microspheres.
(4) The microsphere shape in the microporous plate is observed under the condition of adding a P microscope into the micropores of a 96 microporous plate in advance, and the whole is shot to be subjected to image analysis.
And step 3: preparing the target enzyme solution into calcium alginate microspheres with uniform size under the same condition, reacting with a glucose substrate according to a certain group, adding a color-developing agent, observing and analyzing the result.
(1) The reaction volume was first determined to be 100. mu.L for all experimental groups, with substrate excess. Three groups of enzyme encapsulation mode experimental groups were compared with each other. According to the prepared solution and the experimental method for standby in the step 2, in the preparation of the microspheres of the group A, an internal phase sample inlet is connected with a mixed solution of GOX and HRP with the concentration of 5 mu g/mL; in the preparation of the group B microspheres, internal phase sample inlets are respectively sequentially used for refining a 5 mu g/mL Dex-sodium alginate solution of GOX and HRP. In the preparation of group C microspheres, the internal phase injection port is connected with a mixed solution of GOX and HRP with the concentration of 2.5 mug/mL. The medium quality enzymes GOX and HRP in group A were encapsulated in the same calcium alginate microspheres, with the number of microspheres set to a multiple of 30. The influence of different microsphere numbers (reaction contact area and enzyme concentration in the system) on the reaction efficiency is conveniently compared and analyzed. The same mass of enzymes GOX and HRP in group B were encapsulated in different calcium alginate microspheres, and the number of both microspheres was equal to the number of microspheres in group A. In group C, A, B half the mass of the enzymes GOX and HRP were encapsulated in the same calcium alginate microspheres, and the number of microspheres was equal to twice the number of microspheres in group a.
(2) In A, B and C, four groups of different amounts of calcium alginate gel microspheres were collected to form lateral controls. Taking group A as an example, 30, 60, 90 and 120 microspheres simultaneously encapsulating two enzymes of GOX and HRP are collected within 51s, 102s, 153s and 204s of time by using the nitrogen pump working principle described above. Similarly, in the B, C two groups, four different groups of microspheres were collected in this manner to form a gradient in the number of microspheres. In order to form longitudinal contrast between A, B, C groups, B, C groups of microspheres were collected and arranged according to group A. Specifically, the results are shown in Table 1.
TABLE 1 control set-up in enzyme reaction efficiency determination experiments
(3) Respectively adding PEG-CaCl into different micropores of 96 microporous plate2Solution, the microspheres of the experimental group were collected and left to stand at room temperature for 4h to allow the microspheres to settle and the Dex internal phase to diffuse to stability.
(4) Carefully aspirate excess PEG-CaCl above the microspheres in each well using a syringe fitted with a 0.4mm outer diameter needle2And (3) solution. Preparing proper amount of 0.5mg/mL developing solution Amplex Red solution, 1mg/mL HRP-Tris solution and 0.218mmol/L H2O2Aqueous solution and 23.25mmol/L PEG-glucose solution for use.
(5) mu.L of the Amplex Red solution and 96. mu.L of the PEG-glucose solution (excess) were added to each well in this order using a pipette gun, and the reaction volume in each well was 100. mu.L, and the wells were left to react at room temperature (about 23 ℃ C.) in the absence of light. And timed after the reaction started. And measuring the absorbance value of each reaction system at 560nm by using a microplate reader every 5min, and continuously recording the change of the absorbance value to react for 50min in total. And (5) sorting data, drawing a curve and analyzing and processing.
In order to quantitatively characterize the reaction rate of GOX and HRP cascaded enzymes, H is prepared2O2Concentration-absorbance standard curve and conversion processing thereof, by measuring known H2O2And (5) obtaining corresponding data according to the absorbance of the reaction system at 560nm under the concentration and after full reaction. According to which H is made2O2The concentration-absorbance standard curve can obtain a trend line with the R2 of 0.9995 and the equation of y being 0.0114x +0.0014, namely, the absorbance A and the absorbance H2O2The linear equation of the concentration relationship is shown in FIG. 5. According to making H2O2Linear equation obtained from concentration-absorbance standard curve, and converting absorbance values of each group measured in reaction process into product O2And is given as O2Graph of concentration as a function of reaction time. The reaction volume for each set of experiments was 100. mu.L at room temperature (about 23 ℃ C.)) The following reaction is carried out. After the reaction is carried out for 40min, the absorbance measured at 560nm shows a relatively obvious change trend, and a relatively obvious contrast is formed between each group of curves, so that the analysis and observation are facilitated, and therefore, the data of the reaction for 40min are taken as a graph. O is2The ordinate in the graph of concentration-time relationship is the product O2The magnitude of the ordinate value and the slope of the curve in the same time can both represent the speed of the reaction rate.
As shown in FIG. 6, in the three groups a, b and c, the enzymatic reaction rate of A, B, C becomes faster with the increase of the number of microspheres (i.e. the increase of the enzyme concentration in the reaction system and the increase of the reaction contact area) whether the two enzymes GOX and HRP are mixed and packaged in one microsphere or packaged in different microspheres. As shown in FIG. 6d, the relationship between the reaction rates of A, B, C in each of group 4 is: group B > group C > group a. The enzyme reaction rate of the group B is higher than that of the group C, which indicates that the reaction rate of packaging the GOX and the HRP in different microspheres is higher than that of packaging the GOX and the HRP in the same microspheres under the condition that the enzyme concentration and the reaction contact area in the system are the same; the enzyme reaction rate of group C is higher than that of group A, which shows that the larger the reaction contact area is, the faster the enzymatic reaction rate is, under the same conditions of enzyme concentration and two-enzyme encapsulation mode in the system.
Drawing trend lines according to the curve in FIG. 6d, obtaining the slope k of the three trend linesA＝0.0738mmol·L-1·min-1，kB＝0.7305mmol·L-1·min-1，kC0.1191 mmol. L-1. min-1. Since the slope represents the average enzyme reaction rate, it is known that the reaction is within the first 40min from the start of the reaction. The average enzyme reaction rate in group B, d was about 9.90 times that in group A, 4 and 6.13 times that in group C, 4. It is shown that in the double aqueous phase-based cascade enzyme reaction, the group B enzyme encapsulation mode can ensure that the average enzyme reaction rate is far greater than that of the group A and the group C.
According to A, B, C in FIG. 6d from O in the respective group 4 reaction systems2Relative concentration at the same time point, further take O in three groups 4 when the reaction proceeded for 40min2The concentration value data (Table 2) was subjected to calculation analysis to obtain O shown in FIG. 72Relative relationship of concentration. Group B, 4O in the system when the reaction is carried out for 40min2The concentration was 7.66 times that of group A, 4 and 509 times that of group C, 4. This indicates that, when the reaction proceeded for 40min, the product O formed was accumulated in the system2The total amount relationship is that A, B and C are 1:7.66:1.51, and the reaction efficiency of the enzyme encapsulation mode of the B group is proved to be far higher than that of the A group and the C group in the period of time.
TABLE 2O in A, B, C group d at 40min reaction2Concentration value
Therefore, the two enzymes GOX and HRP are respectively packaged in different calcium alginate microspheres, and the method has a very obvious effect on improving the cascade enzymatic reaction rate in a PEG-Dex aqueous two-phase system. The target enzyme is encapsulated in the calcium alginate microspheres, so that the reaction contact area of the substrate and the enzyme is increased. In the double water phase system, Dex has relatively slow mass transfer speed and PEG has relatively fast mass transfer speed, so that most of the reaction occurs in the interface of the two water phases and intermediate product H is reduced2O2The mass transfer distance of (a); due to the high concentration of H2O2Inhibit HRP activity, so that the enzyme-carrying mode is divided into2O2The inhibition effect on the GOX enzyme activity is effectively reduced, the local enzyme concentration is also improved, and the reaction efficiency of the cascade enzyme is obviously improved.
The invention is based on alginate to catalyze cascade enzymatic reaction in a PEG/Dex aqueous two-phase system (the reagent of the reaction system is cheap), calcium alginate microspheres are used for actively discharging liquid to simulate the interaction of the internal environment and the external environment of original cells, and based on a microfluidic technology, the invention develops the bionic catalytic research which has high flux, good biocompatibility (no surfactant is added) and strengthened enzymatic reaction, and is the target continuously pursued in the field of biochemical engineering. The calcium alginate microspheres are generated based on an aqueous two-phase system by utilizing a coaxial microfluidic technology to strengthen the cascade enzymatic reaction, the calcium alginate microspheres encapsulating the target enzyme have the advantages of good biocompatibility, high enzyme activity, large specific surface area and high mass transfer rate, and compared with the common enzymatic reaction, the method can strengthen the enzymatic reaction by 7 times.
Claims (8)
1. A preparation method of enzyme-loaded calcium alginate microspheres based on an aqueous two-phase system is characterized by comprising the following steps:
step 1: respectively preparing a PEG aqueous solution with the mass concentration of 8% and a Dex aqueous solution with the mass concentration of 8%; fully mixing and then carrying out phase separation, wherein the upper phase is a PEG solution, and the lower phase is a Dex solution;
step 2: adding sodium alginate and a target enzyme solution into the Dex solution to form a Dex-sodium alginate solution containing the target enzyme, wherein the mass concentration of the sodium alginate is 0.5%;
and step 3: taking the PEG solution prepared in the step 1 as an external aqueous phase, taking the Dex-sodium alginate solution containing the target enzyme obtained in the step 2 as an internal aqueous phase, and forming a double aqueous phase sodium alginate liquid drop at the conical tip of the coaxial capillary;
and 4, step 4: and (4) introducing the aqueous two-phase sodium alginate droplets obtained in the step (3) into a PEG-calcium chloride aqueous solution to generate the enzyme-loaded calcium alginate microspheres.
2. The method for strengthening the cascade enzymatic reaction by adopting the enzyme-loaded calcium alginate microspheres based on the aqueous two-phase system as claimed in claim 1, wherein the enzyme-loaded calcium alginate microspheres obtained in step 4 are added into a PEG-glucose solution to generate the cascade enzymatic reaction.
3. The preparation method of the enzyme-loaded calcium alginate microspheres based on the aqueous two-phase system according to claim 1, wherein the PEG aqueous solution and the Dex aqueous solution in the step 1 are fully mixed by a rotary incubator, and phase separation is performed after the mixture is left for 12 hours.
4. The preparation method of the enzyme-loaded calcium alginate microspheres based on the aqueous two-phase system according to claim 1, wherein the target enzymes in the step 2 are glucose oxidase and horseradish peroxidase; in the Dex-sodium alginate solution of the target enzyme, the concentration of glucose oxidase is 5 mug/mL, the concentration of horseradish peroxidase is 5 mug/mL, the concentrations of the glucose oxidase and the horseradish peroxidase are both 5 mug/mL or the concentrations of the glucose oxidase and the horseradish peroxidase are both 2.5 mug/mL.
5. The preparation method of the enzyme-loaded calcium alginate microspheres based on the aqueous two-phase system according to claim 1, wherein in the step 3, the external aqueous phase and the internal aqueous phase are injected by an air pump, the injection pressure of the external aqueous phase is 0.030MPa, and the injection pressure of the internal aqueous phase is 0.015-0.030 MPa; and in the process of sampling the internal water phase, starting an air pump for 0.2s, and closing the air pump for 1.5s to serve as a sampling period.
6. The preparation method of the enzyme-loaded calcium alginate microspheres based on the aqueous two-phase system according to claim 1, wherein the diameter range of the enzyme-loaded calcium alginate microspheres in the step 4 is 200-400 μm.
7. The preparation method of the enzyme-loaded calcium alginate microspheres based on the aqueous two-phase system according to claim 1, wherein the aqueous solution of PEG-calcium chloride in step 4 is formed by adding calcium chloride to the PEG solution in step 1, wherein the concentration of the calcium chloride is 10% by mass.
8. The method for the reinforced cascade enzymatic reaction of the enzyme-loaded calcium alginate microspheres based on the aqueous two-phase system of claim 2, wherein the PEG-glucose solution is formed by adding glucose to the PEG solution in the step 1, and the concentration of the glucose is 23.25 mmol/L.
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