Preparation technology of double-encapsulated activated lipase-containing gel microspheres

Abstract

The present invention discloses a preparation technology for double-encapsulated inner flower gel microspheres for lipase activation. The present invention first uses a surfactant to activate lipase, then combines two carrier materials, inorganic salt crystals and alginate gel, and uses a double-encapsulation technology to give the lipase a double layer of "armor" to prepare a white inner flower gel microsphere encapsulating the activated lipase. Due to the strong network structure of the outer layer gel and its high mechanical strength, the integrity of the crystal and enzyme molecular structure in the microsphere can be effectively protected during the catalysis and recovery process; the crystal flowers are firmly confined in the pores of the gel grid, and the Ca on the surface of the crystal is 2+ Residual ‑COO on the gel surface ‑ The cross-linking effect can effectively prevent the loss of lipase, fully ensuring the catalytic activity and operational stability of the lipase. The present invention provides a new immobilization strategy for improving the efficiency of lipase, especially the green and mild preparation conditions, which is particularly suitable for the production and processing of food and medicine.

Description

Technical Field

[0001] The invention relates to the technical field of biological immobilized enzymes, in particular to a preparation technology of double-embedded activated lipase inner gel microspheres. Background Art

[0002] In the food industry, many food production and processing processes involve ester-related reactions, such as baking, grains and oils, beverages, and liquor. Lipases (Triacylglycerol acylhydrolases, EC 3.1.1.3) can be used to efficiently catalyze ester hydrolysis, esterification, and transesterification reactions. Their mild catalytic conditions, environmental friendliness, and adherence to the concept of green catalysis have made them popular in the food processing industry. However, natural lipases (free lipases) are susceptible to environmental factors and are unstable. Furthermore, they are difficult to recycle and reuse in practical applications. Therefore, research on lipase immobilization technology has become a hot topic in food development.

[0003] In recent years, with the gradual interpenetration of disciplines, breakthroughs have been made in enzyme immobilization technology, with metal phosphate crystals attracting attention as a new type of immobilization carrier. Researchers have attempted to construct enzyme-inorganic crystal complexes with flower-like micro-nanostructures using phosphate crystals of different single metals or composite metals, such as copper, zinc, calcium, and manganese. The microscopic flower-like micro-nanostructures of the crystals possess structural rigidity, a large number of voids, and a large specific surface area. This not only effectively improves the stability of the encapsulated enzyme protein molecules, but also facilitates the transfer of substrates into the crystals, ensuring full contact with the enzyme molecules. Furthermore, the metal ions in the crystals can act as activators, promoting the catalytic activity of the enzyme.

[0004] The active site of lipase is usually covered by an α-helical "lid", which is in a closed conformation and has low catalytic activity. A suitable surfactant will help open the "lid" and convert the lipase into an open conformation, thereby showing higher catalytic activity. In the early stage of investigating the factors affecting enzyme catalysis, the applicant found that surfactant (PEG 8000) and metal ions (calcium) can act as activating factors to synergistically promote the catalytic activity of lipase. The reason is that PEG 8000 first opens the α-helical "lid" covering the active site of lipase, and the channel for calcium ions to enter the active site of the enzyme is opened, which promotes the catalytic activity of lipase. Therefore, the applicant envisioned that PEG 8000 can be used to activate the lipase first, so that it is in an open conformation; then the activated lipase can be combined with another activating factor, calcium ions, to make a flower-like micro-nanostructured lipase-calcium phosphate crystal complex (Ca3(PO4)2@PEG-Lip), and the enzyme is embedded in the crystal flower. However, previous experimental studies have found that although the initially prepared Ca3(PO4)2@PEG-Lip promoted enzyme activity and achieved enzyme recycling, the self-assembled Ca3(PO4)2@PEG-Lip crystal flower structure was relatively fragile. During the centrifugal recycling process, the structure was easily destroyed and the enzyme activity loss was still large.

[0005] Sodium alginate (SA) is a natural linear polymer that can react with Ca 2+ Cross-linking to form a three-dimensional network gel structure (preparation process as Figure 2 As shown in the figure, this cross-linked structure is firmly bonded and not easily damaged. Therefore, embedding the fragile Ca3(PO4)2@PEG-Lip crystal flowers in alginate gel microspheres can effectively protect the integrity of the crystal flower structure during recycling operations, thereby improving the operational stability and reuse times of the immobilized lipase. Summary of the Invention

[0006] In order to further solve the above problems, the present application adopts a double encapsulation technology to immobilize lipase: first, PEG8000 is used to activate the lipase; then, calcium phosphate crystal flowers are used to encapsulate the activated lipase to make Ca3(PO4)2@PEG-Lip; then, the green and mild calcium alginate (CA) gel (Gel) technology is used to coat the Ca3(PO4)2@PEG-Lip crystal flowers to make a double-encapsulated activated lipase inner flower gel microsphere (CA-Ge1@Ca3(PO4)2@PEG-Lip). The purpose is to effectively protect the structural integrity of the enzyme molecules during the recycling operation, facilitate the recycling of the immobilized enzyme, reduce the loss of lipase, and give full play to the catalytic activity of the lipase, providing new technical support for improving the efficiency of lipase use in the food industry.

[0007] The technical solutions of the present invention are as follows:

[0008] The present invention provides a preparation technology of double-encapsulated lipase-containing gel microspheres, comprising the following steps:

[0009] (1) PBS buffer solution containing lipase is mixed with a surfactant to obtain a mixed solution, and CaCl2 solution is added to the mixed solution for crystallization to obtain calcium phosphate crystal flowers (Ca3(PO4)2@PEG-Lip) encapsulating activated lipase;

[0010] (2) dissolving sodium alginate in PBS buffer to obtain a sodium alginate solution;

[0011] (3) The calcium phosphate crystal flowers encapsulating activated lipase are dispersed in a sodium alginate solution to obtain a mixed system; the mixed system is then added dropwise into a cross-linking agent CaCl2 solution for cross-linking to obtain an inner flower gel microsphere with double encapsulation of activated lipase.

[0012] Preferably, the lipase in the PBS buffer containing the lipase in step (1) is Lipase F-AP 15.

[0013] Preferably, the concentration of lipase in the PBS buffer containing lipase in step (1) is 0.025-0.5 mg / mL; the surfactant in step (1) is Triton X-100, SDS, PEG 8000, SXS, CAB 35, Span 80, PVA or CTAB; the concentration of the added surfactant is 0-250 mM; the concentration of the CaCl2 solution added in step (1) is 62.5-2500 mM; the crystallization time in step (1) is 3-15 h, and the crystallization temperature is below 4°C.

[0014] In step (1), PBS buffer solution containing lipase is mixed with a surfactant, the surfactant is used to activate the lipase, and after adding the surfactant, the mixture is mixed and shaken to allow the surfactant to fully activate the lipase.

[0015] Most preferably, the concentration of lipase in the PBS buffer in which lipase is dissolved in step (1) is 0.15 mg / mL; the surfactant in step (1) is PEG 8000, and the concentration of the added surfactant is 75 mM; the concentration of the CaCl2 solution added in step (1) is 250 mM; and the crystallization time in step (1) is 12 h.

[0016] Preferably, the mass volume percentage concentration (m / v, g / 100 mL) of the sodium alginate solution in step (2) is 0.5%-2.5%.

[0017] Most preferably, the mass volume percentage concentration (m / v, g / 100 mL) of the sodium alginate solution in step (2) is 1.5%.

[0018] The mass volume percentage concentration (m / v, g / 100 mL) of the cross-linking agent CaCl2 solution in step (3) is 2%.

[0019] The present invention also provides inner flower gel microspheres with double embedded activated lipase prepared by the preparation technology.

[0020] Beneficial effects of the present invention:

[0021] In the preparation method of the present invention, during the immobilization process, a surfactant is first used to activate the lipase, and then two carrier materials, inorganic salt crystals and alginate gel, are combined. The lipase is given a double-layer "armor" through a double-encapsulation technique to prepare an inner-flowered gel microsphere with double-encapsulated activated lipase. Since the network structure of the outer gel layer is firm and has high mechanical strength, the integrity of the crystal flower structure within the microsphere can be effectively protected during the catalytic and recovery processes; the calcium phosphate crystal flowers of the inner layer that encapsulate the lipase are firmly confined in the pores of the gel grid, effectively preventing the loss of the lipase and ensuring the catalytic activity and operational stability of the lipase. This article provides a new immobilization strategy for improving the utilization efficiency of lipase, especially green and mild preparation conditions, which are particularly suitable for the production and processing of food and medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 CD spectrum scanning diagram of Lipase F-AP 15 and PEG-Lipase F-AP 15.

[0023] Figure 2 Diagram of the process of Lipase transitioning from a closed conformation to an open conformation.

[0024] Figure 3 This is a diagram showing the effect of different PEG8000 concentrations on the preparation of Ca3(PO4)2@PEG-Lip.

[0025] Figure 4 This is a diagram showing the effect of different lipase concentrations on the preparation of Ca3(PO4)2@PEG-Lip.

[0026] Figure 5 This figure shows the effect of different CaCl2 addition concentrations on the preparation of Ca3(PO4)2@PEG-Lip.

[0027] Figure 6 This is a diagram showing the influence of different crystallization times on the preparation of Ca3(PO4)2@PEG-Lip.

[0028] Figure 7This is a diagram showing the effect of different sodium alginate concentrations on the preparation of CA-Gel@Ca3(PO4)2@PEG-Lip.

[0029] Figure 8 These are macroscopic images of three types of immobilized lipases; among them, a is the crystal flower Ca3(PO4)2@PEG-Lip that encapsulates activated lipase; b is the gel microsphere CA-Gel@PEG-Lip that encapsulates activated lipase; c is the inner flower gel microsphere CA-Gel@Ca3(PO4)2@PEG-Lip that double encapsulates activated lipase.

[0030] Figure 9 These are SEM images of Ca3(PO4)2@PEG-Lip crystal flowers; among them, a is a macroscopic image of dahlia; b is a SEM image at 9.3mm×35.0K; c is a SEM image at 9.3mm×60.0K.

[0031] Figure 10 Schematic diagram of the preparation process of Ca3(PO4)2@PEG-Lip crystal flowers.

[0032] Figure 11 SEM images of CA-Gel@Ca3(PO4)2@PEG-Lip gel microspheres; a is the SEM image at 8.6 mm × 5.00 K; b is the SEM image at 8.0 mm × 40 K; c is the SEM image at 8.0 mm × 150 K.

[0033] Figure 12 Schematic diagram of the preparation process of CA-Gel@Ca3(PO4)2@PEG-Lip gel microspheres.

[0034] Figure 13 The microscopic composition characterization diagram of Ca3(PO4)2@PEG-Lip crystal flower; among them, a is the EDS image of Ca3(PO4)2@PEG-Lip; b is the CLSM image of Ca3(PO4)2@PEG-Lip.

[0035] Figure 14 This is the infrared spectrum of Ca3(PO4)2@PEG-Lip crystal flower.

[0036] Figure 15 Figure 2 is the reuse rate diagram of three immobilized lipases.

[0037] Figure 16 This is an investigation diagram of the stability of CA-Gel@Ca3(PO4)2@PEG-Lip; among them, a is the effect of temperature on the stability of CA-Gel@Ca3(PO4)2@PEG-Lip; b is the effect of pH on the stability of CA-Gel@Ca3(PO4)2@PEG-Lip. DETAILED DESCRIPTION

[0038] Experimental reagents and instruments.

[0039] Lipase F-AP 15 (from Rhizopus oryzae) was purchased from Sigma-Aldrich; dinitrophenyl p-palmitate (pNPP), p-nitrophenol (pNP), Nile blue, tris (hydroxymethylaminomethane) (Tris), and polyethylene glycol 8000 (PEG 8000) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; sodium alginate (SA) and calcium chloride were purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. All reagents were of analytical grade except pNP, which was used as an analytical standard.

[0040] Cary-60 ultraviolet spectrophotometer (UV), FEI Company, USA; FV-3000 confocal microscope (CLSM), Olympus Corporation, Japan; X-Max energy dispersive spectrometer (EDS), Oxford Instruments Ltd., UK; ME-204E microbalance, Mettler-Toledo Instruments (Shanghai) Co., Ltd.; TG-16G desktop high-speed centrifuge, Beihong Instrument Co., Ltd.; VS-802A freeze drying oven, Wuxi Woxin Instrument Manufacturing Co., Ltd.; Multiskan GO full-wavelength microplate reader, Thermo Scientific Corporation, USA; 470FI-IK Fourier transform infrared spectrometer (FT-IR), Nicolet Instruments, USA; BT101L peristaltic pump, Baoding Refu Fluid Technology Co., Ltd.

[0041] Example 1: Preparation of CA-Gel@Ca3(PO4)2@PEG-Lip

[0042] (1) Preparation of Ca3(PO4)2@PEG-Lip crystal flowers

[0043] Accurately weigh a certain amount of lipase powder into a 100mL volumetric flask and dilute to volume with PBS buffer (pH 7.5, 10mM) to prepare a lipase solution. Pipette 5.00mL of the lipase solution into a centrifuge tube, then add 100μL of PEG 8000 solution and shake at room temperature for 3h to allow PEG 8000 to fully activate the lipase Lipase F-AP 15. Then, continue to add 100μL of CaCl2 solution, mix well, and let it stand in a 4℃ refrigerator for crystallization for a period of time. Centrifuge (6000rpm, 5min), discard the supernatant, and wash the crystal precipitate three times with a small amount of PBS buffer (10mM, pH7.5). Collect the precipitate to obtain calcium phosphate crystals encapsulating activated lipase (Ca3(PO4)2@PEG-Lip).

[0044] (2) Prepare SA solution using PBS buffer (10 mM pH 7.5);

[0045] (3) Accurately weigh the prepared Ca3(PO4)2@PEG-Lip, disperse it in 5 mL of SA solution, vortex mix it, and then drip it dropwise into the CaCl2 solution (2%, m / v) that is being magnetically stirred using a peristaltic pump (4000 μL / min). The calcium alginate gel coats the Ca3(PO4)2@PEG-Lip crystal flowers to form gel microspheres. The gel microspheres are then removed using a filter with a mesh size of approximately 0.5 mm and washed with a small amount of purified water to obtain double-encapsulated activated lipase inner flower gel microspheres CA-Gel@Ca3(PO4)2@PEG-Lip.

[0046] Preparation of gel microspheres encapsulating activated lipase (CA-Gel@Lip): Accurately weigh the lipase solution with an amount equivalent to the above-mentioned immobilized enzyme Ca3(PO4)2@PEG-Lip and disperse it in 5 mL of SA solution. The remaining steps refer to the preparation method of the above-mentioned CA-Gel@Ca3(PO4)2@PEG-Lip to prepare gel microspheres encapsulating activated lipase (CA-Ge1@Lip).

[0047] Example 2: Determination of lipase activity, enzyme immobilization rate, and enzyme kinetic constant

[0048] (1) Enzyme activity assay: In a 10 mL centrifuge tube, accurately add 4.50 mL of Tris-HCl buffer (50 mM, pH 8.0) and 0.50 mL of 6 mM pNPP solution to form the substrate solution. Then, add 10 μL of 1.00 mg / mL lipase solution, or an equivalent amount of CA-Gel@Lip, CA-Gel@Ca3(PO4)2@PEG-Lip, and Ca3(PO4)2@PEG-Lip. Incubate at 30°C for 5 min. Rapidly add 2.50 mL of anhydrous ethanol to terminate the reaction. Measure the absorbance at λ = 405 nm using UV light.

[0049] Definition of lipase activity unit (U): Under the above conditions, the amount of enzyme required to hydrolyze the substrate pNPP to produce 1 μmol pNP per minute is 1 unit of enzyme activity. The unit activity (X) of lipase is expressed as U / mg of enzyme activity per milligram of pure lipase:

[0050]

[0051] In formula (1): ΔOD 405 is the absorbance change at λ = 405 nm; Vtotal is the total volume of the reaction solution; n is the dilution factor; Δt is the reaction time (min); ε is the extinction coefficient, ε = 15670 L / (mol·cm); and m is the amount of enzyme added (mg).

[0052] (2) Calculation of relative enzyme activity:

[0053]

[0054] In formula (2): X I To determine enzyme activity; X S It is the reference enzyme activity of the same group.

[0055] (3) Determination of enzyme immobilization rate: The protein content was determined by BCA method. That is, the protein in the enzyme molecule can immobilize Cu at pH 8. 2+ Reduction to Cu + , Cu + It appears blue and absorbs at λ=562nm.

[0056] Prepare BCA working solution: Mix BCA reagent and copper sulfate reagent in a volume ratio of 50:1 according to the kit to prepare BCA working solution, which should be prepared before use.

[0057] The standard curve equation was established by preparing a gradient solution of bovine serum albumin (BSA) with a concentration of 10-100 μg / mL using Tris-HCl buffer (50 mM, pH 8.0). 25 μL of the gradient solution was pipetted into a 96-well plate, and then 200 μL of the prepared BCA working solution was added. The plates were incubated at 37°C for 30 min, and the absorbance was measured at λ = 562 nm using a microplate reader. The standard curve equation was obtained as follows: y = 0.005x - 0.0713, R 2 =0.9991.

[0058] Protein content determination: Centrifuge the prepared mixed solution containing immobilized enzyme. Pipette 25 μL of the supernatant into a 96-well plate, then add 200 μL of BCA working solution. Incubate at 37°C for 30 min. Measure the absorbance at λ = 562 nm using a microplate reader. Calculate the protein content of the supernatant using a standard curve.

[0059] Calculation of enzyme immobilization rate:

[0060]

[0061] In formula (3): C T is the total protein content of the lipase solution before immobilization; C s The protein content of the supernatant after centrifugation.

[0062] (4) Determination of lipase kinetic constant: Derived according to the Lineweaver-Burk double reciprocal plotting method.

[0063] The enzyme activities of free lipase and two immobilized enzymes, Ca3(PO4)2@PEG-Lip and CA-Gel@Ca3(PO4)2@PEG-Lip, were determined at different substrate concentrations (0.5mM, 0.5mM, 2.5mM, 5mM, 8mM, 10mM, 30mM, and 50mM) according to method (1). The reciprocal of the substrate concentration (1 / [S]) was plotted as the horizontal axis and the reciprocal of the reaction rate (1 / [V]) was plotted as the vertical axis. The K values ​​of Free Lipase, Ca3(PO4)2@PEG-Lip, and CA-Gel@Ca3(PO4)2@PEG-Lip were calculated using the Michaelis-Menten equation. m and V max .

[0064]

[0065] In formula (4), V is the rate of enzymatic reaction; V m is the maximum reaction rate; K m is the Michaelis constant; [S] is the substrate concentration.

[0066] Example 3: Preparation of CA-Gel@Ca3(PO4)2@PEG-Lip

[0067] (1) Preparation of Ca3(PO4)2@PEG-Lip

[0068] 1) Selection of surfactant

[0069] A 50 mM surfactant solution was prepared in purified water. The surfactants screened included Triton X-100, SDS, PEG 8000, SXS, CAB 35, Span 80, PVA, and CTAB.

[0070] Take eight 10mL centrifuge tubes and add 100μL of each of the eight surfactant solutions (50mM concentration), 4.4mL of Tris-HCl buffer (50mM, pH 8.0), and 10μL of 1.00mg / mL lipase solution. Vortex thoroughly to mix, then add 0.50mL of 6mM pNPP solution. Incubate at 30°C for 5 minutes, and quickly add 2.50mL of anhydrous ethanol to terminate the reaction. The lipase activity was determined using the method in Example 2. In the control group, 100μL of purified water was used instead of the surfactant solution and the same method was used for determination. The enzyme activity in the control group was used as the reference enzyme activity, and the relative enzyme activity was calculated. By comparing the relative enzyme activities, the effects of the eight surfactants on the enzyme activity of Lipase F-AP 15 were investigated, and the optimal surfactant was determined. The experimental results are shown in Table 1.

[0071] Table 1 Effect of surfactants on the activity of Lipase F-AP15

[0072]

[0073] Based on the comparison of relative enzyme activities in Table 1, the present application selected PEG8000 as the most suitable surfactant with a stronger promoting effect on lipase.

[0074] In order to further explore the mechanism of action of PEG 8000 in promoting lipase activity, the present invention scanned Lipase F-AP 15 enzyme solution (0.10 mg / mL) and Lipase F-AP 15 enzyme solution activated by the optimal surfactant (0.10 mg / mL), recorded the wavelength of 190-260 nm, compared the changes in the secondary structure of Lipase F-AP 15 protein in the two spectra, and analyzed the mechanism of action of surfactant on lipase. The experimental results are as follows Figure 1 shown.

[0075] Depend on Figure 1 It can be found that compared with free lipase, the height of the positive peak at 191nm, which represents the α-helix, decreased after adding PEG 8000, and the intensity of the negative peaks at 208nm and 222nm decreased, indicating that the α-helix content in the protein secondary structure decreased. This indicates that the surfactant interacts with the lipase α-helix, causing the α-helix structure to rearrange, opening the "lid" covering the lipase active site, exposing the lipase active center, and converting the lipase from a closed conformation to an open conformation ( Figure 2 ).

[0076] From the above analysis, it can be seen that if a lipase in a closed conformation is immobilized in a phosphate crystal, the α-helix covering the enzyme's active site will be more difficult to open. However, if the lipase is first activated to an open conformation with a surfactant before immobilization, the lipase will be stabilized in the open conformation in the crystal, maintaining high enzyme activity. Therefore, the present invention uses PEG 8000 to activate the lipase before immobilization.

[0077] 2) Selection of PEG 8000 concentration

[0078] Use PBS buffer (10mM, pH 7.5) to prepare PEG8000 solutions with concentrations of 0mM, 25mM, 50mM, 75mM, 100mM, 125mM, 150mM, and 250mM. Then, 100μL of the above-mentioned PEG8000 solutions of each concentration were accurately added to eight 5mL lipase solutions in sequence, and the remaining steps were carried out according to the preparation method of Ca3(PO4)2@PEG-Lip crystal flowers in Example 1. The enzyme activity of the crystal flower immobilized enzyme was determined by the method of Example 2, and the enzyme activity of the crystal flower immobilized enzyme prepared without adding PEG8000 was used as the reference enzyme activity to calculate the relative enzyme activity; the immobilization rate of lipase in the crystal flower was determined and calculated by the method of Example 2. The effect of different concentrations of PEG 8000 on the immobilization effect of Ca3(PO4)2@PEG-Lip was investigated by relative enzyme activity and enzyme immobilization rate. The experimental results are as follows. Figure 3 shown.

[0079] PEG 8000 is an activating factor of lipase and helps lipase maintain its active conformation when immobilized. Therefore, this application introduces PEG 8000 into the preparation of Ca3(PO4)2@PEG-Lip. Figure 3 As shown in the figure, compared with the crystal flower immobilized enzyme prepared without adding PEG 8000, the relative enzyme activity of the immobilized lipase gradually increased with the increase of PEG8000 concentration. When the PEG8000 concentration increased to 75mM, the enzyme activity increased by 8%, reaching the highest level. As the PEG8000 concentration continued to increase, the relative enzyme activity began to decline. When the PEG 8000 concentration increased to 250mM, the relative enzyme activity of the immobilized enzyme decreased to 73.21%. The reason is that excessive PEG 8000 will enter and occupy the active site of the immobilized lipase, inhibiting the adsorption catalysis of the lipase on the substrate. In addition, Figure 3 It can also be seen that when the PEG 8000 concentration ranges from 0 to 250 mM, the immobilization rate of lipase in the Ca3(PO4)2 crystal flowers is basically stable.

[0080] In summary, this application selected PEG 8000 with an addition concentration of 75 mM for the preparation of Ca3(PO4)2@PEG-Lip.

[0081] 3) Selection of lipase concentration

[0082] The lyophilized lipase F-AP 15 powder was fully dissolved in PBS buffer (10 mM, pH 7.5) to prepare lipase solutions with concentrations of 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, and 0.5 mg / mL, respectively.

[0083] Accurately add 5 mL of the above-mentioned lipase solution of different concentrations to six centrifuge tubes in sequence, and prepare Ca3(PO4)2@PEG-Lip crystal flowers with reference to Example 1 for the remaining preparation steps. Then, refer to the method of Example 2 to determine the enzyme activity of the crystal flower immobilized enzyme, and take the highest enzyme activity measured as the reference enzyme activity to calculate the relative enzyme activity of the crystal flower immobilized enzyme prepared at different lipase addition concentrations; and refer to the method of Example 2 to determine and calculate the enzyme immobilization rate. The effect of the addition concentration of lipase on the immobilization effect of Ca3(PO4)2@PEG-Lip was investigated by comparing the relative enzyme activity and the enzyme immobilization rate. The experimental results are as follows. Figure 4 shown.

[0084] Depend on Figure 4 As can be seen, when the lipase concentration increased from 0.025 mg / mL to 0.15 mg / mL, the relative enzyme activity of the immobilized enzyme increased dramatically, from 24% to 89%, while the average immobilization rate of lipase showed a downward trend, from 67% to 48%. Analysis suggests that as the lipase concentration increased, the amount of immobilized enzyme in the crystals increased, leading to a sharp increase in the relative enzyme activity of the immobilized enzyme. However, the available space for lipase immobilization in the crystals is limited and fixed. As the lipase concentration increased, the competition for enzyme molecules to enter the immobilization space also increased, resulting in a downward trend in the average immobilization rate of lipase. When the lipase concentration continued to increase from 0.15 mg / mL to 0.5 mg / mL, the increase in the relative enzyme activity of the immobilized enzyme decreased significantly, increasing by only 11%, while the average immobilization rate of lipase continued to show a significant downward trend. After adding high concentration of lipase, although the amount of immobilized enzyme continues to increase, the arrangement of lipase in the crystal will become more and more dense or even accumulate, resulting in more and more active sites of the enzyme being covered, thus affecting the full play of the enzyme's catalytic activity. Therefore, the enzymatic activity of the immobilized enzyme does not always increase in a gradient.

[0085] Based on the above analysis, comprehensive consideration Figure 4 The immobilization rate and relative enzyme activity of the immobilized lipase changed with the increase of the lipase addition concentration. Taking into account the cost of lipase immobilization, this application selected a lipase addition concentration of 0.15 mg / mL for the preparation of Ca3(PO4)2@PEG-Lip crystal flowers.

[0086] 4) Selection of CaCl2 concentration

[0087] Use purified water to prepare CaCl2 solutions with concentrations of 62.5mM, 125mM, 250mM, 500mM, 1M, and 2.5M.

[0088] According to the above optimization results, six 5mL portions of PBS buffer solution containing lipase and PEG 8000 were taken respectively, and 100μL of the above-mentioned CaCl2 solutions of each concentration were accurately added in turn. The remaining preparation steps were prepared according to Example 1 to prepare Ca3(PO4)2@PEG-Lip crystal flowers. Then, the enzyme activity of the crystal flower immobilized enzyme was determined respectively according to the method of Example 2, and the highest enzyme activity measured was used as the reference enzyme activity. The relative enzyme activity of the crystal flower immobilized enzyme prepared at different CaCl2 addition concentrations was calculated respectively; and the enzyme immobilization rate was determined and calculated according to the method of Example 2. The effect of the added concentration of CaCl2 on the immobilization effect of Ca3(PO4)2@PEG-Lip was investigated by comparing the relative enzyme activity and the enzyme immobilization rate. The experimental results are as follows. Figure 5 shown.

[0089] Ca 2+ As another activating factor of lipase, a part of it forms a complex with lipase to promote enzyme activity, and the other part precipitates with phosphate ions to form crystals to embed lipase. Therefore, the immobilization rate and enzyme activity of Ca3(PO4)2@PEG-Lip are similar to those of Ca 2+ Close relationship. Figure 5 It can be seen that in Ca 2+ When the addition concentration was in the range of 62.5mM to 250mM, the enzyme immobilization rate increased rapidly from 42% to 48.5%, and the relative enzyme activity of the immobilized enzyme reached the maximum from 72%, i.e. 100% (here, Ca 2+ The highest enzyme activity of the immobilized enzymes prepared with different addition concentrations is the reference enzyme activity, i.e. 100%). 2+ With the addition of Ca3(PO4)2 crystals encapsulating lipase, the solution gradually formed a precipitate of Ca3(PO4)2 crystals. 2+ As the concentration of added increases, more Ca3(PO4)2 crystals are formed in the solution to encapsulate lipase, so the enzyme immobilization rate and the relative enzyme activity of the immobilized enzyme also increase. 2+ When the concentration was in the range of 250mM to 2.5M, the increasing trend of the enzyme immobilization rate began to flatten out, and the relative enzyme activity of the immobilized enzyme began to decrease from 100%. 2+ When the concentration reached 2.5M, the relative enzyme activity dropped to 81.43%, and the immobilization rate slowly increased to 55.02%. 2+ With the continuous addition of Ca, the solution gradually reaches precipitation equilibrium, so the enzyme immobilization rate gradually tends to be flat. 2+ Appropriate addition concentration can promote the catalytic activity of lipase, and if Ca 2+ Adding too high a concentration will have the opposite effect. Excessive metal ions will destroy the charge distribution of the surface groups of the enzyme molecules and inhibit the catalytic activity of the enzyme.

[0090] Based on the above analysis, comprehensive consideration Figure 5 Medium Ca 2+ The relationship between the addition concentration and the immobilization rate and relative enzyme activity of immobilized lipase. In this study, Ca 2+ Add Ca3(PO4)2@PEG-Lip crystal flowers at a concentration of 250 mM.

[0091] 5) Selection of crystallization time

[0092] According to the above optimization results, referring to the method of Example 1, five samples were prepared, and the samples were allowed to stand for crystallization for 3h, 6h, 9h, 12h, and 15h in a 4°C refrigerator to prepare Ca3(PO4)2@PEG-Lip. Then, referring to the method of Example 2, the enzyme activity of the crystal flower immobilized enzyme was determined respectively, and the highest enzyme activity measured was used as the reference enzyme activity. The relative enzyme activity of the crystal flower immobilized enzyme obtained at different crystallization times was calculated respectively; and the enzyme immobilization rate was determined and calculated referring to the method of Example 2. The effect of different crystallization times on the immobilization effect of Ca3(PO4)2@PEG-Lip was investigated by comparing the relative enzyme activity and enzyme immobilization rate. The experimental results are as follows. Figure 6 shown.

[0093] Depend on Figure 6 As shown, when the crystallization time was extended from 3 hours to 12 hours, the relative enzyme activity of the corresponding Ca3(PO4)2@PEG-Lip increased from 50% to 89%, and the enzyme immobilization rate increased from 35% to 48%. Further extension of the crystallization time did not significantly increase the relative enzyme activity and immobilization rate. Therefore, this application selected a crystallization time of 12 hours for the preparation of Ca3(PO4)2@PEG-Lip.

[0094] (2) Preparation of CA-Gel@Ca3(PO4)2@PEG-Lip

[0095] 1) Selection of sodium alginate concentration

[0096] Sodium alginate (SA) solutions (m / v) with mass percentages of 0.5%, 1%, 1.5%, 2%, and 2.5% were prepared using PBS buffer (10 mM, pH 7.5).

[0097] Prepare Ca3(PO4)2@PEG-Lip crystal flowers according to the optimization results in (1). Accurately weigh five portions of 0.15g of Ca3(PO4)2@PEG-Lip crystal flowers and disperse them in 5mL of the above-prepared SA solution of different concentrations. The remaining steps refer to the preparation method of double-embedded activated lipase inner flower gel microspheres CA-Gel@Ca3(PO4)2@PEG-Lip in Example 1. Then, refer to the method of Example 2 to measure the enzyme activity of CA-Gel@Ca3(PO4)2@PEG-Lip gel microspheres respectively. The highest enzyme activity measured is used as the reference enzyme activity, and the relative enzyme activity of the gel microspheres prepared at different SA concentrations is calculated respectively.

[0098] Mechanical strength determination of gel microspheres: 20 CA-Gel@Ca3(PO4)2@PEG-Lip gel microspheres prepared at different SA concentrations were randomly selected, their surface moisture was dried, and they were placed on an analytical balance in turn. The sharp surface of a steel ruler was used to press the microspheres. When the gel microspheres broke, the balance reading was recorded and the average value was calculated.

[0099] The effect of different SA concentrations on the immobilization effect of CA-Gel@Ca3(PO4)2@PEG-Lip was investigated by measuring the relative enzyme activity and mechanical strength of CA-Gel@Ca3(PO4)2@PEG-Lip gel microspheres. Figure 7 shown.

[0100] The gel is composed of divalent Ca 2+ The -COOCaCOO- network structure is formed by cross-linking with the -COO- in sodium alginate (SA). When the SA solution is dropped dropwise into the CaCl2 solution (with a mass volume percentage concentration of 2%), the Ca in the CaCl2 solution 2+ First, it contacts with SA on the outer surface of the droplet and quickly cross-links to form a film, forming gel microspheres, and then Ca 2+ The cross-linking continues gradually within the membrane, and the core gradually solidifies into a gel network, which improves the mechanical strength of the gel microspheres and provides operational stability. The concentration of the SA solution will affect the density of the gel network structure formed, thereby affecting the mechanical strength of the gel microspheres and the relative enzyme activity of the immobilized lipase. Figure 7 It can be seen that when the SA concentration is in the range of 0.5%-1.5%, the mechanical strength of CA-Gel@Ca3(PO4)2@PEG-Lip gel microspheres increases gradually with the increase of SA concentration; the relative enzyme activity also increases gradually. This is because as the SA concentration increases, the interaction between SA and Ca 2+Crosslinking increases the density of the gel mesh structure, thereby enhancing the mechanical strength of the gel microspheres. This also strengthens the Ca₃(PO₄)₂@PEG-Lip crystals immobilized within the gel mesh, reducing their loss from the gel and leading to an upward trend in the relative activity of the immobilized enzyme. When the SA concentration continues to increase to the 1.0%-1.5% range, the mechanical strength of the gel microspheres rises from 9.31 to 10.50, while the relative activity of the immobilized enzyme increases slightly, though not significantly.

[0101] In summary, this application selected a 1.5% by mass SA solution (m / v) to prepare CA-Gel@Ca3(PO4)2@PEG-Lip gel microspheres.

[0102] Example 4: Characterization of the morphology and composition of immobilized lipase

[0103] (1) Preparation of CA-Gel@Ca3(PO4)2@PEG-Lip

[0104] 1) Using the preferred preparation method in Example 3, accurately weigh 0.15 g of lipase powder into a 100 mL volumetric flask, dilute to volume with PBS buffer (pH 7.5, 10 mM) to prepare a 0.15 mg / mL lipase solution. Pipette 5.00 mL of the lipase solution into a centrifuge tube, add 100 μL of 75 mM PEG 8000 solution, and shake at room temperature for 3 hours to allow PEG 8000 to fully activate the lipase Lipase F-AP 15. Then, continue to add 100 μL of 250 mM CaCl2 solution, mix well, and let stand in a 4 ° C refrigerator for 12 hours to crystallize. Centrifuge (6000 rpm, 5 min), discard the supernatant, wash the crystal precipitate 3 times with a small amount of PBS buffer (10 mM, pH 7.5), collect the precipitate, and obtain calcium phosphate crystal flowers (Ca3(PO4)2@PEG-Lip) that encapsulate activated lipase;

[0105] 2) Prepare a 1.5% SA solution using PBS buffer (10 mM, pH 7.5);

[0106] 3) Accurately weigh 25 mg of the prepared Ca3(PO4)2@PEG-Lip, disperse it in 5 mL of SA solution, vortex mix it, and then add it dropwise into a magnetically stirred CaCl2 solution (2%, m / v) using a peristaltic pump (4000 μL / min). The calcium alginate gel coats the Ca3(PO4)2@PEG-Lip crystals to form gel microspheres. The gel microspheres are then removed using a filter with a mesh size of approximately 0.5 mm and washed with a small amount of purified water to obtain CA-Gel@Ca3(PO4)2@PEG-Lip gel microspheres with dual embedded activated lipase.

[0107] Accurately weigh the lipase solution equivalent to 25 mg of the prepared Ca3(PO4)2@PEG-Lip enzyme and disperse it in 5 mL of SA solution. The remaining steps and methods refer to the preparation method of CA-Gel@Ca3(PO4)2@PEG-Lip mentioned above to prepare gel microspheres encapsulating activated lipase (CA-Gel@PEG-Lip).

[0108] (2) Characterization of macroscopic morphology

[0109] According to the three methods in Example 1, we prepared the activated lipase-encapsulating crystal flower Ca3(PO4)2@PEG-Lip, the activated lipase-encapsulating gel microspheres CA-Gel@PEG-Lip, and the double-encapsulated activated lipase inner flower gel microspheres CA-Gel@Ca3(PO4)2@PEG-Lip. We used a mobile phone camera to photograph the appearance of each of the three and observe their macroscopic morphology. The experimental results are shown in Figure 1. Figure 8 shown.

[0110] Twenty CA-Gel@PEG-Lip or CA-Gel@Ca3(PO4)2@PEG-Lip gel microspheres were randomly selected from the same batch of prepared microspheres. The surface moisture was dried and the diameter of each microsphere was measured at three different positions using a vernier caliper to calculate the average value.

[0111] The picture taken by the camera is displayed ( Figure 8 ), activated lipase was embedded in Ca3(PO4)2 crystals to prepare Ca3(PO4)2@PEG-Lip crystal flowers, which had a white powdery macroscopic appearance ( Figure 8 a); The activated free lipase was embedded in the calcium alginate gel microspheres to prepare CA-Gel@PEG-Lip gel microspheres, whose macroscopic morphology was transparent ellipsoidal ( Figure 8 b); If the calcium phosphate crystal flower (Ca3(PO4)2@PEG-Lip) embedded in the activated lipase is coated in the calcium alginate gel microsphere to prepare the double embedded activated lipase inner flower gel microsphere CA-Gel@Ca3(PO4)2@PEG-Lip, its macroscopic morphology is white elliptical sphere ( Figure 8 c), this is because the white crystal flowers of Ca3(PO4)2@PEG-Lip are embedded in the gel network inside the microspheres, making the gel microspheres appear white.

[0112] The experimental results show that the diameter of single gel microspheres prepared by the two methods is about 2 mm ( Figure 8 ), a filter with a mesh size of about 0.5 mm can be used to achieve rapid recovery.

[0113] (3) Scanning electron microscope (SEM) observation

[0114] The cross-sectional micromorphology of the crystal flowers or gel microspheres was observed using a scanning electron microscope (SEM).

[0115] 1) Morphological characterization of Ca3(PO4)2@PEG-Lip crystal flowers

[0116] Through SEM observation ( Figure 9 ), Ca3(PO4)2@PEG-Lip crystal powder microscopically resembles a dahlia ( Figure 9 a), the crystal flower is composed of a lamellar structure with a diameter of about 1 μm ( Figure 9 b, c). The dense pores between the flower-shaped lamellae provide a large specific surface area. This not only provides ample physical space for the lipase to be immobilized, but also facilitates mass transfer of the substrate through the pores into the crystal flower, allowing it to fully contact the enzyme molecules within and promote the catalytic reaction.

[0117] The formation process of Ca3(PO4)2@PEG-Lip can be described by Figure 10 Description: In a mixed system containing lipase, PEG 8000, CaCl2 and PBS buffer, PEG 8000 enables lipase to maintain and stabilize in an open conformation; CaCl2 2+ With PO4 3- Ca2(PO4)3 nanocrystals are generated; Ca on the surface of the crystals 2+ Coordinates with the amide groups in the lipase protein backbone to form Ca3(PO4)2-Lip + The complex surface repeats the process of crystallization and coordination, and then grows into a layered structure. These layered structures depend on Ca 2+ The enzyme protein molecules self-assemble with the binding sites of lipase, acting as adhesives, and eventually assemble the lamellar structure into Ca3(PO4)2@PEG-Lip crystal particles with a microscopic flower-like structure.

[0118] Through SEM observation ( Figure 11 ), the capsule surface of CA-Gel@Ca3(PO4)2@PEG-Lip gel microspheres is uneven ( Figure 11 a), peeling off the capsule membrane reveals a multi-layer gel network inside ( Figure 11 b), the pores between the networks range from 0 to 100 μm ( Figure 11 b, c), which provides sufficient physical space for the lipase embedded therein, and is also beneficial to the mass transfer of substrate and full contact with enzyme molecules during the catalytic process, as well as the release of catalytic products.

[0119] The complete preparation process of CA-Gel@Ca3(PO4)2@PEG-Lip can be Figure 12First, the free lipase is activated to an activated conformation by a surfactant and then embedded in Ca3(PO4)2 crystals to produce Ca3(PO4)2@PEG-Lip crystal flowers. This is then mixed with a 1.5% by weight SA solution and then dripped with a 2% by weight calcium chloride solution, ultimately producing CA-Gel@Ca3(PO4)2@PEG-Lip gel microspheres that double-encapsulate the activated lipase.

[0120] In the technical route of lipase immobilization in this application, in addition to introducing the surfactant PEG 8000 as the lipase activation factor, Ca 2+ It also plays three crucial roles: First, Ca 2+ It is also an activation factor that promotes the catalytic ability of lipase; the second is Ca 2+ It is the key element to form Ca3(PO4)2-Lip complex; thirdly, Ca 2+ It is the key element in forming a strong cross-linked structure of the gel.

[0121] (4) Composition analysis

[0122] The microstructure of the immobilized enzyme was analyzed by confocal laser scanning electron microscopy (CLSM), Fourier transform infrared spectroscopy (FTIR) and energy dispersive spectroscopy (EDS).

[0123] EDS detection of Ca3(PO4)2@PEG-Lip crystal flowers ( Figure 13 ), it is not difficult to find C, N, O, Na, P and Ca 2+ The existence of Figure 13 a). The carbon and nitrogen elements are components of lipase; the oxygen content is as high as 44.31%, derived partly from the lipase and partly from the Ca₃(PO₄)₂ crystals. In addition to the above three elements, calcium accounts for 26.95%, followed by phosphorus at 13.5%.

[0124] The Ca3(PO4)2@PEG-Lip crystal flower was prepared using Nile blue labeled lipase, and the red fluorescence ( Figure 13 b), indicating that the crystal flowers contain lipase protein.

[0125] The composition of the immobilized enzyme was characterized by FTIR spectroscopy. Figure 14 The a, b, and c lines are the infrared spectra of Ca3(PO4)3, free lipase, and Ca3(PO4)2@PEG-Lip, respectively. Free Lipase has a wavelength of 1700-1600 cm -1The peaks in the region are caused by the stretching vibration of acyl groups in the protein structure, among which 1655 cm -1 It is a typical absorption peak of lipase, attributed to the -C=O stretching vibration of the amide bond; 2800-3200cm -1 The broad and strong band at 1028cm is attributed to the stretching vibration of -CH2 and -CH3. -1 The broad peak at 561 cm is attributed to the stretching vibration of PO. -1 and 602cm -1 The peak at is attributed to the bending vibration absorption of O=PO. Compared with spectra a and b, spectrum c has no new absorption peaks and obvious peak shifts, and has the typical absorption peaks in a and b.

[0126] The combined analysis results of SEM, EDS, CLSM and FTIR show that this paper successfully prepared the crystal flower Ca3(PO4)2@PEG-Lip encapsulating activated lipase, which laid the foundation for the preparation of inner flower gel microspheres CA-Gel@Ca3(PO4)2@PEG-Lip with dual encapsulation of activated lipase.

[0127] Example 5: Analysis of enzymatic properties of immobilized lipase

[0128] According to the determination method in Example 2, the kinetics of lipase at three stages in the preparation process were studied. As shown in Table 2, the K m 0.13mM, V max is 73.69 μm / min; the K of the crystal flower Ca3(PO4)2@PEG-Lip embedded with activated lipase is m 0.14mM, V max The kinetic parameters of the two are similar, indicating that the catalytic activity of lipase is almost lost during the process of being embedded in Ca3(PO4)2 crystals. The reason for this is that during the preparation of Ca3(PO4)2@PEG-Lip crystals, two activating factors of the enzyme, Ca 2+ and PEG8000, and the preparation process is self-assembled and the operating conditions are mild, so that the structure and enzyme activity of the lipase protein molecule are effectively protected.

[0129] According to Table 2, the K m 0.16mM, V maxIt is 58.82 μm / min. Compared with Free Lipase and Ca3(PO4)2@PEG-Lip, the substrate affinity is reduced and the reaction rate is reduced. The reason is that after the lipase is secondary embedded in the gel, the mass transfer resistance between the substrate and the enzyme molecules fixed in the double-layer "armor" increases, and thus the reaction rate decreases.

[0130] Table 2

[0131] enzymes <![CDATA[K m (mM)]]> <![CDATA[V max (μM / min)]]> Free Lipase 0.13 73.69 <![CDATA[Ca3(PO4)2@PEG-Lip]]> 0.14 71.07 <![CDATA[CA-Gel@Ca3(PO4)2@PEG-Lip]]> 0.16 58.82

[0132] Example 6: Reusability of immobilized lipase

[0133] Enzyme activity was measured using the same enzyme batch for activated lipase-encapsulating crystals (Ca3(PO4)2@PEG-Lip), dual-encapsulating activated lipase-encapsulating gel microspheres (CA-Gel@Ca3(PO4)2@PEG-Lip), and activated lipase-encapsulating gel microspheres (CA-Gel@Ca3(PO4)2@PEG-Lip). After the reaction, the Ca3(PO4)2@PEG-Lip crystals were recovered by high-speed centrifugation. The CA-Gel@Lip and CA-Gel@Ca3(PO4)2@PEG-Lip gel microspheres were recovered using a 0.5 mm filter. The three immobilized lipases were recycled 10 times, and the changes in enzyme activity during recycling were compared using relative enzyme activity.

[0134] Depend on Figure 15 It can be seen that after 10 cycles, the enzyme activity retention rates of the three immobilized lipases are as follows from large to small: CA-Gel@Ca3(PO4)2@PEG-Lip (83.93%) > CA-Gel@Lip (58.04%) > Ca3(PO4)2@PEG-Lip (51.07%).

[0135] It is obvious that CA-Gel@Ca3(PO4)2@PEG-Lip has the highest enzyme activity retention rate. Analysis shows that in calcium alginate gel microspheres, the Ca3(PO4)2 crystal structure is effectively protected; under the "double-layer armor" protection of Ca3(PO4)2 crystals and calcium alginate gel, lipase is not easily lost from the gel microspheres, and operational stability is significantly enhanced. In addition, the particle size of CA-Ge1@Ca3(PO4)2@PEG-Lip gel microspheres is large (about 2mm), and they can be quickly recovered and reused using a filter with a pore size of about 0.5mm. The recovery operation is convenient and fast, and does not require the use of special instruments and equipment such as low-temperature centrifuges. This not only avoids the damage of centrifugal force to enzyme molecules and carrier materials, but also reduces the cost of recovery operations.

[0136] In addition, from Figure 15It can also be seen that after 10 cycles, the activated lipase-encapsulating crystal flower, Ca3(PO4)2@PEG-Lip, retained the lowest enzyme activity, only about 51.07%. This is because the Ca3(PO4)2@PEG-Lip crystal powder cannot be recovered by filtration and must be recovered in a low-temperature centrifuge. During centrifugation and removal of the supernatant, the fragile crystal structure of Ca3(PO4)2@PEG-Lip is easily broken by centrifugal force, resulting in a large loss of lipase.

[0137] The diameter of the gel microspheres CA-Gel@PEG-Lip, which encapsulates free activated lipase, is similar to that of the inner flower gel microspheres CA-Gel@Ca3(PO4)2@PEG-Lip, which double-encapsulates activated lipase, both of which are about 2 mm. Therefore, a filter with a pore size of about 0.5 mm is also used for recovery. After 10 cycles, the enzyme activity retention rate of CA-Gel@Lip (58.04%) is significantly lower than that of CA-Gel@Ca3(PO4)2@PEG-Lip (83.93%), and only slightly higher than that of Ca3(PO4)2@PEG-Lip (51.07%). Analysis shows that when free lipase is immobilized using gel microspheres alone, the pores formed by the gel network are of different sizes, and the enzyme molecules embedded in the large pores are easily lost during the catalytic process. However, if the crystal flowers encapsulating activated lipase are coated in gel microspheres, the crystal flowers are firmly confined in the pores of the gel grid, and the Ca2+ on the surface of the crystals is also confined to the pores of the gel grid. 2+ The cross-linking effect with the residual -COO- on the surface of the gel can effectively prevent the loss of lipase and ensure the catalytic activity and operational stability of the lipase.

[0138] Example 7: Stability of immobilized lipase

[0139] Thermal stability determination: Free lipase and double-encapsulated activated lipase intracellular gel microspheres CA-Gel@Ca3(PO4)2@PEG-Lip were placed at 20-60°C, kept warm for 12 hours, and then quickly cooled to 4°C. The residual enzyme activity was determined according to the method in Example 2 and compared with the initial enzyme activity before the thermal stability determination to investigate its temperature stability.

[0140] Acid-base stability determination: Free Lipase and CA-Gel@Ca3(PO4)2@PEG-Lip were stored in PBS buffer at pH 6-10 for 12 h, and the residual enzyme activity was determined according to the method in Example 2. The residual enzyme activity was compared with the initial enzyme activity before the acid-base stability determination to investigate the pH stability.

[0141] like Figure 16a shows that after CA-Gel@Ca3(PO4)2@PEG-Lip was stored in the temperature range of 30-70℃ for 12h, its enzyme activity retention rate was higher than that of the free enzyme, which was the result of the dual protection of Ca3(PO4)2 crystals and calcium alginate gel on lipase; especially in the range of 40-50℃, the enzyme activity of CA-Gel@Ca3(PO4)2@PEG-Lip was relatively stable; after the temperature rose to 55℃, the enzyme activity of CA-Gel@Ca3(PO4)2@PEG-Lip began to drop sharply, which was related to the destruction of the enzyme protein molecular structure caused by high temperature.

[0142] Depend on Figure 16 Figure b shows that within the pH range of 6-10, the enzyme activity retention rate of CA-Gel@Ca3(PO4)2@PEG-Lip after 12 hours of storage is also higher than that of the free enzyme to varying degrees. Clearly, under the "double armor" of Ca3(PO4)2 crystals and calcium alginate gel, the lipase embedded therein has better pH and thermal stability than the free enzyme. Therefore, the preparation of double-encapsulated activated lipase-containing hydrogel microspheres CA-Gel@Ca3(PO4)2@PEG-Lip technology is meaningful for the industrial production of food and medicine.

Claims

1. A preparation technology for double-encapsulated activated lipase inner gel microspheres, characterized in that: The following steps are involved: (1) A PBS buffer solution containing lipase F-AP 15 was mixed with a surfactant to obtain a mixed solution, and a 250 mM CaCl2 solution was added to the mixed solution for crystallization to obtain calcium phosphate crystal flowers encapsulating activated lipase; the surfactant was PEG 8000, and the concentration of the added surfactant was 75 mM; The concentration of lipase in the PBS buffer containing lipase was 0.15 mg / mL; The crystallization time was 12 h and the crystallization temperature was below 4°C; (2) Dissolving sodium alginate in PBS buffer to obtain a sodium alginate solution; The mass volume percentage concentration of sodium alginate solution is 1.5%; (3) Calcium phosphate crystal flowers encapsulating activated lipase are dispersed in a sodium alginate solution to obtain a mixed system; the mixed system is then added dropwise into a cross-linking agent CaCl2 solution with a mass volume percentage concentration of 2% to cross-link, thereby obtaining inner flower gel microspheres with double encapsulation of activated lipase, wherein the diameter of the inner flower gel microspheres is 2 mm.

2. Double-encapsulated inner flower gel microspheres with activated lipase prepared by the preparation technology as described in claim 1.

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