Immobilized lipase catalytic hydrolysis reaction system based on Pickering emulsion as well as construction method and application of immobilized lipase catalytic hydrolysis reaction system

Abstract

The invention discloses an immobilized lipase catalytic hydrolysis reaction system based on Pickering emulsion as well as a construction method and application of the immobilized lipase catalytic hydrolysis reaction system, and belongs to the technical field of biological enzymes. The immobilized lipase which is long in storage time and good in emulsifying property is obtained through a large number of experimental optimization by utilizing immobilized adsorption of bentonite on lipase; the immobilized lipase catalytic hydrolysis reaction system based on the Pickering emulsion is prepared by further optimizing the process parameters, the optimized catalytic hydrolysis reaction system has a high hydrolysis rate on tuna oil, and the content of n-3 PUFAs after hydrolysis is remarkably increased; the optimized catalytic hydrolysis reaction system also realizes stable recovery of the immobilized lipase, the relative activity of the constructed catalytic hydrolysis reaction system is still kept at 65% or above after the constructed catalytic hydrolysis reaction system is circulated for 6 times, and the system can be applied to the field of grease hydrolysis and has the potential of industrial popularization and application.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioenzymes, and particularly relates to an immobilized lipase-catalyzed hydrolysis reaction system based on Pickering emulsion, a construction method thereof, and an application thereof. Background Art

[0002] Lipase (EC3.1.1.3), also known as triglyceride hydrolase, belongs to the serine hydrolase class and can catalyze various reactions such as ester hydrolysis, ester synthesis, and transesterification. Lipases are widely distributed in animals, plants, and microorganisms, and among them, microorganisms are the main source of industrial lipases. The typical catalytic active center of lipase has a "lid" structure, and the α-helix in this structure is amphiphilic, with its outer surface being relatively hydrophilic and the inner surface facing the catalytic center being relatively hydrophobic. When lipase binds to the oil / water interface, the "lid" opens, the catalytic active center is exposed, and the substrate enters this hydrophobic channel to bind to the active center, thereby activating the catalytic activity of lipase. This phenomenon is called "interface activation" and is a unique property of lipase.

[0003] Due to the "interface activation" property, lipase can only exert its maximum activity at the oil / water interface. Therefore, the size of the interface area determines the efficiency of the catalytic hydrolysis reaction, but most substrates of lipase are insoluble in water. In the traditional oil / water two-phase system, due to the very small interface area, lipase and the substrate do not contact sufficiently, resulting in low catalytic efficiency. The suspension system constructed by immobilized enzymes is an important medium for lipase reactions. However, currently, the methods for immobilizing lipase usually require chemical modification of solid particles or / and enzyme molecules, which not only increases the complexity of the process but also has an adverse effect on enzyme activity, and problems such as enzyme molecule shedding, enzyme activity reduction, and limited diffusion of substrates and products are likely to occur during the catalytic process. The Pickering emulsion stabilized by lipase-loaded solid particles is a new type of two-phase catalytic reaction system. Its oil / water interface area is huge and lipase is directly anchored on the oil / water interface, so the catalytic efficiency is extremely high, and it is a new type of two-phase catalytic reaction system with great potential. However, common commercial immobilized enzymes lack emulsifying ability, and currently, constructing immobilized lipase with emulsifying properties usually requires a complex process and involves various physical and chemical reactions such as activation and crosslinking. The process is cumbersome, costly, and prone to enzyme activity loss, which limits the practical application of this new type of two-phase catalytic system. Summary of the Invention

[0004] Based on the above problems, the object of the present invention is to provide an immobilized lipase-catalyzed hydrolysis reaction system based on Pickering emulsion, its construction method and application. The present invention screens a cheap porous carrier with emulsifying ability, optimizes experimental parameters to obtain an immobilized lipase with relatively strong activity and good emulsifying ability, and the constructed Pickering emulsion-catalyzed hydrolysis reaction system. The hydrolysis system can not only realize the recycling of the immobilized lipase, but also significantly increase the content of n-3 PUFAs in the tuna oil hydrolysis system.

[0005] To achieve the above object of the invention, the present invention adopts the following technical solutions: The present invention provides a construction method of an immobilized lipase-catalyzed hydrolysis reaction system based on Pickering emulsion, and the construction method specifically includes the following steps: (1) Add lipase to a buffer to form a mixed lipase solution; add a porous carrier to the mixed lipase solution to immobilize and adsorb the lipase, centrifuge to remove the supernatant; then wash with the buffer, centrifuge twice and dry to obtain the immobilized lipase; (2) Disperse the immobilized lipase in a buffer solution, add an oil-phase substrate, and perform high-speed dispersion to obtain a catalytically active Pickering emulsion; (3) Hydrolyze the catalytically active Pickering emulsion in a water bath to construct an immobilized lipase-catalyzed hydrolysis reaction system based on Pickering emulsion; (4) After catalytic hydrolysis, centrifuge to precipitate, recover the immobilized lipase, and continue to cycle steps (2) and (3) to realize the recycling of the immobilized lipase.

[0006] Further, in the step (1), the type of the porous carrier is bentonite, the mass-volume ratio of the bentonite to the lipase is 1:1 to 1:2.5, and the immobilization adsorption time of the lipase is 8 to 16 h.

[0007] Further, in the step (1), the pH value range of the buffer is 5.0 to 8.0.

[0008] Further, in the step (2), the oil-phase substrate is at least one of n-heptane, soybean oil, peanut oil, and tuna oil, and the oil-phase fraction φ of the oil-phase substrate is 0.1 to 0.6.

[0009] Further, in the step (2), based on the volume of the Pickering emulsion-catalyzed hydrolysis reaction system, the mass-volume ratio of the immobilized lipase is 3% to 8%.

[0010] Further, in step (3), the hydrolysis temperature is 40 - 60 °C, and the hydrolysis pH value is 5.0 - 8.0.

[0011] Further, in step (4), the number of cyclic operations is 1 - 8 times.

[0012] Further, in step (1), the optimal preparation process of the immobilized lipase is as follows: the volume - mass ratio of CalB to bentonite is 2:1, the buffer pH = 7.0, and the fixed adsorption time is 12 h.

[0013] The present invention also provides the immobilized lipase prepared by the above - mentioned method, whose application temperature range is 20 - 65 °C, and the pH is 6.5 - 8.5; the stable storage time of the immobilized lipase is 1 - 60 days.

[0014] The present invention also provides the application of the immobilized lipase - catalyzed hydrolysis reaction system based on Pickering emulsion obtained by the above - mentioned construction method in hydrolyzing tuna oil.

[0015] Further, when the immobilized lipase hydrolysis system based on Pickering emulsion hydrolyzes tuna oil, the optimal conditions are: pH is 7.5, temperature is 55 °C, the mass - volume ratio of immobilized CalB is 6%, and the hydrolysis time is 24 h.

[0016] Further, the immobilized lipase - catalyzed hydrolysis reaction system based on Pickering emulsion significantly increases the content of n - 3 PUFAs in tuna oil.

[0017] Compared with the prior art, the advantages and positive effects of the present invention are as follows: 1. The method for preparing the immobilized lipase with emulsifying properties in the present invention is simple and economical. The carrier is green, safe, easy to obtain, and very cheap. The immobilization method is simple and effective, and has strong industrial operability, especially suitable for large - scale industrial application.

[0018] 2. The immobilized CalB prepared in the present invention has emulsifying ability, can form a stable Pickering emulsion, effectively increases the interfacial area required for lipase reaction, solves the problem of low catalytic efficiency caused by interfacial activation of lipase, and thus improves the catalytic efficiency of lipase.

[0019] 3. The immobilized lipase prepared by the present invention can form a Pickering emulsion with good stability at a homogenization time of 3 min and a homogenization speed of 11000 rpm, with no obvious oil leakage phenomenon, uniform droplet morphology, and a cream - separation index as high as 95%. And the emulsion can maintain good stability within the range of oil - phase fraction φ = 0.1 - 0.6.

[0020] 4. The immobilized lipase-catalyzed hydrolysis reaction system based on Pickering emulsion provided by the present invention realizes the efficient hydrolysis of tuna oil by optimizing the construction method through a large number of experiments. During the efficient hydrolysis, the content of n-3 PUFAs is 195.3 times that of the tuna oil before hydrolysis and 4.98 times that of the n-3 PUFAs content in the commercially available immobilized lipase-catalyzed hydrolysis of fish oil. Moreover, the immobilized lipase can be reused multiple times.

[0021] 5. The immobilized lipase-catalyzed hydrolysis reaction system based on Pickering emulsion constructed by the present invention. In the novel reaction system, each droplet can act as a microreactor, which has significant advantages such as a large oil / water interface area, environmental friendliness, simple product separation and purification, and reusability. It is a very promising novel two-phase catalytic hydrolysis reaction system for lipase. Brief Description of the Drawings

[0022] Figure 1 Appearance diagrams of emulsions formed by different porous carriers and commercially available immobilized lipases; Figure 2 Influence diagrams of different porous carriers on the creaming index and oil leakage rate of emulsions; Figure 3 Type diagram of the Pickering emulsion formed by bentonite; Figure 4 Standard curve diagram of bovine serum albumin; Figure 5 Standard curve diagram of p-NP; Figure 6 Influence diagrams of different lipase concentrations, pH values, and adsorption times on the immobilization rate of lipase; Figure 7 Fourier transform infrared spectrum diagram of immobilized CalB; Figure 8 Atomic force microscope diagram of bentonite and immobilized lipase; Figure 9 Data diagrams of the optimal pH and pH stability of free CalB and immobilized CalB; Figure 10 Data diagrams of the optimal temperature and temperature stability of free CalB and immobilized CalB; Figure 11 Data diagrams of the thermal inactivation kinetics of immobilized CalB and free CalB; Figure 12 Enzymatic reaction kinetic parameter diagrams of immobilized CalB and free CalB; Figure 13 Influence of metal ions, organic reagents, denaturants, and ultrasound on the activities of three lipases; Figure 14 Data analysis chart for the storage stability of different lipases; Figure 15 Chart showing the effects of different homogenization times on the appearance, emulsion microstructure, CI, and average droplet size of Pickering emulsions with catalytic activity; Figure 16 Chart showing the effects of different homogenization speeds on the appearance, emulsion microstructure, CI, and average droplet size of Pickering emulsions with catalytic activity; Figure 17 Chart showing the effects of different oil phase fractions on the appearance, emulsion microstructure, CI, and average droplet size of Pickering emulsions with catalytic activity; Figure 18 Chart showing the effects of pH, temperature, and immobilized CalB content in the hydrolysis process system on the hydrolysis rate of tuna oil; Figure 19 Data chart for the reusability evaluation of the immobilized lipase-catalyzed hydrolysis reaction system based on Pickering emulsions; Figure 20 Chart showing the effects of the immobilized lipase-catalyzed hydrolysis reaction system based on Pickering emulsions, commercially available immobilized lipase based on traditional two-phase systems, and free CalB on the hydrolysis rate of tuna oil. Detailed implementation manners

[0023] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0024] In the present invention, the mass-volume ratio w / v, the mass unit is g, and the volume unit is mL.

[0025] Example 1: Screening of porous carriers and evaluation method for indicators of immobilized lipases (1) Screening of porous carriers Select 0.5 g of porous carriers: bentonite (1 μm grade), diatomite (7 μm grade, 13 μm grade, 22 μm grade, 29 μm grade), silica (20 nm grade, 30 nm grade, 50 nm grade), and place them in a 50 mL test tube. Then, add sodium acetate buffer solution and paraffin oil, and the volume ratio of the buffer solution to paraffin oil is 4:1. Use a homogenizer to homogenize at 12,000 rpm for 3 min, observe the appearance of the emulsion, and analyze the emulsion type, creaming index, and oil leakage rate.

[0026] From Figure 1As can be seen from a, except for bentonite which can form Pickering emulsions, other porous carriers cannot form stable Pickering emulsions or even the water and oil phases are completely separated without other complex pretreatment; Figure 2 This conclusion was further confirmed by the oil leakage rate and creaming index: the emulsion formed by bentonite has a uniform texture and strong stability, with the lowest oil leakage rate of only 5%, while the emulsions formed by other carriers have poor stability and high oil leakage rates. Figure 1 As can be seen from b, commercially available immobilized lipase not only cannot form emulsions, but also the particles are broken due to homogenization. Figure 3 It can be seen that the emulsion type formed by bentonite is oil-in-water.

[0027] 2. Index evaluation method of immobilized lipase (1)Determination of lipase loading efficiency Lipase loading efficiency is a key index to measure the binding effect between lipase and carrier during the immobilization process. The lipase loading efficiency is determined by the Bradford protein quantification method and the following steps.

[0028] After lipase is immobilized on bentonite, the supernatant collected by the last centrifugation is placed in a sample bottle and stored at 4 °C. The initial protein content of each system and the protein content in the supernatant after centrifugation are measured by the Bradford method. Then, the lipase loading efficiency is calculated according to the following formula:

[0029] Dissolve 100 mg of Coomassie Brilliant Blue G250 in 50 mL of 95% ethanol, add 100 mL of 85% W / V phosphoric acid, dilute to 1000 mL with distilled water, and filter for later use. Then prepare a standard protein solution, and use 0.2 mol / L pH 7.0 phosphate buffered saline (PBS) to prepare a 1 mg / mL standard protein solution of crystalline bovine serum albumin. Dilute the standard protein solution with PBS to protein concentration gradient standard solutions with protein concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, and 1.0 mg / mL. Take 0.1 mL from each standard solution into a test tube, add 5 mL of Coomassie Brilliant Blue solution and shake well. Then, within 5 - 20 min, with the blank tube as the control, measure the absorbance at a wavelength of 595 nm. With the standard protein concentration as the abscissa and A595 as the ordinate, draw a protein standard curve as Figure 4 shown. The established standard curve of protein concentration is Y = 0.55132X + 0.12311, and the regression coefficient R2 = 0.99089.

[0030] (2)Determination of the recovery rate of lipase activity Lipase can catalyze the hydrolysis of p-nitrophenyl palmitate (p-NPP) to produce p-nitrophenol (p-NP). p-Nitrophenol exhibits yellow under alkaline conditions and has a characteristic absorption peak at a wavelength of 410 nm. By measuring the change in absorbance, the lipase activity can be calculated. Mix 0.4 mL of the substrate p-NPP solution (15 mmol / L, dissolved in isopropanol) with 3.5 mL of 50 mmol / L Tris-HCl buffer (pH 7.02, containing 1 g / L gum arabic). After preheating the mixture in a constant temperature water bath shaker at 37 °C for 5 min, add 0.1 mL of the enzyme solution to initiate the reaction. After 5 min, add 1.5 mL of a chloroform and isoamyl alcohol mixed solution (volume ratio 24:1) to terminate the reaction. Centrifuge the resulting mixture at 10000 rpm at 4 °C for 5 min, collect the supernatant, measure the absorbance at 410 nm using a spectrophotometer, and compare it with the enzyme-free group. The lipase activity unit is defined as the amount of enzyme required to catalyze the formation of 1 μmol of p-NP from p-NPP per minute. Calculate the lipase activity recovery rate according to the following formula:

[0031] Prepare p-NP ethanol standard solutions with different concentration ranges (20 μmol / L, 40 μmol / L, 60 μmol / L, 80 μmol / L, 100 μmol / L, 120 μmol / L, 140 μmol / L), perform colorimetry at 410 nm, use the concentration as the abscissa, and plot the standard curve of p-NP with the measured absorbance value as the ordinate as Figure 5 shown. The established standard curve of p-NP is Y = 0.01039X - 0.00164, and the regression coefficient R 2 = 0.9999.

[0032] Example 2: A method for constructing an immobilized lipase-catalyzed hydrolysis reaction system based on Pickering emulsion (1) Add 1 mL CalB and 9 mL sodium acetate buffer (pH = 4) to a conical flask to form a mixed enzyme solution, and add 1 g bentonite. Place on a magnetic stirrer (800 rpm, 25°C) and stir for more than 2 h, remove and place in a constant temperature environment at 4°C to allow the bentonite to swell. The fixation time is 2 h to fully adsorb the lipase. Use a high-speed refrigerated centrifuge (TGL 16M) to centrifuge at 5000 rpm, 4°C, and 5 min. After centrifugation, add buffer to wash the precipitate and centrifuge again. The precipitate is freeze-dried to obtain immobilized CalB.

[0033] (2) Prepare 20 mL of catalytically active Pickering emulsion: Take 0.2 g of immobilized CalB in a test tube, the mass volume ratio of immobilized CalB to the entire catalytically active Pickering emulsion is 1% (w / v), then add n-heptane with an oil phase fraction φ=0.1, and add pH=5 sodium acetate buffer until the total solution in the test tube is 20 mL, use a homogenizer to disperse at 5000 rpm for 1 min to obtain a catalytically active Pickering emulsion. Observe the appearance and microscopic image of the emulsion, and measure its creaming index and particle size.

[0034] (3) Prepare 20 mL of Pickering emulsion-based immobilized lipase-catalyzed hydrolysis reaction system solution: 0.4 g of immobilized CalB was placed in a test tube, the immobilized CalB accounted for 2% (w / v) of the entire Pickering emulsion-based lipase-catalyzed hydrolysis reaction system, add tuna oil with an oil phase fraction of φ=0.2, and add sodium acetate buffer with a pH of 7.5 to a total volume of 20 mL, use a homogenizer to disperse at high speed at 11000 rpm for 3 minutes to prepare the Pickering emulsion-based immobilized lipase-catalyzed hydrolysis reaction system, and take samples after 24 hours of water bath at 35°C for sufficient hydrolysis. Determine the hydrolysis rate of tuna oil.

[0035] (4) Experiment on the reusability of immobilized enzyme in the Pickering emulsion-based immobilized lipase catalytic hydrolysis system (20 mL): tuna oil was added to make the oil phase volume fraction reach φ = 0.2; 1.2 g of immobilized CalB was added to make its mass ratio account for 6% (w / v) of the volume of the entire Pickering emulsion-based immobilized lipase catalytic hydrolysis system; finally, sodium acetate buffer (pH = 7.5) was added to make the total volume of the mixed system reach 20 mL. The immobilized lipase catalytic hydrolysis system based on Pickering emulsion was prepared by high-speed dispersion at 11000 rpm for 3 min using a homogenizer. After being incubated in a water bath at 55 °C for 6 h, the mixture was passed through 10000 rpm and stirred for 1 h. gCentrifuge for 5 min, collect the precipitated immobilized CalB to determine its activity change, and collect the oil phase to determine the hydrolysis rate of the system. This is considered as one cycle.

[0036] (5) Repeat the operation steps in (4). The reusability of the immobilized lipase in the Pickering emulsion-based catalytic reaction system is investigated using the relative activity (%). The relative activity (%) is defined as the percentage of the hydrolysis rate of tuna oil in the nth reaction cycle to the hydrolysis rate of tuna oil in the first reaction.

[0037] Example 3: Compare the effects of different ratios of the amount of CalB enzyme solution to bentonite on the immobilization rate of the immobilized lipase The difference between this example and Example 2 is that in step (1), different amounts of CalB and sodium acetate buffer solution are added to the conical flask to form a mixed enzyme solution, and the others are the same as in Example 2. The specific amounts are shown in Table 1: Table 1: Data table of different CalB addition amounts

[0038] Determine the total protein immobilization amount value and the immobilization rate of the immobilized CalB obtained with different amounts of CalB. According to Figure 6 a in, when choosing 2 mL of CalB enzyme solution and 1 g of bentonite, the total protein immobilization amount is 7.65 mg, and there is no obvious change in the total protein immobilization amount of the 2.5E+M group. It can be seen that the volume-to-mass ratio of CalB to bentonite of 2:1 (w / v) is the best choice, and the immobilization rate is 53.95% at this time.

[0039] Example 4: Compare the effects of sodium acetate buffer solutions with different pH values on the immobilization rate of lipase Add 2 mL of CalB to conical flasks containing sodium acetate buffer solutions with pH = 4.0, 5.0, 6.0, 7.0, and 8.0 respectively. Each group of sodium acetate buffer solution is 8 mL to form a mixed enzyme solution, and then add 1 g of bentonite. Place it on a magnetic stirrer (800 rpm, 25 °C) and stir for more than 2 h. Take it off and place it in a constant temperature environment at 4 °C. The bentonite swells, and the fixing time is 2 h to fully adsorb the lipase. Use a high-speed refrigerated centrifuge (TGL 16M) to perform centrifugation at 5000 rpm, 4 °C, and 5 min. After centrifugation, add buffer solution to wash the precipitate and then centrifuge again, and freeze-dry the precipitate to obtain immobilized CalB.

[0040] According to Figure 6 it can be known that Figure 6 b in shows that when the pH is 7.0, the immobilization rate is the highest, which is 81.02%.

[0041] Example 5: Effect of different immobilization adsorption times on the immobilization rate of immobilized lipase Add 2 mL of CalB and 8 mL of sodium acetate buffer (pH = 7.0) into a conical flask to form a mixed enzyme solution, and add 1 g of bentonite. Place it on a magnetic stirrer (800 rpm, 25 °C) and stir for more than 2 h. Take it off and place it in a constant temperature environment at 4 °C. The bentonite swells and adsorbs and immobilizes the lipase. The immobilization adsorption times are 1 h, 2 h, 4 h, 8 h, 10 h, 12 h, and 16 h respectively. Use a high-speed refrigerated centrifuge (TGL 16M) to perform centrifugation at 5000 rpm, 4 °C, and 5 min. After centrifugation, add buffer to wash the precipitate and then centrifuge again, and freeze-dry the precipitate to obtain immobilized CalB.

[0042] According to Figure 6 the data of the immobilization rate of lipase with the immobilization adsorption time shown in c, when the immobilization adsorption time is 12 h, the immobilization rate is 87.45%. However, the immobilization adsorption time of 16 h does not produce an obvious effect. Therefore, in this experiment, the optimized immobilization adsorption time is 12 h as the best choice.

[0043] Combined with Examples 2-5, it can be seen that the ratio of different CalB dosages to bentonite, pH, and immobilization adsorption time have a significant impact on the immobilization rate of lipase. Combining the experimental data, the optimal process for immobilizing lipase is: the volume-mass ratio of CalB to bentonite is 2:1, the buffer pH = 7.0, and the immobilization adsorption time is selected as 12 h. At this time, the lipase immobilization rate can reach 87.45%.

[0044] Example 6: Fourier transform infrared spectroscopy (FT-IR) analysis of immobilized CalB The immobilized CalB in this example uses the immobilized CalB obtained with an immobilization adsorption time of 12 h in Example 5.

[0045] Take samples of immobilized CalB, bentonite, and free CalB respectively, mix them with dry potassium bromide (KBr), grind them thoroughly in a mortar until uniform, put the mixture into a tablet pressing mold, and press it into a transparent thin film with a tablet press. Use a Fourier transform infrared spectrometer for measurement. Preheat for 15-30 min and select the Smart iTR diamond ART mode. Use a pure KBr tablet as a reference and scan within 4000-400 cm -1 with a resolution set to 4 cm -1 and the number of scans is 64 times.

[0046] The results are as Figure 7 Bentonite has mainly two characteristic peaks at 1018.2 cm -1The strong absorption peak at [[]] is attributed to the antisymmetric stretching vibration of Si-O-Si in the silicate skeleton. The peak of the lipase amide I band shifts from 1638 cm －1 to 1655.6 cm －1 . However, the amide I band in immobilized CalB indicates that the binding of CalB to bentonite does not affect the structure of the lipase, preserving the native conformation of the lipase and maintaining the bioactive conformation of the lipase. Free CalB has two main amide bands. The peak at 1655.6 cm -1 is the protein amide I band, reflecting the α-helix or β-sheet structure. The peak near 1500 cm -1 is the amide II band, usually corresponding to N-H bending and C-N stretching vibrations. Immobilized CalB retains the amide I band at 1655.6 cm -1 . However, compared with free CalB, if the peak position shifts (such as shifting to a lower wavenumber), it may indicate that the enzyme molecule forms hydrogen bonds with the surface groups of bentonite (such as hydroxyl groups), resulting in a change in the electron cloud density of the C=O bond. The weakening of the peak intensity or the shift of the wavenumber at 1018.2 cm -1 in immobilized CalB suggests that the enzyme molecule loading may affect the vibration mode of the bentonite silicate skeleton, reflecting the physical adsorption or electrostatic interaction between the enzyme and the carrier. The appearance of new peaks at 995.1 cm -1 or 670 cm -1 may correspond to the chemical bonds formed at the enzyme-carrier interface (such as Si-O-C), or the interaction between the interlayer cations of bentonite and the charged groups of the enzyme.

[0047] Example 7: Atomic Force Microscopy Analysis of Immobilized CalB The immobilized CalB in this example is the immobilized CalB obtained in Example 5 with a fixed adsorption time of 12 h.

[0048] Take the powder to be tested obtained by freeze-drying the immobilized CalB and bentonite, dilute the sample concentration to 0.5 mg / mL with sodium acetate buffer, pipette 20 μL of the sample dilution, drop it on a mica sheet, dry the sample with a baking lamp, and then place it on the instrument sample stage to observe the sample. The measurement parameters are set as follows: the scanning range is 5 μm × 5 μm, and the scanning rate is 0.5 Hz.

[0049] As Figure 8 , bentonite swells and agglomerates easily when encountering water, while the immobilized CalB particles are more dispersed, which is more beneficial to constructing a stable immobilized lipase-catalyzed hydrolysis reaction system based on Pickering emulsion.

[0050] Example 8: Study on the Optimal pH and pH Stability of Free CalB and Immobilized CalB The immobilized CalB in this example is the immobilized CalB obtained with an immobilization adsorption time of 12 h in Example 5.

[0051] The immobilized CalB and the substrate p-NPP were placed in a phosphate buffer solution (50 mmol / L) with a pH range of 6.0 - 8.0 and reacted for 10 min under the condition of a 37°C water bath to measure the activity of the immobilized CalB. The optimal pH of the immobilized CalB was studied. The highest enzyme activity value was expressed as 100% in relative activity, and the pH at which the enzyme activity reached the highest was the optimal pH. The pH stability of the immobilized CalB was measured. The immobilized CalB was placed in a phosphate buffer solution (50 mmol / L) with a pH range of 6.0 - 8.0 and maintained for 2 h under the condition of a 25°C water bath to measure the residual activity of the immobilized CalB. Free CalB was used as a control in both measurements.

[0052] As Figure 9 shown in the figure on the left, as the pH value changed, the relative activities of both enzymes showed a certain fluctuating trend. Near pH 7.5, the immobilized CalB reached the peak of relative activity, while the relative activity of free CalB was relatively low. This indicates that the adaptability of CalB alone in an acidic or alkaline environment is limited, and the conformation of its active center changes greatly when deviating from the optimal pH, resulting in a decrease in catalytic efficiency.

[0053] The stability of the two enzymes under different pH conditions is obvious ( Figure 9 in the figure on the right). The immobilized CalB showed higher residual activity than CalB in a wider pH range. Especially at pH 7.0 and pH 7.5, its residual activity was significantly higher than that of CalB. This indicates that the immobilization treatment with bentonite effectively improved the stability of CalB, enabling it to maintain more persistent activity under complex environmental conditions. This enhanced stability may be due to the interaction between bentonite and CalB, which restricts the conformational changes of the enzyme protein, reduces the damage of pH to the active site, and thus maintains the higher activity of the enzyme.

[0054] Example 9: Study on the Optimal Temperature and Temperature Stability of Free CalB and Immobilized CalB The immobilized CalB in this example is the immobilized CalB obtained with an immobilization adsorption time of 12 h in Example 5.

[0055] The immobilized CalB was placed in a phosphate buffer (50 mmol / L) of uniform pH together with the substrate p-NPP and reacted for 10 min under the water bath condition of 20~70°C. The activity of the immobilized CalB was measured to study the optimal temperature of the immobilized CalB. The highest value of enzyme activity was expressed as 100% in relative activity, and the temperature at which the enzyme activity reached the highest was the optimal temperature. The temperature stability of the immobilized CalB was measured. The immobilized CalB was placed in a phosphate buffer (50 mmol / L) of uniform pH and maintained for 2 h under the water bath condition of 20~70°C, and the residual activity of CalB was measured. Free CalB was used as a control in both measurements.

[0056] As Figure 10 (left figure), between 20°C and 70°C, both free CalB and immobilized CalB showed a trend of first increasing and then decreasing. From the relative activity changes under different temperature conditions, it can be seen that as the temperature increased, the relative activities of both enzymes showed a trend of first rising and then falling.

[0057] The change trend of the relative activity of the immobilized CalB was similar to that of free CalB, but at high temperatures, the decline in its activity was smaller. This indicates that the immobilization of bentonite provided a relatively stable microenvironment for CalB, enabling the enzyme to maintain good activity at high temperatures.

[0058] Under the condition that other identical conditions were the same, observe Figure 10 (right figure), between 20~60°C, the residual activities of both free CalB and immobilized CalB were above 50%, and both decreased rapidly after exceeding 50°C. The residual activity of the immobilized CalB was slightly higher than that of free CalB.

[0059] Example 10: Study on thermal inactivation kinetics The immobilized CalB in this example was the immobilized CalB obtained with a fixed adsorption time of 12 h in Example 5.

[0060] The immobilized CalB was incubated in a phosphate buffer (50 mmol / L, pH 7.02) at 60°C, 70°C, and 80°C for 120 min. Then, corresponding samples to be measured were taken out every 20 min at each temperature. After cooling to room temperature, the remaining enzyme activity was measured. According to the remaining enzyme activity data, the inactivation kinetics model was fitted, and a semi-logarithmic plot of the percentage of remaining activity versus time was drawn. The inactivation rate constant (k d ) and half-life (t 1 / 2 ) at different temperatures were calculated through the fitted curve; using the Arrhenius equation, the inactivation energy (E d ) was calculated through the inactivation rate constants at different temperatures and analyzed as shown in Table 2.

[0061] Table 2 Thermal inactivation constants (K d ), half-life (t 1 / 2 ), and inactivation energy (E d ) of free and immobilized CalB

[0062] K d represents the thermal inactivation rate, and a lower value indicates better heat resistance. t 1 / 2 is the time required for the enzyme activity to decrease to 50% of its initial activity, and E d is the inactivation energy.

[0063] As Figure 11 can be seen, compared with free CalB, immobilized CalB significantly reduced the K d value, and it was more significant at 60 °C, that is, K d was reduced by 43%, and t 1 / 2 increased by 49%; however, at higher temperatures, especially at 80 °C, this effect decreased, which may be because at this temperature, the effects of both were weakened, and the protection ability of the carrier for CalB was weakened. Binding to bentonite increased the E d of CalB from 12.49 kJ / mol to 31.21 kJ / mol, an increase of 150%, indicating that binding to bentonite can effectively improve the thermal stability of CalB.

[0064] Example 11: Study on kinetic parameters of enzymatic reactions The immobilized CalB in this example was the immobilized CalB obtained with a fixed time of 12 h in Example 5.

[0065] The reaction kinetic parameters of free CalB and immobilized lipase were determined using the p-NPP hydrolysis method, as Figure 12 shown.

[0066] First, a series of p-NPP solutions with different concentrations (0.5, 1.0, 2.0, 3.0, 4.0, 8.0, 16.0, 24.0 mmol / L) were prepared. Using p-NPP with different concentrations as substrates, the activities of free CalB lipase and immobilized CalB were measured at the optimal pH and optimal temperature of the enzyme, and the kinetic parameters K m and V max were calculated by fitting the Lineweaver Burk equation. The results are shown in Table 3.

[0067] V is the rate of enzymatic reaction. By taking the derivative of absorbance with respect to time, V can be obtained as the rate of enzymatic reaction.

[0068]

[0069]

[0070] In formulas (2-1) and (2-2): V —— Initial rate of enzymatic reaction; [S] —— Substrate concentration; V max —— Maximum reaction rate; K m —— Michaelis constant.

[0071] Table 3 Michaelis constants (K m ) and maximum reaction rates (V max ) of free CalB and immobilized CalB

[0072] As shown in Table 3, compared with free CalB, the increase in K m of immobilized CalB indicates that its affinity weakens after binding to bentonite, because the physical adsorption of lipase by bentonite reduces the contact between the enzyme active center and the substrate P-NPP. V max is 0.699 and decreases to 0.1680 after immobilization.

[0073] Example 12: Exploring the effects of metal ions, organic solvents, denaturants, and ultrasound on the activity of immobilized lipase The immobilized CalB in this example is the immobilized CalB obtained with a fixation time of 12 h in Example 5.

[0074] (1) Commercially available immobilized lipase, free CalB, and immobilized CalB were separately added to several common metal ion solutions (with a concentration of 0.1 mmol / L each). The metal salts used in this study were NiCl2, MgCl2, FeCl3, MnCl2, and CuCl2. After incubating for 2 h at 25°C, the residual enzyme activity of CalB was measured for the three lipases at 37.5°C and pH = 7.0. The effect of metal ions on enzyme activity was expressed as relative activity. The enzyme activity measured for the lipase sample without metal ions was set as 100%, and the blank control group was without added lipase.

[0075] (2) Commercial immobilized lipase, free CalB, and immobilized CalB were separately added to several common organic solvents. The organic solvents used in this study were ethanol, carbon tetrachloride, n - heptane, and hexanediol. After incubation at 25 °C for 2 h, the residual enzyme activity of the three lipases was measured at 37.5 °C and pH = 7.0. The effect of the organic reagent on enzyme activity was expressed as relative activity. The enzyme activity measured for the lipase sample without the organic reagent was set as 100%, and the blank control group was the sample without added lipase.

[0076] (3) Commercial immobilized lipase, free CalB, and immobilized CalB were separately added to several denaturants. The denaturants used in this study were 95% methanol, sodium dodecyl sulfate (SDS), and urea. After incubation at 25 °C for 2 h, the residual enzyme activity of the lipase was measured at 37.5 °C and pH = 7. The effect of the organic reagent on enzyme activity was expressed as relative enzyme activity. The enzyme activity measured for the lipase sample without the organic reagent was set as 100%, and the blank control group was the sample without added lipase.

[0077] (4) 0.4 mL of p - NPP solution (15 mmol / L, dissolved in 2 - propanol) was added to 3.5 mL of Tris - HCl buffer solution with a concentration of 50 mmol / L (pH = 7.02, containing 1 g / L arabic gum). After mixing and incubation at 37 °C for 10 min, commercial immobilized lipase, free CalB, and immobilized CalB were added, and the lipase hydrolysis reaction was carried out under ultrasonic conditions (power of 1000 W) for 10 min. The effect of ultrasound on the lipase catalytic process was expressed as relative activity. The enzyme activity measured for the lipase sample without ultrasonic treatment was set as 100%, and the blank control group was the sample without added lipase.

[0078] The above are the specific operations of the experiments on the effects of four factors on lipase activity. The experimental results are as Figure 13 shown.

[0079] As Figure 13 shown in a, after the commercial immobilized lipase was soaked in the metal ion solution for 1 h, the lipase enzyme fell off into the solution, and the commercial immobilized lipase was greatly affected by metal ions. Figure 13 As shown in b, carbon tetrachloride had a greater impact on the commercial immobilized lipase. During the experiment, the carrier particles of the soaked commercial immobilized lipase gradually became transparent and melted, and the enzyme activity also decreased significantly. Figure 13Figure d in the figure shows that 95% methanol causes the greatest damage to lipase. The highly polar environment may have changed the conformation and stability of the enzyme. The enzyme activity of free CalB is only 20% of that before treatment. Urea has little effect on both, while SDS has a greater effect on free CalB. SDS combines with free CalB to form a complex, which hinders the binding of the substrate to the active site. However, due to the low concentration, it is not enough to completely denature the enzyme protein structure. Figure 13 Figure c shows that the hydrolysis ability of lipase is improved under ultrasonic conditions. This is because ultrasonic treatment can promote the diffusion of reactants, thereby improving the catalytic activity of the enzyme.

[0080] Example 13: Study on the storage stability of immobilized lipase The immobilized CalB in this example is the immobilized CalB obtained in Example 5 with an immobilization time of 12 h.

[0081] Specific operation: Store commercially available immobilized lipase, free CalB and immobilized CalB at 25°C under the same conditions, take samples every 7 days to measure the activity until the enzymatic activity of one of the three lipases is completely lost, and then continue to detect the activity of other enzymes for at least 4 weeks.

[0082] The results are as follows Figure 14 As shown, when stored at 25°C, although there is a protective agent in the free CalB enzyme solution, the activity of free CalB will gradually decrease at this temperature. In contrast, the stability of commercially available immobilized lipase and immobilized CalB does not change much. The protection of the carrier allows it to be maintained for a long time at room temperature. Although the enzyme activity decreases, the degree of decrease does not exceed 20%.

[0083] Example 14: Investigating the effects of different homogenization times on the appearance, emulsion microstructure, CI and average droplet size of the Pickering emulsion with catalytic activity The difference between this example and Example 2 is that the homogenization time in step (2) is 1 min, 2 min, 4 min, 5 min, and 6 min, and the other steps are the same as step (2) in Example 2. The effect of different homogenization times on the Pickering emulsion with catalytic activity is observed. Figure 15 As far as we know, when the homogenization time is 3 min, all emulsions show good stability and no oil leakage occurs. From a microscopic perspective, the emulsion droplets present a highly uniform round morphology; the average droplet size of the emulsion droplets reaches a minimum of 1394 nm, and the CI is as high as 96.19% after 24 h, showing excellent emulsion stability. Therefore, the homogenization time was optimized to 3 min in this experiment.

[0084] Example 15: Investigate the effects of different homogenization speeds on the appearance of the catalytically active Pickering emulsion, the emulsion microstructure, CI, and the average droplet size of the emulsion This example uses the example in Step (2) of Example 14 with a homogenization time of 3 min. The difference is that the homogenization speeds in Step (2) are 5000 rpm, 8000 rpm, 11000 rpm, 14000 rpm, and 17000 rpm, and the effects of different homogenization speeds on the catalytically active Pickering emulsion are observed.

[0085] As Figure 16 is known, at homogenization speeds of 5000 rpm, 8000 rpm, 11000 rpm, and 14000 rpm, the appearance of the emulsion is uniform without obvious stratification, and no severe aggregation of droplets is observed under an optical microscope. However, at 17000 rpm, obvious stratification and droplet aggregation phenomena occur in the emulsion. At rotation speeds of 8000 rpm, 11000 rpm, and 14000 rpm, the CI of the emulsion all reaches 100%, and the average droplet size of the emulsion further decreases. Especially under the homogenization condition of 11000 rpm, the average droplet size of the emulsion decreases to 1366 nm. Therefore, a homogenization speed of 11000 rpm is selected to ensure a better emulsification effect.

[0086] Example 16: Investigate the effects of different oil phase fractions on the appearance of the catalytically active Pickering emulsion, the emulsion microstructure, CI, and the average droplet size of the emulsion This example uses the operation method of the example in Step (2) of Example 15 with a homogenization speed of 11000 rpm. The difference is that the oil phase fractions in Step (2) are φ = 0.1, φ = 0.2, φ = 0.4, φ = 0.6, and φ = 0.8, and the effects of different oil phase fractions on the Pickering emulsion are observed.

[0087] According to Figure 17 shown, specifically, when the oil phase fraction φ increases from 0.1 to 0.6, the CI increases significantly and reaches a maximum value of approximately 95% at φ = 0.6. The average droplet size of the emulsion also increases with the increase of the oil phase fraction, gradually increasing from 754.73 nm to 1105.19 nm, and there is no oil leakage phenomenon. However, when the oil phase fraction reaches φ = 0.8, obvious stratification occurs in the emulsion, with a clear oil phase on the upper layer and a small amount of water phase on the lower layer, indicating that the stability of the emulsion has been damaged at this time, and the average droplet size of the emulsion also increases to 1552.85 nm.

[0088] Combined with Examples 14 - 16, it can be seen that different homogenization times, homogenization speeds, and oil phase fractions have a significant impact on the Pickering emulsion with catalytic activity. Among them, the emulsion formation effect is the best when the homogenization time is 3 min and the homogenization speed is 11,000 rpm. On this basis, within the range of oil phase fraction φ = 0.1 - 0.6, the emulsion can maintain good stability. The operating parameters and the range of oil phase fraction of the lipase-catalyzed hydrolysis reaction system based on the Pickering emulsion are preliminarily determined to be φ = 0.1 - 0.6. Therefore, when preparing a Pickering emulsion with catalytic activity, it is necessary to comprehensively consider the influence of the oil phase fraction on the emulsion stability and droplet size, and optimize the emulsion performance. The experimental results of this study not only provide a key experimental basis for the subsequent construction of a two-phase lipase catalytic system, but also lay a foundation for a deeper understanding of the mechanism of action of various operating conditions in emulsion formation.

[0089] Example 17: In the catalytic hydrolysis reaction system, compare the effects of different pH values on the hydrolysis rate of tuna oil The difference between this example and step (3) of Example 2 is that the sodium acetate buffer solutions are pH = 6.0, pH = 7.0, pH = 7.5, and pH = 8.0 respectively, and the others are the same as step (3) of Example 2. Observe the effects of different pH values on the hydrolysis rate of tuna oil.

[0090] According to Figure 18 a in, under the hydrolysis conditions of 35 °C and pH = 6.0, the hydrolysis rate of tuna oil is only 20.19%. Under the same temperature conditions, it is found that the hydrolysis rate is the highest at pH = 7.5, which is 35.15%. Therefore, pH = 7.5 is selected as the optimal pH of the immobilized lipase-catalyzed hydrolysis reaction system based on the Pickering emulsion.

[0091] Example 18: During the catalytic hydrolysis process, compare the effects of different hydrolysis temperatures on the hydrolysis rate of tuna oil This example adopts the operation method of using the sodium acetate buffer solution with pH = 7.5 in step (3) of Example 17. The difference is that the hydrolysis is carried out at hydrolysis temperatures of 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, and 60 °C respectively, and the effects of different hydrolysis temperatures on the hydrolysis rate of tuna oil are tested.

[0092] Combined with Figure 18 As shown in b, within the range of 35 °C to 60 °C, the hydrolysis rate of tuna oil first increases and then decreases. The hydrolysis rate is the highest at 55 °C, reaching 49.34%. This temperature is the optimal hydrolysis temperature.

[0093] Example 19: During the catalytic hydrolysis process, compare the effects of different immobilized CalB mass-to-volume ratios on the hydrolysis rate of tuna oil This example adopts the operation method with a hydrolysis temperature of 55 °C in step (3) of Example 18. The mass-volume ratio of immobilized CalB, that is, the content of immobilized lipase (w / v), is 2%, 3%, 4%, 5%, and 6% respectively, to determine the effect of different mass-volume ratios of immobilized CalB on the hydrolysis rate of tuna oil. According to Figure 18 As shown in c of

[0094] Combined with Example 2 and Examples 17 - 19, explore the effects of the hydrolysis process, including the pH of the buffer, hydrolysis temperature, and mass-volume ratio of immobilized CalB, on the hydrolysis rate of tuna oil. Combined with Figure 18 data analysis, it can be known that the optimal conditions for the immobilized lipase hydrolysis system based on Pickering emulsion are a pH of 7.5, a temperature of 55 °C, and a mass-volume ratio of immobilized CalB of 6%. The hydrolysis rate of the hydrolysis system at 24 h is 76.09%, and the hydrolysis rate of the system before process optimization is 20%, which is increased by 3 times.

[0095] Example 20: Evaluation of the reusability of the immobilized lipase-catalyzed hydrolysis reaction system based on Pickering emulsion This example uses the relative activities (%) obtained from cycles 1 to 6 described in Example 2 to evaluate the reusability of the immobilized lipase in the immobilized lipase-catalyzed hydrolysis reaction system based on Pickering emulsion, and directly compares the effect of the number of hydrolysis cycles on the hydrolysis ability of the immobilized lipase through the relative activities.

[0096] The results are as Figure 19 shown. It is found that as the number of cycles increases, the relative activity gradually decreases, and the relative activity after 6 cycles is 68.1%.

[0097] Example 21: Effects of the immobilized lipase-catalyzed hydrolysis reaction system based on Pickering emulsion, commercially available immobilized lipase based on traditional two-phase system, and free CalB on the hydrolysis rate of tuna oil The immobilized CalB in this example is the immobilized CalB prepared in Example 2.

[0098] Prepare a 20 mL Pickering emulsion-based immobilized lipase-catalyzed hydrolysis reaction system: Add tuna oil (oil phase fraction φ = 0.2) and 1.2 g of immobilized CalB into a test tube. The immobilized CalB accounts for 6% of the mass volume ratio of the Pickering emulsion. Finally, add sodium acetate buffer (pH = 7.5) to a volume of 20 mL, and homogenize for 3 min at a homogenizer speed of 11,000 rpm to prepare the Pickering emulsion. After water bath at 55 °C for 24 h, centrifuge at 10,000 g centrifuge for 5 min to recover the immobilized CalB, and collect the oil phase to measure the hydrolysis rate of the system.

[0099] Take 1.2 g of immobilized CalB, 1.2 g of commercially available immobilized lipase, and 1.2 mL of free CalB and place them in tuna oil with an oil phase fraction φ = 0.2, and add sodium acetate buffer (pH = 7.5) to a volume of 20 mL. After water bath at 55 °C for 24 h, centrifuge at 10,000 g centrifuge for 5 min, and collect the oil phase to measure the hydrolysis rate of the system. Compare the hydrolysis rates of the Pickering emulsion-based immobilized lipase-catalyzed hydrolysis reaction system, commercially available immobilized lipase, and free CalB on tuna oil.

[0100] As Figure 20 shown, it was found that the hydrolysis rate of the Pickering emulsion-based immobilized lipase-catalyzed hydrolysis reaction system on tuna oil was 72.41%, which was 16.7 times that of free CalB and 4.6 times that of commercially available immobilized lipase. The significant increase in the hydrolysis rate shown by the above results can be attributed to the increase in the interfacial area in the Pickering emulsion system. Compared with the traditional two-phase system, the Pickering emulsion-based immobilized lipase-catalyzed hydrolysis reaction system constructed in the present invention has a huge interfacial area provided by its fine emulsion droplets. And the lipase has the characteristic of interfacial activation, which increases the contact area between the active center of the lipase and the substrate, greatly increases the contact opportunity between the lipase and the substrate, and thus improves the hydrolysis rate of the lipase on tuna oil.

[0101] Example 22: Explore the hydrolysis effects of the lipase-catalyzed hydrolysis reaction system based on Pickering emulsion, free CalB, and commercially available immobilized lipase on tuna oil in the traditional two-phase system In this example, the immobilized CalB used was the immobilized CalB obtained in Example 5 with a fixation time of 12 h.

[0102] In this example, the hydrolysis method used the optimized process method of the best catalytic hydrolysis reaction system obtained in Example 19 to hydrolyze the immobilized CalB, commercially available immobilized lipase, and free lipase respectively.

[0103] Take 25 mg of the oil samples hydrolyzed by the above three lipases respectively, add 2 mL of 0.5 mol / L sodium hydroxide methanol solution, keep it at a constant temperature in a water bath at 65 °C for 30 min. After cooling, add 2 mL of boron trifluoride / methanol solution (1:3, v / v), keep it in a water bath at 70 °C for 5 min, take it out and let it cool naturally, add 2 mL of n-hexane, shake it, finally add 4 mL of saturated sodium chloride solution, take the upper organic phase, add anhydrous sodium sulfate, and then perform GC analysis after passing through a 0.22 µm organic filter membrane.

[0104] Detection conditions: Chromatographic column (Trace TRFAME, 60 m×0.25 mm×0.25 μm), hydrogen flame ionization detector. Set the nitrogen flow rate to 1.0 mL / min, and set the inlet and detector temperatures to 250 °C.

[0105] Gas chromatography program: First, keep it at 80 °C for 0.5 min, then quickly heat it from 80 °C to 180 °C at a rate of 40 °C / min, then heat it to 230 °C at a rate of 4 °C / min, and keep it for 4 min. By comparing the retention times of fatty acid standards, the fatty acids in the product are qualitatively analyzed, and the peak area normalization method is used for quantification. The analysis of the fatty acid composition before and after hydrolyzing tuna oil in different systems is shown in Table 4.

[0106] Table 4 Comparison of the compositions of free fatty acids (mg / kg) before and after hydrolysis catalyzed by different lipases ,

[0107] Through the analysis of the compositions of free fatty acids before and after hydrolyzing tuna oil by comparing free CalB, commercially available immobilized lipase, and immobilized CalB, it can be seen that after hydrolyzing tuna oil with different lipases, the free fatty acids have all increased. By comparing the contents of the target product n-3 polyunsaturated fatty acids (n-3PUFAs), it is found that the content of n-3PUFAs obtained by the immobilized lipase prepared in the present invention through the Pickering emulsion catalytic hydrolysis reaction system is 195.3 times that of the n-3PUFAs in the fish oil before hydrolysis, and 4.98 times that of the n-3PUFAs in the fish oil hydrolyzed by the commercially available immobilized lipase.

[0108] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A method for constructing an immobilized lipase-catalyzed hydrolysis reaction system based on Pickering emulsion, characterized in that, The construction method specifically includes the following steps: (1) Add lipase to a buffer to form a mixed lipase solution; add a porous carrier to the mixed lipase solution to immobilize and adsorb the lipase, and centrifuge to remove the supernatant; then wash with a buffer, centrifuge again, and dry to obtain immobilized lipase; (2) Disperse the immobilized lipase in a buffer solution, add an oil-phase substrate, and perform high-speed dispersion to obtain a catalytically active Pickering emulsion; (3) Hydrolyze the catalytically active Pickering emulsion in a water bath to construct a catalytic hydrolysis reaction system of immobilized lipase based on the Pickering emulsion; (4) After catalytic hydrolysis, centrifuge to precipitate, recover the immobilized lipase, and continue to cycle steps (2) and (3) to achieve the recycling of the immobilized lipase.

2. The construction method of an immobilized lipase-catalyzed hydrolysis reaction system based on Pickering emulsion according to claim 1, characterized in that, In the step (1), the type of the porous carrier is bentonite, the mass-volume ratio of the bentonite to the lipase is 1:1 to 1:2.5, and the immobilization adsorption time of the lipase is 8 to 16 h.

3. The construction method of an immobilized lipase-catalyzed hydrolysis reaction system based on Pickering emulsion according to claim 1, wherein In the step (1), the pH value range of the buffer is 5.0 to 8.

0.

4. The construction method of an immobilized lipase-catalyzed hydrolysis reaction system based on Pickering emulsion according to claim 1, wherein, In the step (2), the oil-phase substrate is at least one of n-heptane, soybean oil, peanut oil, and tuna oil, and the oil-phase fraction φ of the oil-phase substrate is 0.1 to 0.

6.

5. The construction method of an immobilized lipase-catalyzed hydrolysis reaction system based on Pickering emulsion according to claim 1, wherein, In the step (2), based on the volume of the catalytic hydrolysis reaction system of immobilized lipase based on the Pickering emulsion, the mass-volume ratio of the immobilized lipase is 3% to 8%.

6. The construction method of an immobilized lipase-catalyzed hydrolysis reaction system based on Pickering emulsion according to claim 1, characterized in that, In the step (3), the hydrolysis temperature is 40 to 60 °C, and the hydrolysis pH value is 5.0 to 8.

0.

7. The construction method of an immobilized lipase-catalyzed hydrolysis reaction system based on Pickering emulsion according to claim 1, wherein The optimal preparation process of the immobilized lipase in the step (1) is: the volume-mass ratio of CalB to bentonite is 2:1, the buffer pH = 7.0, and the immobilization adsorption time is selected as 12 h.

8. The immobilized lipase obtained in step (1) of the preparation method according to claim 1, characterized in that, The temperature range for the application of the immobilized lipase is 20 - 65 °C, and the pH is 6.5 - 8.5; the stable storage time of the immobilized lipase is 1 - 60 days.

9. Application of the catalytic hydrolysis reaction system of immobilized lipase based on the Pickering emulsion obtained by the construction method according to any one of claims 1 - 7 in the hydrolysis of tuna oil.

10. The application according to claim 9, wherein The optimal conditions for the hydrolysis of tuna oil by the immobilized lipase hydrolysis system based on the Pickering emulsion are: pH is 7.5, temperature is 55 °C, the mass-volume ratio of immobilized CalB is 6%, and the hydrolysis time is 24 h.

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