Organic-inorganic hybrid nanoflower immobilized lipase with interface activation effect, its preparation method and application in catalytic preparation of 1,3-diglyceride

Abstract

The application provides an organic-inorganic hybrid nanoflower immobilized lipase with an interface activation effect, a preparation method thereof and application thereof in catalytic preparation of 1,3-glycerol diester, belongs to the technical field of biological enzymes, and introduces a biological surfactant sodium cocoyl glutamate on the basis of traditional nanoflower immobilized enzyme technology, so as to provide an interface activation effect for the lipase, then the lipase activated by the interface is used as an organic component to form an organic-inorganic hybrid nanoflower immobilized lipase with a metal inorganic salt, and high-efficiency preparation of 1,3-glycerol diester is realized; the activation effect of the sodium cocoyl glutamate on the lipase is still retained after the enzyme is immobilized, and the addition of the sodium cocoyl glutamate can also improve the immobilization capacity and reusability of the immobilized lipase, and greatly reduces the use cost of the lipase.

Description

Technical Field

[0001] This invention relates to the field of bioenzyme technology, specifically to an organic-inorganic hybrid nanoflower immobilized lipase with interfacial activation effect, its preparation method, and its application in the catalytic preparation of 1,3-diglycerides. Background Technology

[0002] Lipases (triacylglycerol acyl hydrolases, EC 3.1.1.3) are naturally occurring enzymes that hydrolyze triacylglycerol ester bonds and are widely used in the food, pharmaceutical, and chemical industries. Lipases have a "lid" structure, and their conformational changes are closely related to their activation. In the closed conformational isomer of the enzyme, the active site is completely buried under the "lid" of the α-helix; when the "lid" is opened, the active site of the enzyme is exposed, exhibiting catalytic activity, a process known as "interfacial activation."

[0003] Adding 1,3-diglycerides to edible oils can improve blood lipid levels and reduce obesity. Enzymatic catalysis, which uses glycerol and fatty acids as substrates and directly esterifies them via lipase to obtain high-purity 1,3-diglycerides, is widely used. However, the low activity, poor stability, and difficulty in reusing free lipases during the enzymatic catalysis process lead to high costs, limiting the further industrial application of lipases.

[0004] Immobilized enzyme technology is a technique that immobilizes enzymes on a support to improve their catalytic stability and reusability. Combining enzymes and supports effectively enhances enzyme stability and enables continuous operation. While traditional carrier-immobilized enzymes partially improve performance, they still require sacrificing loading capacity or complex surface modifications to achieve immobilization. From a nanotechnology perspective, nanostructured supports have emerged as a novel technology to address the issues of loading capacity and complex processes associated with traditional immobilized enzymes. The formation of inorganic hybrid nanoflowers to immobilize enzymes using the complexation of heavy metal ions with enzyme molecules can shorten the immobilized enzyme preparation process while increasing the loading rate.

[0005] However, after interfacial activation of lipases, the activation conformation depends on the dynamic flexibility and hydrophobic exposure of the "cap" structure. If the immobilization process causes the hydrophobic group face of the lipase towards the aqueous phase, the "cap" may not be able to contact the oil substrate phase, resulting in the "cap" failing to open and the activation effect being lost. Therefore, ensuring that the lipase retains its activation effect after interfacial activation and immobilization places strict requirements on the selection of interfacial activators and immobilization methods, as well as their synergistic effect. Furthermore, in the enzymatic catalytic preparation of 1,3-diglycerides, increasing the lipase loading, improving the stability and reusability of the immobilized enzyme, will significantly improve the utilization rate of lipase and reduce its usage cost. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides an organic-inorganic hybrid nanoflower immobilized lipase with an interfacial activation effect. Based on traditional nanoflower immobilized enzyme technology, the biosurfactant sodium cocoyl glutamate is introduced to provide an interfacial activation effect for the lipase. Then, the interfacially activated lipase is used as an organic component to form an organic-inorganic hybrid nanoflower immobilized lipase with a metal-inorganic salt. The activation effect of sodium cocoyl glutamate on the lipase is retained after enzyme immobilization, and the addition of sodium cocoyl glutamate also improves the immobilization capacity and reusability of the immobilized lipase, achieving efficient preparation of 1,3-diglyceride.

[0007] The technical solution of this invention is as follows: An organic-inorganic hybrid nanoflower immobilized lipase with an interfacial activation effect is obtained by interfacial activation of the lipase with sodium cocoyl glutamate, followed by binding of the interfacially activated lipase with a metal inorganic salt to form an organic-inorganic hybrid nanoflower immobilized lipase.

[0008] Preferably, the lipase is Novozymes lipase TL100L.

[0009] The above-mentioned method for preparing organic-inorganic hybrid nanoflower immobilized lipase firstly uses sodium cocoyl glutamate to activate the lipase at the interface, and then combines the interface-activated lipase with a metal inorganic salt to form organic-inorganic hybrid nanoflower immobilized lipase.

[0010] Preferably, the inorganic metal salt is a copper sulfate.

[0011] Preferably, the above method specifically includes the following steps: (1) Using phosphate buffer solution as solvent, prepare lipase solution and sodium cocoyl glutamate solution respectively. Mix the lipase solution and sodium cocoyl glutamate solution and incubate to obtain solution 1; (2) Add a metal inorganic salt solution to solution 1 from step (1), incubate, and obtain solution 2; (3) Centrifuge the solution 2 from step (2), collect the precipitate, wash the precipitate with water, and dry it to obtain organic-inorganic hybrid nanoflower immobilized lipase.

[0012] Preferably, in the phosphate buffer solution described in step (1), dihydrogen phosphate (H2PO4) is present. - ) and hydrogen phosphate (HPO4) 2- The total concentration of the substance was 0.05~0.1M, and the pH value was 6.8~7.4.

[0013] Preferably, the concentration of lipase in the lipase solution in step (1) is 0.2~0.3 g / L, and the concentration of sodium cocoyl glutamate in the sodium cocoyl glutamate solution is 0.15~0.35 mM.

[0014] Preferably, the incubation conditions in step (1) are: incubation at 3~5℃ for 10~14h.

[0015] Preferably, the volume ratio between the lipase solution and the sodium cocoyl glutamate solution in step (1) is 1:(8.5~9.5).

[0016] Preferably, the metal inorganic salt solution in step (2) is a copper sulfate solution with a concentration of 120~200mM.

[0017] Preferably, the amount of metal inorganic salt solution added in step (2) is 1 / (1450~1550) of the volume of solution 1.

[0018] Preferably, the incubation conditions in step (2) are: incubation at 3~5℃ for 10~72h.

[0019] Preferably, the centrifugation conditions in step (3) are: centrifugation at 8000~12000 r / min for 3~8 min.

[0020] Preferably, the drying conditions in step (3) are: freeze drying at -80℃ for 10~14h.

[0021] The above-mentioned organic-inorganic hybrid nanoflower immobilized lipase with interfacial activation effect is used in the catalytic preparation of 1,3-glycerol diester.

[0022] Preferably, the application method is as follows: using glycerol and oleic acid as substrates, 1,3-diglyceride is prepared by catalysis of the immobilized lipase.

[0023] More preferably, the catalytic conditions are: catalytic reaction at 45~55℃ for 1.5~3h.

[0024] Beneficial effects: (1) This invention provides an organic-inorganic hybrid nanoflower immobilized lipase with an interfacial activation effect. Based on traditional nanoflower immobilized enzyme technology, sodium cocoyl glutamate is introduced to provide an interfacial activation effect for the lipase. Then, the interfacially activated lipase is used as an organic component and forms an organic-inorganic hybrid nanoflower immobilized lipase through complexation with a metal-inorganic salt. The activation effect of sodium cocoyl glutamate on the lipase is retained after the enzyme is immobilized. Furthermore, the addition of sodium cocoyl glutamate can improve the immobilization loading and reusability of the immobilized lipase, with an immobilization loading of up to 13.013 mg / g. After five reuses, the enzyme activity retention rate is 77.4%.

[0025] (2) Using the above-mentioned organic-inorganic hybrid nanoflowers to immobilize lipase, 1,3-diglyceride was prepared by catalysis with glycerol and oleic acid as substrates. This achieved efficient preparation of 1,3-diglyceride, greatly reduced the cost of using lipase, and is suitable for industrial application.

[0026] (3) Sodium cocoyl glutamate is a safe, mild and biodegradable surfactant that is suitable for interfacial activation of lipases and catalytic preparation of 1,3-glycerol diesters. It has broad application prospects in the fields of food, medicine and chemical industry. Attached Figure Description

[0027] Figure 1 Scanning electron microscope image of lipase immobilized in nanoflowers; Figure 2 This is a standard curve for protein concentration. Figure 3 The protein loading of the nanoflower-immobilized lipases prepared in Examples 1-5 and Comparative Example 1 is shown. Figure 4 This is a standard curve for the concentration of p-nitrophenol; Figure 5 The enzyme activity of the nanoflower-immobilized lipases prepared in Examples 1-5 and Comparative Example 1 was measured. Figure 6 The conversion rates of 1,3-diglyceride prepared by immobilized lipase from nanoflowers in Examples 1-5 and Comparative Example 1 are shown. Figure 7 The enzyme activity retention rate of the nanoflower-immobilized lipase prepared in Example 1 and Comparative Example 1 after five reuses. Detailed Implementation

[0028] The present invention will now be described in detail with reference to specific embodiments. The description in this section is merely illustrative and explanatory, and should not be construed as limiting the scope of protection of the present invention.

[0029] Explanation of the source of experimental materials: Lipase: Novozymes lipase TL100L, purchased from Novozymes (China) Biotechnology Co., Ltd.

[0030] Example 1: Preparation of an organic-inorganic hybrid nanoflower immobilized lipase with interfacial activation effect Follow these steps: (1) Prepare dihydrogen phosphate (H2PO4) solution using 5.075 g / L NaH2PO4 solution and 10.988 g / L Na2HPO4 solution. - ) and hydrogen phosphate (HPO4) 2-A phosphate buffer solution with a total concentration of 0.1 M and a pH of 7.4 was prepared. Using the phosphate buffer solution as a solvent, a lipase solution with a concentration of 0.25 g / L and a sodium cocoyl glutamate solution with a concentration of 0.15 mM were prepared. 30 mL of lipase solution and 270 mL of sodium cocoyl glutamate solution were mixed and incubated at 4 °C for 12 h to obtain 300 mL of solution 1. (2) Add 0.2 mL of 120 mM CuSO4 solution to solution 1 from step (1), and incubate at 4 °C for 72 h to obtain solution 2; (3) Centrifuge the solution 2 from step (2) at 10000 r / min for 5 min, collect the precipitate, resuspend the precipitate in pure water and wash it 3 times, combine the supernatants from the 3 washes and count them as the total supernatant for later use; freeze-dry the washed precipitate at -80℃ for 12 h to obtain an organic-inorganic hybrid nanoflower immobilized lipase with interfacial activation effect.

[0031] Example 2: Preparation of an organic-inorganic hybrid nanoflower immobilized lipase with interfacial activation effect Unlike Example 1, the concentration of the sodium cocoyl glutamate solution in step (1) is 0.35 mM. The remaining steps are the same as in Example 1, resulting in an organic-inorganic hybrid nanoflower immobilized lipase with an interface activation effect, which is then ready for use.

[0032] Example 3: Preparation of an organic-inorganic hybrid nanoflower immobilized lipase with interfacial activation effect Unlike Example 1, the concentration of CuSO4 solution in step (2) is 200 mM, and the remaining steps are the same as in Example 1, resulting in an organic-inorganic hybrid nanoflower immobilized lipase with interfacial activation effect.

[0033] Example 4: Preparation of an organic-inorganic hybrid nanoflower immobilized lipase with interfacial activation effect Unlike Example 1, the pH of the phosphate buffer solution was changed in step (1). Specifically, the solution was prepared using a 6.443 g / L NaH₂PO₄ solution and a 6.573 g / L Na₂HPO₄ solution to prepare dihydrogen phosphate (H₂PO₄) solution. - ) and hydrogen phosphate (HPO4) 2- A phosphate buffer solution with a total concentration of 0.1 M and a pH of 6.8 was prepared; the remaining steps were the same as in Example 1, to obtain an organic-inorganic hybrid nanoflower immobilized lipase with an interface activation effect.

[0034] Example 5: Preparation of an organic-inorganic hybrid nanoflower immobilized lipase with interfacial activation effect Unlike Example 1, the concentration of the phosphate buffer solution was changed in step (1). Specifically, the dihydrogen phosphate (H2PO4) solution was prepared using a 2.538 g / L NaH2PO4 solution and a 5.494 g / L Na2HPO4 solution. - ) and hydrogen phosphate (HPO4) 2- A phosphate buffer solution with a total concentration of 0.05 M and a pH of 7.4 was prepared; the remaining steps were the same as in Example 1, to obtain an organic-inorganic hybrid nanoflower immobilized lipase with an interfacial activation effect.

[0035] Comparative Example 1: Unlike Example 1, step (1) does not involve the addition of sodium cocoyl glutamate. The specific steps are as follows: dihydrogen phosphate (H2PO4) is prepared using a 5.075 g / L NaH2PO4 solution and a 10.988 g / L Na2HPO4 solution. - ) and hydrogen phosphate (HPO4) 2- A phosphate buffer solution with a total concentration of 0.1 M and a pH of 7.4 was prepared. A lipase solution with a concentration of 0.25 g / L was prepared using the phosphate buffer solution as a solvent. 30 mL of the lipase solution and 270 mL of the phosphate buffer solution were mixed and vortexed to obtain 300 mL of the mixture. 0.2 mL of CuSO4 solution with a concentration of 120 mM was added to the mixture and incubated at 4 °C for 72 h. The remaining steps were the same as in Example 1 to obtain an organic-inorganic hybrid nanoflower immobilized lipase that was not activated by the sodium cocoyl glutamate interface.

[0036] Comparative Example 2: Unlike Example 1, in step (1), sodium cocoyl glutamate was replaced with an equal mass of sodium oleoyl taurate, and the remaining steps were the same as in Example 1, to obtain organic-inorganic hybrid nanoflower immobilized lipase.

[0037] Comparative Example 3: Unlike Example 1, in step (1), sodium cocoyl glutamate was replaced with an equal mass of sucrose fatty acid ester, and the remaining steps were the same as in Example 1, to obtain organic-inorganic hybrid nanoflower immobilized lipase.

[0038] Comparative Example 4: Unlike Example 1, in step (1), sodium cocoyl glutamate was replaced with an equal mass of propylene glycol fatty acid ester, and the remaining steps were the same as in Example 1, to obtain organic-inorganic hybrid nanoflower immobilized lipase.

[0039] Comparative Example 5: Unlike Example 1, in step (1), sodium cocoyl glutamate was replaced with an equal mass of sodium stearoyl lactylate, and the remaining steps were the same as in Example 1, to obtain organic-inorganic hybrid nanoflower immobilized lipase.

[0040] Experimental Example 1: SEM Characterization The organic-inorganic hybrid nanoflower-immobilized lipase with interfacial activation effect prepared in Example 1 was characterized by scanning electron microscopy, and the results are as follows: Figure 1 As shown. By Figure 1 It can be seen that the nanoflower immobilized lipase prepared by the present invention has a diameter of about 13.27µm, is spherical in appearance, has a uniform petal-like surface, and multiple nanoflower immobilized enzymes are in a dispersed state.

[0041] Experimental Example 2: Determination of Protein Immobilization Capacity The protein loading of the nanoflower-immobilized lipases prepared in Examples 1-5 and Comparative Examples 1-5 was determined, and the specific procedures were as follows: First, the protein concentration of the "total supernatant" collected in step (3) above was determined using the BCA method: 10 μL each of the standard solutions (500 μg / mL, 1000 μg / mL, 1500 μg / mL, 2000 μg / mL) and the sample to be tested (total supernatant) were added to a microplate, with 3 replicate wells for each sample; then 200 μL of BCA working solution was added to each well and mixed thoroughly; the mixed reaction plate was incubated at 37℃ for 30 min, and after the reaction was completed, it was removed and cooled to room temperature; the absorbance value of each reaction well was measured at a wavelength of 562 nm using an enzyme-linked immunosorbent assay (ELISA) reader; based on the absorbance value measurement results, a standard curve was plotted with absorbance value on the x-axis and standard concentration on the y-axis, and the curve was obtained by linear fitting as shown in the figure. Figure 2 The protein concentration standard curve shown is used to calculate the total protein concentration (C1) of the supernatant.

[0042] The protein loading (Q) of the nanoflower-immobilized lipases prepared in Examples 1-5 and Comparative Examples 1-5 was calculated using the following formula: Q = (C0V0 - C1V1) / m; where: Q is the immobilized enzyme protein loading (mg / g), C0 is the initial concentration of the lipase solution (g / L), V0 is the volume of lipase solution added (L), C1 is the protein concentration of the total supernatant (g / L), V1 is the volume of the total supernatant (L), and m is the mass of the organic-inorganic hybrid nanoflower immobilized enzyme (g).

[0043] The calculation results of protein immobilization are as follows: Figure 3 As shown in Table 1. Among them, Figure 3 Table 1 shows the protein loading of the immobilized enzymes prepared in Examples 1-5 and Comparative Example 1. A higher protein loading indicates a higher efficiency of immobilized lipase.

[0044] Table 1. Protein immobilization capacity of the immobilized enzymes prepared in Example 1 and Comparative Examples 1-5

[0045] Depend on Figure 3 As shown in Table 1, in Comparative Example 1, without any surfactant for interfacial activation, the protein loading of the immobilized enzyme was approximately 5.512 mg / g. In Examples 1-5, the protein loading of the lipases after interfacial activation with sodium cocoyl glutamate was all above 10 mg / g, reaching a maximum of 13.013 mg / g, representing an increase of approximately 136% compared to Comparative Example 1. However, Comparative Examples 2-5 used sodium oleoyl taurate, sucrose fatty acid ester, propylene glycol fatty acid ester, and sodium stearoyl lactylate for interfacial activation, respectively. Only Comparative Example 2, using sodium oleoyl taurate for interfacial activation, showed a slightly higher protein loading than the control group (Comparative Example 1), but significantly lower than Example 1. The other surfactants failed to increase the protein loading of the immobilized enzymes, and were even lower than the protein loading of the control group.

[0046] The above results indicate that using sodium cocoyl glutamate to activate lipase at the interface can significantly increase the protein loading of the immobilized enzyme.

[0047] Experimental Example 3: Enzyme Activity Assay The enzyme activity of the nanoflower-immobilized lipases prepared in Examples 1-5 and Comparative Example 1 was determined according to the following steps: 1) Drawing the standard curve Prepare a 0.05 mol / L phosphate buffer solution with pH 7.2. Using this phosphate buffer solution, prepare p-nitrophenol solutions with concentrations of 0.02 mol / L, 0.04 mol / L, 0.06 mol / L, 0.08 mol / L, 0.10 mol / L, and 0.12 mol / L. Take 10 mL of each p-nitrophenol solution and incubate at 50℃ for 10 min. Add 2 mL of 1 mol / L Na₂CO₃. Using the phosphate buffer solution as a blank control, measure the absorbance at 410 nm using a spectrophotometer. Based on the absorbance measurements, plot a standard curve with absorbance on the x-axis and p-nitrophenol concentration on the y-axis. Obtain the standard curve through linear fitting. Figure 4 The standard curve shown.

[0048] 2) Determination of enzyme activity of organic-inorganic hybrid nanoflower immobilized lipase by p-nitrophenol palmitate colorimetric method Prepare a 0.05 mol / L phosphate buffer solution with pH 7.2. Take 1 mL each of the phosphate buffer solution and the p-nitrophenol palmitate solution AB mixture, mix them, and incubate in a 40℃ water bath for 5 min. Then add 0.5 mL of the immobilized lipase solution diluted to a certain ratio, react in a 40℃ water bath for 15 min, and then add 3 mL of phosphate buffer solution to make up the volume to 5.5 mL. Use free lipase inactivated at 99℃ for 10 min as a blank control. Measure the absorbance at 410 nm using a spectrophotometer. Substitute the measured absorbance value into the standard curve in step 1) to calculate the corresponding p-nitrophenol concentration, and then calculate the enzyme activity (U) using the following formula: U = V * N * (C1 - C0) / T / m; where: U is the enzyme activity of the immobilized enzyme (U / g), V is the total reaction volume (5.5 mL), N is the dilution factor of the enzyme solution, C1 is the concentration of p-nitrophenol calculated based on the absorbance value after adding the enzyme solution in step 2) (µmol / mL), C0 is the concentration of p-nitrophenol calculated based on the absorbance value after adding the inactivated free lipase (µmol / mL), T is the reaction time (min), and m is the mass (g) of the added organic-inorganic hybrid nanoflower immobilized enzyme.

[0049] Under the above conditions, the amount of enzyme required to hydrolyze p-nitrophenol palmitate per minute to produce 1 µmol of p-nitrophenol is defined as 1 enzyme activity unit (U).

[0050] The calculation results of enzyme activity are as follows: Figure 5 As shown. By Figure 5 It can be seen that, in Comparative Example 1, without the addition of any surfactant for interfacial activation, the enzyme activity of the immobilized enzyme prepared was approximately 199.703 U / g; in Examples 1-5, after the lipases were immobilized with sodium cocoyl glutamate for interfacial activation, the highest enzyme activity of the immobilized enzymes reached 389.976 U / g, which was about 95.28% higher than that of Comparative Example 1.

[0051] The above results indicate that sodium cocoyl glutamate has an activating effect on the activity of lipase, and this activating effect is retained after immobilization.

[0052] Experimental Example 4: Catalytic Preparation of 1,3-Diglycerides The ability of the nanoflower-immobilized lipases prepared in Examples 1-5 and Comparative Example 1 to catalyze the preparation of 1,3-glycerol diesters was determined by following these steps: 1) Using 10g oleic acid and 2g glycerol as reaction substrates, add 0.24g immobilized lipase and catalyze the reaction at 50℃ for 2h. The volume of the reaction solution is recorded as V1. After the reaction is completed, add the same volume of 10% TCA (trichloroacetic acid) solution as the reaction solution to terminate the catalysis and obtain mixed solution A.

[0053] 2) The content of product 1,3-glycerol diester was determined by high performance liquid chromatography (HPLC). The specific detection conditions were as follows: The chromatographic method used was a differential refractive index detector. The chromatographic column was a ShimNex HE Sil C18 normal phase analytical column (250×4.6mm). The mobile phase was hexane:isopropanol:formic acid (20:1:0.003, V / V / V). The flow rate was 1.0 mL / min. The column temperature was 30℃. The injection volume was 10 μL (the injection solution was 1 μL of mixed solution A + 9 μL of mobile phase, i.e., diluted 10 times).

[0054] 3) Based on the detected 1,3-diglyceride content, calculate the conversion rate (α) using the following formula: α = (C1*10*2*V1 / m)*100%; where: α is the conversion rate of 1,3-diglyceride (%), C1 is the measured content of 1,3-diglyceride (g / L), 10 is the dilution factor at the time of injection, 2 is the dilution factor of the reaction solution; V1 is the volume of the reaction solution (L); m is the mass of the added substrate oleic acid (g).

[0055] The conversion results of 1,3-diglyceride are as follows: Figure 6 As shown. By Figure 6 It can be seen that, in Comparative Example 1, without the addition of any surfactant for interfacial activation, the conversion rate of the immobilized enzyme catalyzing the preparation of 1,3-diglyceride was approximately 29.75%. In Examples 1-5, after the lipases were immobilized with sodium cocoyl glutamate for interfacial activation, the conversion rates of the immobilized enzyme catalyzing the preparation of 1,3-diglyceride were all above 40%, with the conversion rate of Example 1 reaching 46.5%, which is 56.30% higher than that of Comparative Example 1.

[0056] The above results indicate that the nanoflower immobilized enzyme prepared by interfacial activation of lipase with sodium cocoyl glutamate can efficiently prepare 1,3-glycerol diester.

[0057] Experiment Example 5: Reusability Test The reusability of the nanoflower-immobilized lipases prepared in Example 1 and Comparative Example 1 was determined by following these steps: Using 10g oleic acid and 2g glycerol as reaction substrates, 0.24g immobilized lipase was added, and the reaction was catalyzed at 50℃ for 2 hours. After the reaction was completed, the reaction solution was centrifuged, and the precipitate (i.e., the immobilized lipase) was recovered. The enzyme activity of the immobilized lipase was measured using the same method as in Experiment 3 and recorded as U0. The recovered immobilized lipase was then used to continue the catalytic reaction, and the above steps were repeated 4 times. The enzyme activity U1 was measured after each reaction. The enzyme activity of the nanoflower immobilized enzyme measured in the first round was defined as 100%, and the enzyme activity retention rate (η) for the remaining rounds was calculated using the following formula: η=(U1 / U0)*%.

[0058] The calculation results of enzyme activity retention rate are as follows: Figure 7 As shown. By Figure 7 It can be seen that, in Comparative Example 1, without the addition of any surfactant for interfacial activation, the immobilized enzyme prepared had an enzyme activity retention rate of 65.9% after five cycles, while in Example 1, the immobilized enzyme after interfacial activation using sodium cocoyl glutamate still had an enzyme activity retention rate of 77.4% after five cycles, which is about 17.45% higher than that of Comparative Example 1.

[0059] The above results show that the immobilized enzyme provided by the present invention has excellent stability and can still retain more than 75% of its enzyme activity after five reuses.

Claims

1. An organic-inorganic hybrid nanoflower immobilized lipase with interfacial activation effect, characterized in that, Sodium cocoyl glutamate was used to activate the lipase at the interface, and then the activated lipase was combined with a metal inorganic salt to form an organic-inorganic hybrid nanoflower immobilized lipase.

2. The method for preparing the organic-inorganic hybrid nanoflower immobilized lipase according to claim 1, characterized in that, First, sodium cocoyl glutamate was used to activate the lipase at the interface. Then, the activated lipase was combined with a metal-inorganic salt to form an organic-inorganic hybrid nanoflower immobilized lipase.

3. The method as described in claim 2, characterized in that, The inorganic metal salt is a copper sulfate.

4. The method as described in claim 2, characterized in that, Specifically, the steps include the following: (1) Using phosphate buffer solution as solvent, prepare lipase solution and sodium cocoyl glutamate solution respectively. Mix the lipase solution and sodium cocoyl glutamate solution and incubate to obtain solution 1; (2) Add a metal inorganic salt solution to solution 1 from step (1), incubate, and obtain solution 2; (3) Centrifuge the solution 2 from step (2), collect the precipitate, wash the precipitate with water, and dry it to obtain organic-inorganic hybrid nanoflower immobilized lipase.

5. The method as described in claim 4, characterized in that, In the phosphate buffer solution described in step (1), dihydrogen phosphate (H2PO4) ions... - ) and hydrogen phosphate (HPO4) 2- The total concentration of the substance was 0.05~0.1M, and the pH value was 6.8~7.

4.

6. The method as described in claim 4, characterized in that, The concentration of lipase in the lipase solution in step (1) is 0.2~0.3 g / L, and the concentration of sodium cocoyl glutamate in the sodium cocoyl glutamate solution is 0.15~0.35 mM.

7. The method as described in claim 4, characterized in that, The incubation conditions in step (1) are: incubation at 3~5℃ for 10~14h.

8. The method as described in claim 4, characterized in that, The volume ratio between the lipase solution and the sodium cocoyl glutamate solution in step (1) is 1: (8.5~9.5).

9. The method as described in claim 4, characterized in that, The inorganic metal salt solution mentioned in step (2) is a copper sulfate solution with a concentration of 120~200mM; Preferably, the amount of metal inorganic salt solution added in step (2) is 1 / (1450~1550) of the volume of solution 1. Preferably, the incubation conditions in step (2) are: incubation at 3~5℃ for 10~72h.

10. The application of the organic-inorganic hybrid nanoflower immobilized lipase as described in claim 1 in the catalytic preparation of 1,3-diglycerides; Preferably, the application method is as follows: using glycerol and oleic acid as substrates, 1,3-diglyceride is prepared by catalysis of the immobilized lipase; More preferably, the catalytic conditions are: catalytic reaction at 45~55℃ for 1.5~3h.

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