Immobilized enzyme, bio-oil production method and device based on reusable enzyme

Abstract

The application discloses a kind of immobilized enzyme, based on the method and device of bio-oil of the enzyme can be reused, the immobilized enzyme is with polyethylene glycol modified Fe3O4 Nanoparticle as matrix, matrix first adsorbs and loads first part free protease, then it is dispersed in sodium alginate aqueous solution with second part free protease and mixed uniformly, sequentially through Ca 2+ Two-step action process of ion hardening and glutaraldehyde crosslinking reaction, the outer sodium alginate reaction forms reticulated macroporous gel microspheres, finally after washing, drying, the microsphere complex of embedding second part free protease and polyethylene glycol modified nano Fe3O4 load immobilized protease is prepared.The stability of the immobilized enzyme in the non-homogeneous slurry mixing system, in-situ separation convenience greatly improves, immobilized enzyme can be better reused, in addition, the stirring paddle of bio-oil reactor is improved, and the stability of immobilized enzyme use can be further improved.

Description

Technical Field

[0001] This invention relates to an immobilized enzyme, a bio-oil production method and apparatus based on the reusable enzyme. Background Technology

[0002] One of the bottlenecks limiting the large-scale application of aqueous enzymatic extraction of tea seed oil is the high price and difficulty in reusing biocatalytic enzymes. Therefore, using immobilized enzymes to replace free enzymes is an inevitable choice. After immobilization, enzymes exhibit better environmental tolerance, greater stability, and their activity can be continuously maintained after repeated use, making them easy to recover. However, immobilized enzymes are mostly used in homogeneous liquid systems, and there are few reports on heterogeneous systems where pulp and residue coexist. On the one hand, immobilized enzymes are difficult to separate from solid reactants in heterogeneous systems, preventing the reusability of immobilized enzymes; on the other hand, due to the presence of particulate reactants, prolonged collisions and friction between solid particles and immobilized enzymes during stirring and mass transfer lead to enzyme breakage, releasing enzymes into the solution, resulting in severe loss of immobilized enzyme activity and a significant reduction in half-life.

[0003] Immobilized enzyme carriers are rigid and easily broken. While commonly used immobilized catalysts prepared by surface adsorption of diatomaceous earth or ion exchange resins exhibit high activity, frequent collisions with particulate materials during stirring cause the enzyme to detach from the carrier and dissolve into the solution, resulting in rapid deactivation and making recovery impossible. Currently, the most effective method for recovering immobilized enzymes from mixed solutions is to magnetize them. Furthermore, there is a lack of bioreactors specifically designed for enzymatic oil extraction from oilseeds, and the agitators in traditional reactors cause significant damage to immobilized enzymes. Additionally, traditional reactors cannot achieve simultaneous in-situ separation of immobilized enzymes / slurry and oil / water, enabling continuous hydrolysis of oilseeds. Summary of the Invention

[0004] In view of the above-mentioned technical problems existing in the prior art, the purpose of the present invention is to provide an immobilized enzyme, a bio-oil production method and apparatus based on the reusable enzyme.

[0005] The technical solution adopted in this invention is as follows:

[0006] A method for preparing an immobilized enzyme involves using polyethylene glycol-modified Fe3O4 nanoparticles as a matrix. The matrix is ​​stirred with a first portion of free protease in ultrapure water to allow the free protease to fully adsorb onto the matrix. After filtration, the nanoparticles are collected and freeze-dried to obtain polyethylene glycol-modified Fe3O4 nanoparticles loaded with immobilized protease. This immobilized protease is then dispersed together with a second portion of free protease in an aqueous sodium alginate solution and mixed thoroughly to coat the surface with sodium alginate. The mixture is then sequentially passed through a Ca2+ layer. 2+The two-step process of ion hardening and glutaraldehyde cross-linking reaction causes the outer layer of sodium alginate to react and form a network of large-pore gel microspheres. Polyethylene glycol-modified Fe3O4 nanoparticles loaded with immobilized protease and the second part of free protease are co-embedded inside the gel microspheres. Finally, after washing with distilled water and vacuum freeze-drying, a microsphere complex containing the second part of free protease and polyethylene glycol-modified Fe3O4 nanoparticles loaded with immobilized protease is prepared, thus completing the preparation.

[0007] Furthermore, the preparation method of the polyethylene glycol-modified Fe3O4 nanoparticles immobilized with protease specifically includes the following steps:

[0008] Step 1: Fe3O4 nanoparticles are dispersed in a polyethylene glycol aqueous solution. The suspension is ultrasonically dispersed at 30-50℃ for 10-30 min under nitrogen purging. The precipitate is collected by centrifugation and freeze-dried to obtain polyethylene glycol-modified nano-iron oxide. The molecular weight of the polyethylene glycol is 500-2000, and the mass ratio of Fe3O4 nanoparticles to polyethylene glycol is 1:1.5-1.8.

[0009] Step 2: The polyethylene glycol-modified nano-iron oxide obtained in Step 1 and the first part of free protease are dispersed in ultrapure water at a mass ratio of 4-8:1. The mixture is stirred and adsorbed for 10-30 minutes under the condition of adjusting the pH to 7.5-8.5. Then, the mixture is filtered, and the nanoparticles are collected and freeze-dried to obtain the polyethylene glycol-modified nano-Fe3O4-loaded immobilized protease.

[0010] Furthermore, the preparation method of the microsphere complex containing the second part of free protease and polyethylene glycol-modified Fe3O4 nanoparticles immobilized with protease specifically includes the following steps:

[0011] S1: Polyethylene glycol-modified nano-Fe3O4-supported immobilized protease and the second part of free protease are dispersed in sodium alginate aqueous solution at a mass ratio of 0.8-1.2:1, wherein the mass ratio of polyethylene glycol-modified nano-Fe3O4-supported immobilized protease to sodium alginate is 1:1.5-3;

[0012] S2: The mixture obtained in step S1 is sprayed into Ca 2+ After hardening in an ionic aqueous solution at 2-5℃ for 0.5-2 hours, magnetically immobilized microspheres were obtained and washed with distilled water; wherein the sodium alginate in step S1 and the Ca in step S2... 2+ The mass ratio of the ions is 1:0.3-0.5;

[0013] S3: Immerse the magnetically immobilized microspheres obtained in step S2 in an excess of 0.1-1% glutaraldehyde aqueous solution and carry out a cross-linking reaction at room temperature for 1-2 hours. Then, centrifuge to separate the precipitate, wash with distilled water to remove excess glutaraldehyde, and freeze-dry under vacuum to complete the preparation.

[0014] A bio-oil extraction method based on reusable immobilized enzymes involves adding shelled camellia seed powder and soft water to a stirred reactor, adjusting the pH of the reaction solution to alkaline, adding the immobilized enzyme prepared according to this invention, and carrying out a stirred hydrolysis reaction. After the reaction is completed, stirring is stopped, and the mixture is allowed to stand to allow the oil and water to separate. The upper layer of oil is discharged, dehydrated, and dried to obtain the product oil. Then, stirring is restarted, and an annular magnet on the outer side of the stirred reactor is energized to adsorb the immobilized enzyme onto the inner side wall of the stirred reactor. The mixture containing slurry and residue is discharged from the bottom discharge port of the stirred reactor. After rinsing the inside of the stirred reactor with clean water, shelled camellia seed powder and soft water are added again for the next batch of hydrolysis reaction, thus realizing the reuse of the immobilized enzyme.

[0015] Furthermore, the mass ratio of the shelled camellia seed powder to soft water for hydrolysis is 1:4-10, and the mass of the immobilized enzyme is 0.5-1% of the mass of the shelled camellia seed powder.

[0016] Furthermore, the hydrolysis reaction is carried out at a temperature of 50-70℃, for a reaction time of 3-5 hours, and at a stirring speed of 150-300 rpm.

[0017] Furthermore, during hydrolysis, the pH of the reaction solution is adjusted to 8.5-9.5.

[0018] The bio-oil production method of this invention utilizes an apparatus comprising a stirred reactor with a kettle-like structure. The stirred reactor has an overflow port at its upper end and a discharge port at its bottom. A heat exchange jacket for heating the stirred reactor is located on its outer side, through which a heat exchange fluid flows. A ring magnet is mounted on the outer side of the heat exchange jacket, and the ring magnet is connected to a power source via a wire. Both the stirred reactor and the heat exchange jacket are made of non-magnetic stainless steel. An agitator is centrally located inside the stirred reactor, with its upper end extending from the top of the stirred reactor and fixedly connected to a motor. Multiple sets of agitator blades, for example, 2-10 sets, are spaced downwards from top to bottom at the lower end of the agitator. Each set of agitator blades includes 3-4 blades evenly spaced along the circumference. The edges of the agitator blades are smooth arc-shaped, and the blades are inclined relative to the horizontal plane. Small perforations are evenly distributed on the blades. The agitator blades of this invention enable efficient mixing during stirring, reducing fluid resistance and shear damage to the immobilized enzyme. Both the agitator and the agitator blades are made of plastic.

[0019] Furthermore, both the agitator and the agitator blades are made of polytetrafluoroethylene.

[0020] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0021] 1) The stability and in-situ separation of the immobilized enzyme in the heterogeneous slurry-slag mixing system are greatly improved by the present invention. The immobilized enzyme can be reused better. In addition, the stirring blade of the bio-oil reactor is improved. The stirring blade can achieve efficient mixing during stirring, reduce fluid resistance and shear damage to the immobilized enzyme, and further improve the stability of the immobilized enzyme.

[0022] 2) The immobilized enzyme developed in this invention, combined with the device used in the bio-oil production method of this application, facilitates in-situ separation of slurry / immobilized enzyme and oil / water, while better protecting the immobilized enzyme and effectively extending its half-life. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the apparatus used in the bio-oil production method of this invention.

[0024] Figure 2 This is a schematic diagram of the structure of the agitator blades on the agitator.

[0025] Figure 3 This is the result of the total hydrolysis time corresponding to the number of times the immobilized enzyme is used when the hydrolysis reaction is carried out in the stirred reactor designed in this invention in Application Example 1 of this invention, and the immobilized enzyme is one of the immobilized enzymes prepared in Examples 1-6 respectively.

[0026] Figure 4 In Example 2 of this invention, the hydrolysis reaction was carried out using a conventional reactor. The oil yield was determined by the total hydrolysis time with respect to the number of times the immobilized enzyme was used, using the immobilized enzymes prepared in Examples 1-6 respectively. Detailed Implementation

[0027] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0028] The alkaline protease used in the embodiments of the present invention is a liquid alkaline protease purchased from Novozymes (China) Biotechnology Co., Ltd., with an activity of 280,800 U / g.

[0029] The method for determining protease activity refers to GB / T 23527-2009: 1 mL of appropriately diluted enzyme solution and 1 mL of 1 g / mL casein solution are reacted in a constant temperature water bath at 60℃ for 10 min. The reaction is terminated by adding 2 mL of 0.4 mol / L trichloroacetic acid solution, and then allowed to stand for 10 min to precipitate unhydrolyzed casein. The supernatant is filtered out using filter paper. Casein is used as the substrate, and the catalytic activity of the supernatant is determined by the Folin-Ciocalteu method. 1 mL of the supernatant is mixed with 5 mL of 0.4 mol / L Na₂CO₃ solution and 1 mL of Folin-Ciocalteu reagent diluted 3 times with deionized water. The mixture is incubated at 40℃ for 20 min for color development, and the absorbance is measured at 680 nm. An enzyme solution in which trichloroacetic acid is added first to terminate the reaction serves as a blank control; that is, trichloroacetic acid solution is added to the enzyme solution first, followed by the casein solution. The protease activity is determined by treating a standard enzyme solution under the same experimental settings with known activity. One unit of activity (U) of a protease is defined as the amount of enzyme required to release 1 μmol of tyrosine per minute by hydrolyzing casein.

[0030] Blank Example 1: Nano-Fe3O4 was prepared using a solvothermal reduction method. Specifically, 162 g of FeCl3, 90 g of trisodium citrate, and 540 g of sodium acetate were dispersed in 9 L of ethylene glycol and stirred for 30 min. The mixture was then transferred to a high-pressure reactor with a polytetrafluoroethylene liner. The reactor was sealed and heated at 200 °C for 10 h. After the reaction was complete, the reactor was allowed to cool. The solid product in the reaction solution was centrifuged, and 180 g of black Fe3O4 nanoparticles with an average particle size of 90 nm and a specific surface area of ​​36 m² were collected. 2 / g.

[0031] In Examples 1-6 of this invention, immobilized enzymes were prepared using Fe3O4 nanoparticles prepared in Blank Example 1.

[0032] Example 1: Immobilized protease on magnetic chitosan microspheres

[0033] 500 g of chitosan powder was dissolved in 50 L of 0.1% acetic acid aqueous solution to obtain a 1.0% (w / v) chitosan solution. Then, 1 kg of Fe3O4 powder was added to this solution. The resulting mixture was stirred at room temperature to remove air bubbles, and then slowly added dropwise to an excess of 2 mol / L NaOH solution to form microspheres. After soaking in NaOH solution for 10 h, the solid product was magnetically separated and recovered, and washed with deionized water until neutral. The microspheres were collected and soaked in 0.5% (v / v) glutaraldehyde aqueous solution at 25 °C for 2 h. The solid product was magnetically separated and recovered, and excess glutaraldehyde was washed away with deionized water to obtain activated microspheres.

[0034] 50 g of activated microspheres were added to 10 L of an alkaline protease aqueous solution with a concentration of 50 g / L. The mixture was slowly shaken at room temperature for 2 h, and then placed in a refrigerator at 4 ℃ for 16 h. The microspheres were separated by a magnet and repeatedly washed with 0.1 M acetate-sodium acetate buffer (pH 9.0) at 0 ℃. The microspheres were then freeze-dried under vacuum to obtain the chitosan-immobilized protease, referred to as the immobilized enzyme. The protease recovery rate and immobilized enzyme activity were determined.

[0035] Example 2 Sodium alginate immobilized protease

[0036] 500g of sodium alginate was dissolved in 25L of distilled water to obtain a 2.0% (w / v) sodium alginate solution. Fe3O4 powder with a final concentration of 16g / L was added to this solution, and the mixture was stirred for 30 min. The mixture was then heated to 80 °C under nitrogen protection and stirred for 1 h. Subsequently, it was further ultrasonically dispersed for 20 min, and 1250g of alkaline protease was added to bring the final concentration of alkaline protease in the mixture to 50 g / L. The mixture was then atomized and sprayed into 10 L of 10 wt% CaCl2 aqueous solution. The mixture was then soaked and hardened at 4 °C for 1 h to form magnetically immobilized enzyme microspheres. The solid product was magnetically separated and recovered, washed with distilled water, and then added to 5 L of 0.5% glutaraldehyde aqueous solution. The cross-linking reaction was carried out at room temperature for 1.5 h. The solid product was magnetically separated and recovered, and excess glutaraldehyde was washed away with distilled water. The product was then placed in a refrigerator at 4 °C for hardening and vacuum freeze-drying to obtain sodium alginate immobilized protease, referred to as immobilized enzyme. The protease recovery rate and immobilized enzyme activity were determined.

[0037] Example 3: Immobilized protease supported on nano-Fe3O4

[0038] 10 g of Fe3O4 nanoparticles were dispersed in 1000 mL of ultrapure water, the pH was adjusted to 8.0, and 50 g of alkaline protease was added to make the final concentration of alkaline protease in the mixture 50 g / L. The mixture was stirred and adsorbed for 20 min, then filtered and freeze-dried under vacuum to obtain Fe3O4 immobilized protease, referred to as immobilized enzyme. The protease recovery rate and immobilized enzyme activity were determined.

[0039] Example 4: Polyethylene glycol-modified Fe3O4 nanoparticles immobilized with protease

[0040] The average molecular weight of polyethylene glycol is 1000. Dissolve it in water to prepare a polyethylene glycol solution with a mass concentration of 0.8%.

[0041] 500 g of Fe3O4 nanoparticles were dispersed in 10 L of the prepared polyethylene glycol solution. The suspension was incubated at 40 °C under nitrogen for 20 minutes, then sonicated at 19 W and 40 kKz for 20 minutes. The precipitate was collected by centrifugation and freeze-dried to obtain polyethylene glycol-modified iron oxide nanoparticles. 500 g of polyethylene glycol / iron oxide nanoparticles were dispersed in 50 L of ultrapure water, the pH was adjusted to 8.0, and 2500 g of alkaline protease was added to achieve a final concentration of 50 g / L. The mixture was stirred for 20 minutes for adsorption, then filtered, and the nanoparticles were collected and freeze-dried to obtain immobilized iron oxide nanoparticle protease, referred to as immobilized enzyme. The protease recovery rate and immobilized enzyme activity were determined.

[0042] Example 5: Sodium alginate-encapsulated polyethylene glycol-modified nano-Fe3O4 immobilized protease

[0043] Example 5: The preparation steps of the immobilized enzyme include:

[0044] Step 1: Repeat the experimental steps of Example 4 to prepare polyethylene glycol modified Fe3O4 nanoparticles loaded with immobilized protease;

[0045] Step 2: Take 500 g of the polyethylene glycol-modified Fe3O4 nanoparticles immobilized with protease prepared in Step 1, disperse it in 50 L of 2% sodium alginate aqueous solution, mix thoroughly, and then spray it into 10 L of 10 wt% CaCl2 aqueous solution. Soak and harden at 4℃ for 1 h to form magnetically immobilized enzyme microspheres. Magnetically separate and recover the solid product, wash it with distilled water, and add it to 5 L of 0.5% glutaraldehyde aqueous solution. Perform a crosslinking reaction at room temperature for 1.5 h. Magnetically separate and recover the solid product, wash it with distilled water, and freeze-dry it under vacuum to obtain sodium alginate-embedded polyethylene glycol-modified Fe3O4 nanoparticles immobilized with protease, referred to as immobilized enzyme. Determine the protease recovery rate and immobilized enzyme activity.

[0046] Example 6: Sodium alginate-encapsulated polyethylene glycol-modified nano-Fe3O4 immobilized protease

[0047] Example 6: The preparation steps of the immobilized enzyme include:

[0048] Step 1: Repeat the experimental steps of Example 4 to prepare polyethylene glycol modified Fe3O4 nanoparticles loaded with immobilized protease;

[0049] Step 2: Take 500 g of the polyethylene glycol-modified Fe3O4 nanoparticles immobilized with protease prepared in Step 1, and 500 g of alkaline protease, and disperse them together in 50 L of 2% sodium alginate aqueous solution. After mixing evenly, spray the mixture into 10 L of 10 wt% CaCl2 aqueous solution and soak and harden at 4℃ for 1 h to form magnetically immobilized enzyme microspheres. Magnetically separate and recover the solid product, wash it with distilled water, and add it to 5 L of 0.5% glutaraldehyde aqueous solution. Perform a crosslinking reaction at room temperature for 1.5 h. Magnetically separate and recover the solid product, wash it with distilled water, and freeze-dry it under vacuum to obtain sodium alginate-embedded polyethylene glycol-modified Fe3O4 nanoparticles immobilized with protease.

[0050] The enzyme activities of the immobilized enzymes prepared in Examples 1-6 were measured, and the enzyme activity recovery rate during the immobilized enzyme preparation process was calculated according to the following formula: Enzyme activity recovery rate (%) = (Total enzyme activity of immobilized enzyme / Total enzyme activity of free enzyme used) × 100%. Taking the immobilized enzyme prepared in Example 6 as an example, the free enzyme used in the calculation of its enzyme activity recovery rate includes two parts.

[0051] The immobilized enzymes prepared in Examples 1-6 were tested for enzyme activity and enzyme activity recovery rate, and the test results are shown in Table 1.

[0052] Table 1

[0053] .

[0054] The immobilized protease activities and enzyme activity recovery rates obtained by different immobilization methods are shown in Table 1. The enzyme activity recovery rate in Table 1 reflects both the amount of protease loaded onto the carrier from the solution and the activity of the enzyme after immobilization. As can be seen from Table 1:

[0055] 1) The surface immobilization methods in Examples 1 and 4 showed high enzyme activity because the steric hindrance of enzyme adsorption on the carrier surface was small, and the contact area between the enzyme adsorbed on the carrier surface and the casein substrate was large. However, because the surface area of ​​the carrier was relatively small, the enzyme adsorption capacity was limited, so the enzyme activity recovery rate was not high (<10%).

[0056] 2) In Example 3, the high pore size ratio of nano-iron oxide and the large specific surface area significantly increased the enzyme activity recovery rate (35.62%). However, the enzyme activity decreased due to steric hindrance. This is because the pores of nano-iron oxide adsorb the protease. The reaction between the protease and the casein substrate can be divided into the following two cases: ① The casein substrate enters the pores of nano-iron oxide and contacts the protease; ② The protease diffuses out from the pores of nano-iron oxide and enters the solution to contact the casein substrate. Both cases involve steric hindrance.

[0057] 3) In Example 2, the sodium alginate-encapsulated iron oxide and free protease group showed the highest enzyme activity recovery rate, reaching 92.32%. However, due to the increased mass transfer resistance of the substrate from physical encapsulation, the enzyme activity decreased to 16,000 (U / g). In addition, there was a problem of uneven encapsulation of protease and Fe3O4.

[0058] 4) In Example 5, the enzyme was first immobilized on the surface of nano Fe3O4, and then embedded in sodium alginate, which solved the problems of uneven embedding and enzyme stability. However, due to the complex mass transfer, some enzyme activity was sacrificed.

[0059] 5) In Example 6, protease immobilized on the Fe3O4 surface and free enzyme were co-encapsulated inside large-pore sodium alginate microspheres. This solved the problem of low enzyme activity recovery rate. At the same time, the presence of some free enzyme inside the sodium alginate microspheres improved mass transfer and maintained high activity. In addition, the flexible immobilized enzyme had enhanced mechanical collision resistance and was not easily broken. The protease immobilized on the Fe3O4 surface inside was not easily detached and could maintain stable catalysis for a long time. This enabled the repeated catalytic use of enzymes in complex heterogeneous systems and greatly improved production economy.

[0060] Application Example 1: Utilizing the present invention Figures 1-2 The device described above is used for bio-oil production. The device includes a stirred reactor 1 with a kettle-like structure. The stirred reactor 1 has an overflow port 4 at its upper end and a discharge port 5 at its bottom. A heat exchange jacket for heating the stirred reactor 1 is located on its outer side. A heat exchange fluid flows through the heat exchange jacket. A ring magnet 3 is installed on the outer side of the heat exchange jacket and is connected to a power source via a wire. Both the stirred reactor 1 and the heat exchange jacket are made of non-magnetic stainless steel. An agitator 2 is located at the center of the stirred reactor 1. The upper end of the agitator 2 extends from the top of the stirred reactor 1 and is fixedly connected to a motor. Three sets of agitator blades 201 are spaced apart from top to bottom at the lower end of the agitator 2. Each set of agitator blades 201 includes three blades 201 evenly spaced circumferentially. The edges of the agitator blades 201 are smooth arc-shaped. The agitator blades 201 are inclined relative to the horizontal plane and have evenly distributed liquid permeable holes. Figure 2 (Not shown in the image); both the impeller 2 and the impeller blade 201 are made of polytetrafluoroethylene.

[0061] Other comparisons Figure 2 In this design, the end of the stirring blade 201 closest to the stirring paddle 2 is designated as the inner end, and the end of the stirring blade 201 furthest from the stirring paddle 2 is designated as the outer end. The blade width at the inner end of the stirring blade 201 is greater than the blade width at the outer end. This widening of the inner end of the stirring blade 201 increases the liquid discharge capacity of the root blades and improves the stability of the stirring blade 201 during stirring.

[0062] Application Example 1 of the Invention: Utilizing the Invention Figures 1-2 The device described above performs bio-oil production, and the specific process steps are as follows:

[0063] 1) Shelled camellia seeds are mechanically crushed through a 100-mesh sieve. 50 kg of camellia seed powder is dispersed in 350 kg of soft water in a stirred reactor. The temperature of the mixture in stirred reactor 1 is heated to 60°C and then kept at that temperature. NaOH solution is added dropwise to the mixture to adjust the pH to 9.0.

[0064] 2) Add 400g of immobilized enzyme to the mixture described in step 1), and hydrolyze it at 60℃ for 4 hours with stirring at 200rpm. Then stop the reaction, stop stirring, and let it stand to allow the oil and water to separate into an upper oil phase and a lower water phase. The immobilized enzyme is enriched in the lower water phase. Water is introduced into the bottom of the stirred reactor 1, and the upper oil is collected from the overflow port. The collected oil product is dehydrated and weighed. The oil yield obtained from the hydrolysis extraction of camellia seeds is calculated, with the unit being g oil / 100g camellia seeds.

[0065] 3) Then restart the stirring to make the immobilized enzyme evenly dispersed in the aqueous phase. Apply electricity to the ring magnet on the outside of the side of the stirred reactor to make the immobilized enzyme adsorbed on the inner wall of the side of the stirred reactor. The mixture containing slurry is discharged from the bottom discharge port of the stirred reactor. Rinse the inside of the stirred reactor with clean water. Then the immobilized enzyme adsorbed on the inner wall of the side of the stirred reactor is repeatedly recycled.

[0066] 4) Add new camellia seed powder to the stirred reactor and carry out the next batch of hydrolysis reaction according to the operation process of steps 1)-3). Turn off the power to the ring magnet on the outside of the stirred reactor so that the immobilized enzyme is repeatedly dispersed in the reaction solution in the stirred reactor. The hydrolysis reaction time for each batch is 4 hours. This cycle experiment is carried out to test the experimental results of the immobilized enzyme under the cycle.

[0067] Following the experimental process of steps 1)-4) in Application Example 1, the oil yield and enzyme half-life calculation results for different numbers of uses of the immobilized enzyme in the hydrolysis reaction using the immobilized enzymes prepared in Examples 1-6 are summarized in Table 2.

[0068] Table 2

[0069] .

[0070] Furthermore, based on the experimental results in Table 2, a fitting curve equation was established for the hydrolysis reaction using the immobilized enzymes prepared in each embodiment. This fitting curve equation describes the relationship between the number of times the immobilized enzyme is used and the oil yield. In the fitting curve equation, the oil yield is the vertical axis, and the total hydrolysis time for the number of times the immobilized enzyme is used is the horizontal axis. The total hydrolysis time for 1 use of the immobilized enzyme is 4 h, the total hydrolysis time for 2 uses is 8 h, the total hydrolysis time for 3 uses is 12 h, and so on.

[0071] Under the experimental conditions described above, the oil yield was calculated as a function of the total hydrolysis time for each use of the immobilized enzyme, based on the immobilized enzymes prepared in Examples 1-6 during the hydrolysis reaction. The results are summarized below. Figure 3 The corresponding fitted curve equations are as follows:

[0072] 1) When using the immobilized enzyme prepared in Example 1, the established fitting curve equation is y = 49.223e -0.035x Correlation coefficient R 2 =0.9954.

[0073] 2) When using the immobilized enzyme prepared in Example 2, the established fitting curve equation is y = 49.215e -0.029x Correlation coefficient R 2 =0.9807.

[0074] 3) When using the immobilized enzyme prepared in Example 3, the established fitting curve equation is y = 47.952e -0.045x Correlation coefficient R 2 =0.991.

[0075] 4) When using the immobilized enzyme prepared in Example 4, the established fitting curve equation is y = 50.607e -0.032x Correlation coefficient R 2 =0.9918.

[0076] 5) When using the immobilized enzyme prepared in Example 5, the established fitting curve equation is y = 42.625e -0.018x Correlation coefficient R 2 =0.9988.

[0077] 6) When using the immobilized enzyme prepared in Example 1, the established fitting curve equation is y = 49.973e -0.014x Correlation coefficient R 2 =0.9947.

[0078] The calculation process for the half-life in Table 2 is as follows: the hydrolysis time required to achieve 50% of the oil yield when the oil yield reaches the level of the first use of the immobilized enzyme (i.e., a hydrolysis time of 4 hours) is calculated using the established fitted curve equation. This calculated half-life result is recorded as the half-life result.

[0079] The following conclusions can be drawn from the experimental results in Table 2:

[0080] 1) The immobilized enzymes prepared in Examples 1-6 all had a high oil yield after the first hydrolysis reaction, approximately 40g / 100g of camellia seeds.

[0081] 2) Among them, the immobilized enzymes in Examples 2 and 4 showed the best activity and the highest first-batch hydrolyzed oil yield. However, during repeated use, the instability of enzymes immobilized on rigid supports became prominent. For example, the half-life of the immobilized enzyme loaded with Fe3O4 adsorption in Example 3 was only 15.4 h, indicating that the collisions in the heterogeneous slurry-slag mixture were intense, the support was easily broken, and the internal enzyme easily detached from the support, resulting in poor stability. The enzyme immobilized on ethylene glycol-modified nano-Fe3O4 in Example 4 showed slightly better stability, with a half-life of 21.7 h. The enzyme immobilized by sodium alginate magnetic microspheres in Example 2 showed higher stability, with a half-life of 23.9 h. This may be related to the flexibility and collision resistance of the hydrogel surface, but because the physically embedded protease gradually desorbed from the gaps and diffused into the solution after long-term reaction collisions, its stability was still insufficient.

[0082] 3) In Example 6, alkaline protease was first immobilized on the surface of ethylene glycol-modified nano-Fe3O4, and then encapsulated together with some free protease in sodium alginate. This not only solved the enzyme activity problem, but also greatly improved the stability of the immobilized enzyme, with a half-life of 49.51h, which can be used continuously for 10 batches while maintaining relatively high activity.

[0083] Furthermore, the enzyme activity of the sodium alginate immobilized protease prepared in Example 2 of Table 1 is relatively low. This is the result of testing the enzyme activity system according to the method of GB / T 23527-2009, because the mass transfer effect is affected after sodium alginate encapsulation. However, the results in Table 2 are the reaction results in the actual hydrolysis reaction catalyzed by the immobilized enzyme, reflecting the average activity of the immobilized enzyme. High activity indicates that the immobilized enzyme has good stability and slow inactivation, thus indicating a good average catalytic effect.

[0084] Application Example 2: Bio-oil production was carried out using the immobilized enzymes prepared in Examples 1-6. The specific process steps were repeated in Example 1, the only difference being the use of a conventional reactor for the hydrolysis reaction. For the conventional reactor, after each hydrolysis reaction, the immobilized enzyme was manually recovered from the reaction mixture using a magnet for use in the next batch of hydrolysis.

[0085] Table 3 summarizes the oil yield and enzyme half-life calculation results for different numbers of uses of the immobilized enzymes when the immobilized enzymes in the hydrolysis reaction of Example 2 were prepared according to Examples 1-6.

[0086] Table 3

[0087] .

[0088] The results of oil yield versus total hydrolysis time with the number of times the immobilized enzyme was used in the hydrolysis reaction of Example 2 of this invention are summarized below. Figure 4 middle.

[0089] In Table 3 of the results obtained from Application Example 2 of the present invention, the method for calculating the half-life is the same as that in Application Example 1.

[0090] Comparing Table 2 of Application Example 1 with Table 3 of Application Example 2, it can be seen that the stirring blades of the reactor also have a great influence on the enzyme-catalyzed hydrolysis reaction. The special reactor of the present invention can further improve the stability of the immobilized enzyme.

[0091] The contents described in this specification are merely an enumeration of the implementation forms of the inventive concept, and the scope of protection of this invention should not be regarded as limited to the specific forms described in the embodiments.

Claims

1. A bio-oil production method based on reusable immobilized enzymes, characterized by, The camellia seed powder and soft water are added into a stirring reactor, the pH of the reaction solution is adjusted to be alkaline, the immobilized enzyme is added, and a stirring hydrolysis reaction is carried out; The stirring reactor is a kettle structure, a stirring paddle (2) is arranged in the center of the stirring reactor (1), the upper end of the stirring paddle (2) penetrates through the top of the stirring reactor (1) and is fixedly connected with a motor, a plurality of groups of stirring paddle blades (201) are arranged on the lower end of the stirring paddle (2) in a top-to-bottom and interval manner, the edges of the stirring paddle blades (201) are smooth arc-shaped edges, the stirring paddle blades (201) are arranged to be inclined relative to the horizontal plane, and the stirring paddle blades (201) are uniformly distributed with liquid-permeable small holes; the stirring paddle (2) and the stirring paddle blades (201) are both made of plastic material; The preparation method of the immobilized enzyme comprises: taking polyethylene glycol modified Fe3O4 nanoparticles as a matrix, stirring the matrix with the first part of free protease in ultrapure water according to a mass ratio of 4-8:1, so that the free protease is fully adsorbed on the matrix, then filtering, collecting the nanoparticles, freeze-drying to obtain polyethylene glycol modified nano Fe3O4 loaded immobilized protease, then dispersing the second part of free protease in a sodium alginate aqueous solution according to a mass ratio of 0.8-1.2:1, mixing uniformly, so that the sodium alginate is coated on the outer layer, then sequentially performing Ca 2+ The two-step process of ion hardening and glutaraldehyde cross-linking reaction makes the outer layer of sodium alginate react to form a reticular macroporous gel microsphere, and the polyethylene glycol modified nano Fe3O4 loaded immobilized protease and the second part of free protease are co-embedded in the interior of the gel microsphere. Finally, after washing with distilled water and vacuum freeze-drying, the microsphere complex embedded with the second part of free protease and the polyethylene glycol modified nano Fe3O4 loaded immobilized protease is prepared, that is, the preparation is completed. The molecular weight of the polyethylene glycol is 500-2000, and the mass ratio of the Fe3O4 nanoparticles to the polyethylene glycol is 1:1.5-1.

8. The mass ratio of the polyethylene glycol modified nano Fe3O4 to the immobilized protease is 1:1.5-3.

2. The method of producing bio-oil based on reusable immobilized enzyme according to claim 1, wherein, The preparation method of the polyethylene glycol modified nano Fe3O4 immobilized protease specifically includes the following steps: Step 1: Fe3O4 nanoparticles are dispersed in a polyethylene glycol aqueous solution, the suspension is ultrasonically dispersed at 30-50 ℃ for 10-30 min under the condition of nitrogen gas being introduced, the precipitate is collected by centrifugation and freeze-dried, and polyethylene glycol modified nano iron oxide is obtained; Step 2: The polyethylene glycol modified nano iron oxide obtained in step 1 and a first part of free protease are dispersed in ultrapure water in a mass ratio of 4-8:1, stirring adsorption is carried out under the condition of the pH being adjusted to 7.5-8.5 for 10-30 min, then filtration is carried out, the nano particles are collected and freeze-dried, and the polyethylene glycol modified nano Fe3O4 immobilized protease is obtained.

3. The method of producing bio-oil based on reusable immobilized enzyme according to claim 1, wherein, The preparation method of the microsphere complex for embedding a second part of free protease and the polyethylene glycol modified nano Fe3O4 immobilized protease specifically includes the following step: S1: The polyethylene glycol modified nano Fe3O4 immobilized protease and the second part of free protease are dispersed in a sodium alginate aqueous solution; S2: the mixed solution obtained in step S1 is sprayed into Ca 2+ After hardening treatment at a temperature of 2-5℃ in the ionic aqueous solution for 0.5-2h, the magnetically immobilized microspheres are obtained and washed with distilled water; wherein the mass ratio of sodium alginate in step S1 to Ca 2+ is 1:0.3-0.5; S3: The magnetic immobilized microspheres obtained in step S2 are immersed in a 0.1-1% mass fraction of glutaraldehyde aqueous solution, crosslinking reaction is carried out at room temperature for 1-2 h, then the precipitate is separated by centrifugation, the excess glutaraldehyde is removed by washing with distilled water, and vacuum freeze-drying is carried out, and the preparation is completed.

4. The method of producing bio-oil based on reusable immobilized enzyme according to claim 1, wherein, The hydrolysis reaction further includes the following steps: after the reaction is completed, stirring is stopped, oil and water are separated by standing, the upper layer of oil is discharged, and the product oil is obtained after dehydration and drying; then the stirring is restarted, the annular magnet on the side of the stirring reactor is electrified, the immobilized enzyme is adsorbed on the inner wall of the side of the stirring reactor, the mixed solution containing slurry is discharged from the lower discharge port at the bottom of the stirring reactor, the inside of the stirring reactor is washed with clean water, and then the next batch of hydrolysis reaction is carried out by adding the camellia seed powder and soft water again, so that the immobilized enzyme can be reused.

5. The reusable enzyme-immobilized bio-oil production method according to claim 1, wherein The mass ratio of the camellia seed powder to soft water for the hydrolysis reaction is 1:4-10, and the mass of the immobilized enzyme is 0.5-1% of the mass of the camellia seed powder.

6. A method for bio-oil production based on reusable immobilized enzymes according to claim 1, characterized in that The reaction temperature for the hydrolysis reaction is 50-70 DEG C, the reaction time is 3-5h, and the stirring speed is 150-300rpm.

7. A method for bio-oil production based on reusable immobilized enzymes according to claim 1, characterized in that The pH of the reaction solution is adjusted to 8.5-9.

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8. A method for bio-oil production based on reusable immobilized enzymes according to claim 1, characterized in that The upper end of the stirring reactor (1) is provided with an overflow (4), and the bottom is provided with a lower discharge port (5). The outer side of the stirring reactor (1) is provided with a heat exchange jacket for heating the stirring reactor (1). A heat exchange fluid is introduced into the heat exchange jacket. The outer side of the heat exchange jacket is provided with an annular magnet (3). The annular magnet (3) is connected to a power supply through a wire. The stirring reactor (1) and the heat exchange jacket are made of non-magnetic stainless steel.

9. A method for bio-oil production based on reusable immobilized enzymes according to claim 1, characterized in that The stirring paddle (2) and the stirring paddle blade (201) are made of polytetrafluoroethylene.

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