A method for accelerating the enzymatic esterification reaction by using an ultrasonic oscillation rod

Abstract

The present invention relates to the technical field of biodiesel, and discloses a method for accelerating the enzymatic esterification reaction by using an ultrasonic oscillator rod. The method comprises the following steps: adding waste oil and methanol into a beaker, and then respectively adding deionized water and immobilized lipase; heating the beaker in a water bath, then inserting an ultrasonic oscillator rod into the beaker to carry out ultrasonic oscillation reaction to obtain crude biodiesel; performing precision filtration on the crude biodiesel to separate and recover the immobilized lipase, then standing for stratification, and removing the glycerol in the lower layer after stratification to obtain biodiesel. By means of ultrasonic oscillation, the reaction monomers are brought into full contact with the immobilized lipase, thereby shortening the esterification reaction time and improving the synthesis efficiency of biodiesel.

Description

Technical Field

[0001] The present invention relates to the technical field of biodiesel, and particularly to a method for accelerating the enzymatic esterification reaction by using an ultrasonic oscillation rod. Background Art

[0002] With the growth of the population, the demand for fuel is increasing, and the reserves of fossil fuels are decreasing day by day. People have begun to pay attention to the impact of these resources on the environment and the impact of excessive carbon emissions. Finding a better and cleaner fuel is necessary for environmental protection and alleviating the pressure on petrochemical fuels. Biodiesel is a green and safe fuel that can be made from edible and inedible vegetable oils, animal fats, waste cooking oil, and many other inferior-quality oils. Biodiesel is mainly long-chain fatty acid methyl esters or ethyl esters, such as higher fatty acid methyl esters, which can be prepared from biomass such as widely sourced, easily obtainable, and fatty acid-rich animal and vegetable oils, waste frying oil, animal and vegetable waste oil, algae, and cellulose as raw materials and short-chain alcohols such as methanol or ethanol under the action of acids, alkalis, or enzymes. Biodiesel has good combustion performance and does not produce harmful gases such as sulfur dioxide during combustion. It has the characteristics of being green, safe, and renewable, and has gradually become a substitute for traditional petrochemical diesel.

[0003] Currently, the production processes of biodiesel mainly include chemical methods and enzymatic methods. Chemical methods generally use inorganic acids or alkalis as catalysts, which have disadvantages such as complex processes, high energy consumption, large alcohol consumption, and environmental pollution. Enzymatic methods have advantages such as mild reaction conditions, easy separation of products, and environmental friendliness, and are considered to be green processes that are expected to replace chemical methods for producing biodiesel. If free lipase is used as a catalyst, after the reaction, the separation, regeneration, and recycling of the enzyme are very difficult. If immobilized lipase is used, the above-mentioned defects of free enzymes can be overcome, so that the enzyme molecules are protected while effectively playing a catalytic role, are not easily affected by the environment, are easy to separate from the substrate and products, and can be recycled multiple times, thereby reducing costs and showing good application prospects. However, compared with free lipase, immobilized lipase cannot fully contact the reaction monomers, thus affecting the rate of the esterification reaction, and the esterification reaction time is longer, which affects the production efficiency of biodiesel. Summary of the Invention

[0004] In order to overcome the above prior art problems, the present invention provides a method for accelerating the enzymatic esterification reaction by using an ultrasonic oscillation rod. The present invention enables the reaction monomers to fully contact the immobilized lipase through ultrasonic oscillation, thereby shortening the esterification reaction time and improving the synthesis efficiency of biodiesel.

[0005] In order to achieve the above invention purpose, the present invention adopts the following technical solutions:

[0006] A method for accelerating the enzymatic esterification reaction by using an ultrasonic oscillation rod, comprising the following steps:

[0007] 1) Add waste oil and methanol into a beaker, and then add deionized water and immobilized lipase respectively;

[0008] 2) Place the beaker in a water bath and heat it to 40 - 50 °C, then insert an ultrasonic oscillation rod into the beaker and carry out ultrasonic oscillation reaction to obtain crude biodiesel;

[0009] 3) Precisely filter the crude biodiesel, separate and recover the immobilized lipase, then let it stand for stratification, and remove the lower - layer glycerol after stratification to obtain biodiesel.

[0010] In the technical solution of the present invention, waste oil and methanol are used as reaction monomers, and biodiesel is synthesized under the catalytic action of immobilized lipase. The immobilized lipase molecules are protected while effectively playing a catalytic role, are not easily affected by the environment, are easy to separate from substrates and products, and can be recycled multiple times, thereby reducing costs. However, compared with free lipase, the immobilized lipase cannot fully contact the reaction monomers, which affects the rate of the esterification reaction, the esterification reaction time is longer, and the production efficiency of biodiesel is affected. In the present invention, under the action of an ultrasonic oscillation rod during the esterification reaction, the reaction monomers enter the carrier of the immobilized lipase, so that the reaction monomers can fully contact the lipase in the carrier, thereby increasing the rate of the esterification reaction and promoting the synthesis efficiency of biodiesel.

[0011] Preferably, in step 1), the mass ratio of waste oil to methanol is 8:(1 - 2).

[0012] Preferably, in step 1), the addition amount of the immobilized lipase is 1% - 5% of the mass of the waste oil.

[0013] Preferably, in step 2), the oscillation frequency of the ultrasonic oscillation rod is 19 kHz - 20 kHz.

[0014] Preferably, in step 2), the ultrasonic oscillation reaction time is 8 h - 10 h.

[0015] Preferably, in step 1), the preparation method of the immobilized lipase comprises the following steps:

[0016] 11) Add the silane coupling agent KH - 560 into the mixed solution of ethanol and water, heat it to 50 °C, and obtain a coupling agent hydrolysis solution through stirring;

[0017] 12) Add sepiolite into the coupling agent hydrolysis solution, continue stirring and reacting, and obtain coupling agent - modified sepiolite through centrifugal separation, washing and drying;

[0018] 13) Add carboxymethyl chitosan to deionized water and stir to dissolve it to obtain a carboxymethyl chitosan solution. Add the coupling agent-modified sepiolite to the carboxymethyl chitosan solution, heat to 80 °C, and stir and react for 2 h. After filtration and separation, obtain the nascent sepiolite-carboxymethyl chitosan composite particles;

[0019] 14) Add glutaraldehyde to deionized water and stir evenly to obtain a glutaraldehyde solution. Add the nascent sepiolite-carboxymethyl chitosan composite particles to the glutaraldehyde solution, heat and react, and after centrifugal separation, washing and drying, obtain the sepiolite-carboxymethyl chitosan composite particles;

[0020] 15) Add Aspergillus lipase to the phosphate buffer solution to obtain an Aspergillus lipase solution. Add the sepiolite-carboxymethyl chitosan composite particles to deionized water, stir to form a suspension, then add the Aspergillus lipase solution to the suspension, stir evenly, let stand, and after filtration separation, washing and freeze-drying, obtain the immobilized lipase.

[0021] In the technical solution of the present invention, as described above, under the action of an ultrasonic vibration rod, the reaction monomers enter the carrier of the immobilized lipase, so that the reaction monomers are in full contact with the lipase in the carrier, thereby increasing the esterification reaction rate and promoting the synthesis efficiency of biodiesel. In the technical solution of the present invention, Aspergillus lipase is used as the biocatalyst for the esterification reaction, and sepiolite is used as the carrier of Aspergillus lipase. The Aspergillus lipase is bound to the sepiolite by the porous adsorption of sepiolite. However, the problem is that during the esterification reaction, the method of ultrasonic oscillation easily causes the Aspergillus lipase to detach from the sepiolite, and the lipase detached from the sepiolite is difficult to separate from the reaction products, affecting the recycling of the biocatalyst. To solve this problem, the present invention modifies the sepiolite. First, an epoxy silane coupling agent is grafted on the surface and inside the pores of the sepiolite, and then it is immersed in a carboxymethyl chitosan solution. A ring-opening reaction occurs between the amino groups on a part of the carboxymethyl chitosan molecules in the solution and the epoxy groups grafted on the surface of the sepiolite, thereby grafting the carboxymethyl chitosan on the surface and inside the pores of the sepiolite (bonding the carboxymethyl chitosan molecules to the sepiolite through chemical bonds to increase the binding force between the two). Another part of the unreacted carboxymethyl chitosan is adsorbed inside the pores of the sepiolite, and then the carboxymethyl chitosan molecules undergo a cross-linking reaction under the cross-linking action of glutaraldehyde. The cross-linked carboxymethyl chitosan modifies the pores of the sepiolite, which is beneficial to the adsorption of Aspergillus lipase by the sepiolite and increases the binding force between the two. In addition, due to the hydrogen bond force between the hydroxyl groups on the carboxymethyl chitosan molecules and the amino groups on the Aspergillus lipase, the binding force between the sepiolite and the lipase is further increased. Through the above modification of the sepiolite, the problem that the Aspergillus lipase falls off from the sepiolite under the vibration of the ultrasonic vibration rod is solved, and the recycling rate of the biocatalyst is improved. Preferably, in step 13), the mass ratio of the coupling agent-modified sepiolite to carboxymethyl chitosan is 1:(0.5 - 1.3).

[0022] In the technical solution of the present invention, to stably bind the Aspergillus lipase to the sepiolite and prevent the Aspergillus lipase from detaching from the sepiolite under ultrasonic oscillation, a sufficient amount of carboxymethyl chitosan must be grafted and cross-linked on the sepiolite. Therefore, the present invention controls the mass ratio of the coupling agent-modified sepiolite to carboxymethyl chitosan to be less than 1:0.5. The research team of the present invention accidentally found that when the grafting and cross-linking of carboxymethyl chitosan are increased to a certain extent, that is, when the mass ratio of the coupling agent-modified sepiolite to carboxymethyl chitosan is less than 1:1.3, the Aspergillus lipase bound to the sepiolite decreases significantly, resulting in a decrease in the conversion rate of biodiesel. This may be because the excessive cross-linked carboxymethyl chitosan causes the microporous structure of the sepiolite to be blocked, affecting the adsorption of Aspergillus lipase by the sepiolite. Therefore, the present invention controls the mass ratio of the coupling agent-modified sepiolite to carboxymethyl chitosan to be greater than 1:1.3.

[0023] Preferably, in step 14), the heating reaction temperature is 50°C - 60°C.

[0024] Preferably, in the step 14), the heating reaction time is 3 h - 8 h.

[0025] Preferably, in the step 15), the standing time is 10 - 20 h.

[0026] The present invention has the following beneficial effects:

[0027] 1) The immobilized lipase molecules are protected while effectively playing a catalytic role, are not easily affected by the environment, are easy to separate from the substrate and the product, and can be recycled multiple times, thereby reducing costs;

[0028] 2) By using an ultrasonic oscillation rod during the esterification reaction, the reaction monomers enter the carrier of the immobilized lipase, so that the reaction monomers are in full contact with the lipase in the carrier, thereby increasing the esterification reaction rate and promoting the synthesis efficiency of biodiesel;

[0029] 3) Aspergillus lipase has a strong binding force with the modified sepiolite, and Aspergillus lipase is not easily detached from the sepiolite under the vibration of the ultrasonic vibration rod, improving the recovery and utilization rate of the biological enzyme. Specific embodiments

[0030] The following further clearly and detailedly describes the present invention in combination with specific embodiments. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are usually only a part of the embodiments of the present invention, rather than all the embodiments. Therefore, all other embodiments obtained by those of ordinary skill in the art without creative work based on the embodiments of the present invention shall fall within the scope of protection of the present invention.

[0031] Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available or raw materials that can be obtained by those skilled in the art; unless otherwise specified, the methods used in the embodiments of the present invention are all methods mastered by those skilled in the art. The waste oil used in the specific embodiments of the present invention is industrial waste soybean oil from which particulate impurities and moisture have been removed, and the acid value of the waste oil is 83 mgKOH / g.

[0032] Example 1

[0033] A method for accelerating the esterification reaction of biological enzymes by using an ultrasonic oscillation rod, comprising the following steps:

[0034] 1) Add 40 g of waste oil and 9 g of methanol to a beaker, and then add 2 mL of deionized water and 1.8 g of immobilized lipase respectively;

[0035] 2) Place the beaker in a water bath and heat it to 45 °C. Then insert an ultrasonic oscillator rod into the beaker. The oscillation frequency of the ultrasonic oscillator rod is 20 kHz, and carry out ultrasonic oscillation reaction for 9 h to obtain crude biodiesel;

[0036] 3) Precisely filter the crude biodiesel, separate and recover the immobilized lipase, then let it stand for stratification. After stratification, remove the glycerol in the lower layer to obtain biodiesel.

[0037] The preparation method of the immobilized lipase includes the following steps:

[0038] 11) Add 5 g of silane coupling agent KH-560 to a mixed solution of 300 mL of ethanol and 30 mL of water, heat it to 50 °C, and obtain a coupling agent hydrolysis solution through stirring;

[0039] 12) Add 3 g of sepiolite to the coupling agent hydrolysis solution, continue stirring and reacting, and obtain coupling agent-modified sepiolite through centrifugal separation, washing and drying;

[0040] 13) Add 1 g of carboxymethyl chitosan to 200 mL of deionized water and stir to dissolve it to obtain a carboxymethyl chitosan solution. Add the coupling agent-modified sepiolite to the carboxymethyl chitosan solution. The mass ratio of the coupling agent-modified sepiolite to carboxymethyl chitosan is 1:1.2. Heat it to 80 °C, stir and react for 2 h, and obtain primary sepiolite-carboxymethyl chitosan composite particles through filtration and separation;

[0041] 14) Add 0.3 g of glutaraldehyde to 200 mL of deionized water, stir evenly to obtain a glutaraldehyde solution. Add the above-mentioned primary sepiolite-carboxymethyl chitosan composite particles to the glutaraldehyde solution, heat it to 55 °C, react for 7 h, and obtain sepiolite-carboxymethyl chitosan composite particles through centrifugal separation, washing and drying;

[0042] 15) Add 0.5 g of Aspergillus lipase to 50 mL of phosphate buffer solution (pH = 7.5) to obtain an Aspergillus lipase solution. Add 2 g of sepiolite-carboxymethyl chitosan composite particles to 300 mL of deionized water, stir to form a suspension, then add the Aspergillus lipase solution to the suspension, stir evenly, let it stand for 18 h, and obtain the immobilized lipase through filtration and separation, washing and freeze-drying.

[0043] Example 2

[0044] A method for accelerating the enzymatic esterification reaction by using an ultrasonic oscillator rod includes the following steps:

[0045] 1) Add 40 g of waste oil and 6 g of methanol to a beaker, and then add 2 mL of deionized water and 0.8 g of immobilized lipase respectively;

[0046] 2) Place the beaker in a water bath and heat it to 45 °C. Then insert an ultrasonic oscillator rod into the beaker. The oscillation frequency of the ultrasonic oscillator rod is 19 kHz, and carry out ultrasonic oscillation reaction for 9 h to obtain crude biodiesel;

[0047] 3) Carry out precision filtration on the crude biodiesel, separate and recover the immobilized lipase, then let it stand for layering. After layering, remove the glycerol in the lower layer to obtain biodiesel.

[0048] The preparation method of the immobilized lipase includes the following steps:

[0049] 11) Add 5 g of silane coupling agent KH-560 to a mixed solution of 300 mL of ethanol and 30 mL of water, heat it to 50 °C, and obtain a coupling agent hydrolysis solution through stirring;

[0050] 12) Add 3 g of sepiolite to the coupling agent hydrolysis solution, continue stirring and reacting, and obtain coupling agent-modified sepiolite through centrifugal separation, washing and drying;

[0051] 13) Add 1 g of carboxymethyl chitosan to 200 mL of deionized water and stir to dissolve it to obtain a carboxymethyl chitosan solution. Add the coupling agent-modified sepiolite to the carboxymethyl chitosan solution. The mass ratio of the coupling agent-modified sepiolite to carboxymethyl chitosan is 1:0.6. Heat it to 80 °C, stir and react for 2 h, and obtain primary sepiolite-carboxymethyl chitosan composite particles through filtration and separation;

[0052] 14) Add 0.3 g of glutaraldehyde to 200 mL of deionized water, stir evenly to obtain a glutaraldehyde solution. Add the above-mentioned primary sepiolite-carboxymethyl chitosan composite particles to the glutaraldehyde solution, heat it to 55 °C, react for 4 h, and obtain sepiolite-carboxymethyl chitosan composite particles through centrifugal separation, washing and drying;

[0053] 15) Add 0.5 g of Aspergillus lipase to 50 mL of phosphate buffer solution (pH = 7.5) to obtain an Aspergillus lipase solution. Add 2 g of sepiolite-carboxymethyl chitosan composite particles to 300 mL of deionized water, stir to form a suspension, then add the Aspergillus lipase solution to the suspension, stir evenly, let it stand for 12 h, and obtain the immobilized lipase through filtration and separation, washing and freeze-drying.

[0054] Example 3

[0055] A method for accelerating biocatalytic esterification reaction by using an ultrasonic oscillator rod, including the following steps:

[0056] 1) Add 40 g of waste oil and 7 g of methanol to a beaker, and then add 2 mL of deionized water and 1 g of immobilized lipase respectively;

[0057] 2) Place the beaker in a water bath and heat it to 45 °C. Then insert an ultrasonic oscillator rod into the beaker. The oscillation frequency of the ultrasonic oscillator rod is 20 kHz, and carry out ultrasonic oscillation reaction for 9 h to obtain crude biodiesel;

[0058] 3) Precisely filter the crude biodiesel, separate and recover the immobilized lipase, then let it stand for stratification. After stratification, remove the glycerol in the lower layer to obtain biodiesel.

[0059] The preparation method of the immobilized lipase includes the following steps:

[0060] 11) Add 5 g of silane coupling agent KH-560 to a mixed solution of 300 mL of ethanol and 30 mL of water, heat it to 50 °C, and obtain a coupling agent hydrolysis solution through stirring;

[0061] 12) Add 3 g of sepiolite to the coupling agent hydrolysis solution, continue stirring and reacting, and obtain coupling agent-modified sepiolite through centrifugal separation, washing and drying;

[0062] 13) Add 1 g of carboxymethyl chitosan to 200 mL of deionized water and stir to dissolve it to obtain a carboxymethyl chitosan solution. Add the coupling agent-modified sepiolite to the carboxymethyl chitosan solution. The mass ratio of the coupling agent-modified sepiolite to carboxymethyl chitosan is 1:1. Heat it to 80 °C, stir and react for 2 h, and obtain primary sepiolite-carboxymethyl chitosan composite particles through filtration and separation;

[0063] 14) Add 0.3 g of glutaraldehyde to 200 mL of deionized water, stir evenly to obtain a glutaraldehyde solution. Add the above-mentioned primary sepiolite-carboxymethyl chitosan composite particles to the glutaraldehyde solution, heat it to 55 °C, react for 6 h, and obtain sepiolite-carboxymethyl chitosan composite particles through centrifugal separation, washing and drying;

[0064] 15) Add 0.5 g of Aspergillus lipase to 50 mL of phosphate buffer solution (pH = 7.5) to obtain an Aspergillus lipase solution. Add 2 g of sepiolite-carboxymethyl chitosan composite particles to 300 mL of deionized water, stir to form a suspension, then add the Aspergillus lipase solution to the suspension, stir evenly, let it stand for 15 h, and obtain the immobilized lipase through filtration and separation, washing and freeze-drying.

[0065] Example 4

[0066] A method for accelerating the enzymatic esterification reaction by using an ultrasonic oscillator rod includes the following steps:

[0067] 1) Add 40 g of waste oil and 10 g of methanol to a beaker, and then add 2 mL of deionized water and 2 g of immobilized lipase respectively;

[0068] 2) Place the beaker in a water bath and heat it to 50 °C. Then insert an ultrasonic oscillator rod into the beaker. The oscillation frequency of the ultrasonic oscillator rod is 20 kHz, and carry out ultrasonic oscillation reaction for 10 h to obtain crude biodiesel;

[0069] 3) Carry out precision filtration on the crude biodiesel to separate and recover the immobilized lipase, then let it stand for stratification. After stratification, remove the glycerol in the lower layer to obtain biodiesel.

[0070] The preparation method of the immobilized lipase includes the following steps:

[0071] 11) Add 5 g of silane coupling agent KH-560 to a mixed solution of 300 mL of ethanol and 30 mL of water, heat it to 50 °C, and obtain a coupling agent hydrolysis solution through stirring;

[0072] 12) Add 3 g of sepiolite to the coupling agent hydrolysis solution, continue stirring and reacting, and obtain coupling agent-modified sepiolite through centrifugal separation, washing and drying;

[0073] 13) Add 1 g of carboxymethyl chitosan to 200 mL of deionized water and stir to dissolve it to obtain a carboxymethyl chitosan solution. Add the coupling agent-modified sepiolite to the carboxymethyl chitosan solution. The mass ratio of the coupling agent-modified sepiolite to carboxymethyl chitosan is 1:1.3. Heat it to 80 °C, stir and react for 2 h, and obtain primary sepiolite-carboxymethyl chitosan composite particles through filtration and separation;

[0074] 14) Add 0.3 g of glutaraldehyde to 200 mL of deionized water, stir evenly to obtain a glutaraldehyde solution. Add the above-mentioned primary sepiolite-carboxymethyl chitosan composite particles to the glutaraldehyde solution, heat it to 60 °C, react for 8 h, and obtain sepiolite-carboxymethyl chitosan composite particles through centrifugal separation, washing and drying;

[0075] 15) Add 0.5 g of Aspergillus lipase to 50 mL of phosphate buffer solution (pH = 7.5) to obtain an Aspergillus lipase solution. Add 2 g of sepiolite-carboxymethyl chitosan composite particles to 300 mL of deionized water, stir to form a suspension, then add the Aspergillus lipase solution to the suspension, stir evenly, let it stand for 20 h, and obtain the immobilized lipase through filtration and separation, washing and freeze-drying.

[0076] Example 5

[0077] A method for accelerating biocatalytic esterification reaction by using an ultrasonic oscillator rod includes the following steps:

[0078] 1) Add 40 g of waste oil and 5 g of methanol to a beaker, and then add 2 mL of deionized water and 0.4 g of immobilized lipase respectively;

[0079] 2) Place the beaker in a water bath and heat it to 40 °C. Then insert an ultrasonic oscillator into the beaker. The oscillation frequency of the ultrasonic oscillator is 19 kHz, and carry out ultrasonic oscillation reaction for 8 h to obtain crude biodiesel;

[0080] 3) Carry out precision filtration on the crude biodiesel, separate and recover the immobilized lipase, then let it stand for stratification. After stratification, remove the glycerol in the lower layer to obtain biodiesel.

[0081] The preparation method of the immobilized lipase includes the following steps:

[0082] 11) Add 5 g of silane coupling agent KH-560 to a mixed solution of 300 mL of ethanol and 30 mL of water, heat it to 50 °C, and obtain a coupling agent hydrolysis solution through stirring;

[0083] 12) Add 3 g of sepiolite to the coupling agent hydrolysis solution, continue stirring and reacting, and obtain coupling agent-modified sepiolite through centrifugal separation, washing and drying;

[0084] 13) Add 1 g of carboxymethyl chitosan to 200 mL of deionized water and stir to dissolve it to obtain a carboxymethyl chitosan solution. Add the coupling agent-modified sepiolite to the carboxymethyl chitosan solution. The mass ratio of the coupling agent-modified sepiolite to carboxymethyl chitosan is 1:0.5. Heat it to 80 °C, stir and react for 2 h, and obtain nascent sepiolite-carboxymethyl chitosan composite particles through filtration and separation;

[0085] 14) Add 0.3 g of glutaraldehyde to 200 mL of deionized water, stir evenly to obtain a glutaraldehyde solution. Add the above-mentioned nascent sepiolite-carboxymethyl chitosan composite particles to the glutaraldehyde solution, heat it to 50 °C, react for 3 h, and obtain sepiolite-carboxymethyl chitosan composite particles through centrifugal separation, washing and drying;

[0086] 15) Add 0.5 g of Aspergillus lipase to 50 mL of phosphate buffer solution (pH = 7.5) to obtain an Aspergillus lipase solution. Add 2 g of sepiolite-carboxymethyl chitosan composite particles to 300 mL of deionized water, stir to form a suspension, then add the Aspergillus lipase solution to the suspension, stir evenly, let it stand for 10 h, and obtain the immobilized lipase through filtration and separation, washing and freeze-drying.

[0087] Comparative Example 1

[0088] The difference between Comparative Example 1 and Example 1 is that:

[0089] In step 2), the ultrasonic oscillator treatment is replaced by ordinary stirring treatment;

[0090] The remaining operation steps are the same as those in Example 1.

[0091] Comparative Example 2

[0092] The difference between Comparative Example 2 and Example 1 lies in that:

[0093] In the preparation process of the immobilized lipase, steps 11)-14) are omitted,

[0094] The sepiolite-carboxymethyl chitosan composite particles in step 15) are replaced with sepiolite;

[0095] The remaining operation steps are the same as those in Example 1.

[0096] Comparative Example 3

[0097] The difference between Comparative Example 3 and Example 1 lies in that:

[0098] In the preparation process of the immobilized lipase, steps 11) and 12) are omitted,

[0099] The coupling agent-modified sepiolite in step 13) is replaced with sepiolite;

[0100] The remaining operation steps are the same as those in Example 1.

[0101] Comparative Example 4

[0102] The difference between Comparative Example 4 and Example 1 lies in that:

[0103] In the preparation process of the immobilized lipase, step 14) is omitted,

[0104] The sepiolite-carboxymethyl chitosan composite particles in step 15) are replaced with nascent sepiolite-carboxymethyl chitosan composite particles;

[0105] The remaining operation steps are the same as those in Example 1.

[0106] Comparative Example 5

[0107] The difference between Comparative Example 5 and Example 4 lies in that:

[0108] In the preparation process of the immobilized lipase, step 13),

[0109] The mass ratio of the coupling agent-modified sepiolite to carboxymethyl chitosan is 1:1.4,

[0110] The remaining operation steps are the same as those in Example 4.

[0111] Comparative Example 6

[0112] The difference between Comparative Example 6 and Example 5 lies in that:

[0113] In the preparation process of the immobilized lipase, step 13), the mass ratio of the coupling agent-modified sepiolite to carboxymethyl chitosan is 1:0.4,

[0114] The remaining operation steps are the same as those in Example 5.

[0115] Performance Test

[0116] 1. Determination of biodiesel conversion rate: Take the glycerol solution in the clarified lower layer of biodiesel from Examples 1 - 5 and Comparative Examples 1 and 5. Add 3 mL of 0.05 g / mL sodium hydroxide solution and 1 mL of 0.05 g copper sulfate solution to a test tube, then add the glycerol solution in the biodiesel diluted 100 times. After ultrasonic treatment for 5 min, centrifuge at a speed of 4000 r / min for 5 min. Take the supernatant and perform colorimetric analysis at 630 nm with deionized water as the blank. The conversion rate of biodiesel is calculated according to the following formula:

[0117] Conversion rate (%) = actual glycerol concentration / theoretical glycerol concentration × 100%;

[0118] Example 1 Example 2 Example 3 Example 4 Example 5 Comparative Example 1 Comparative Example 5 Conversion rate (%) 98.67 97.86 98.23 98.83 97.55 76.35 83.75

[0119] 2. Determination of the recycling effect of solid lipase:

[0120] After precision filtration of the crude biodiesel from Examples 1 - 5 and Comparative Examples 2, 3, 4, and 6, the immobilized lipase is separated and recovered.

[0121] The immobilized lipase separated and recovered from the examples and comparative examples is used to catalyze the esterification reaction to prepare biodiesel. The method for preparing biodiesel includes the following steps:

[0122] Add 40 g of waste oil and 7 g of methanol to a beaker, then add 2 mL of deionized water and the immobilized lipase respectively; place the beaker in a water bath and heat it to 45 °C, then insert an ultrasonic oscillator rod into the beaker. The oscillation frequency of the ultrasonic oscillator rod is 20 kHz, and perform ultrasonic oscillation reaction for 9 h to obtain crude biodiesel; perform precision filtration on the crude biodiesel to remove the immobilized lipase, then let it stand for stratification. After stratification, remove the glycerol in the lower layer to obtain biodiesel, and test the conversion rate of biodiesel according to the above method for determining the biodiesel conversion rate.

[0123] Example 1 Example 2 Example 3 Example 4 Example 5 Conversion rate (%) 98.65 97.83 98.22 98.81 97.54 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 6 Conversion rate (%) 73.58 79.31 82.36 85.72

[0124] The above are only the preferred embodiments of the present invention. It should be noted that the above - mentioned preferred embodiments should not be regarded as a limitation of the present invention. The protection scope of the present invention should be subject to the scope defined by the claims. For those of ordinary skill in the art, within the spirit and scope of the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for accelerating the enzymatic esterification reaction by using an ultrasonic oscillation rod, characterized in that, It includes the following steps: 1) Add waste oil and methanol into a beaker, and then add deionized water and immobilized lipase respectively; 2) Place the beaker in a water bath and heat it to 40 - 50 °C, then insert an ultrasonic oscillator rod into the beaker and conduct ultrasonic oscillation reaction to obtain crude biodiesel; 3) Conduct precision filtration on the crude biodiesel, separate and recover the immobilized lipase, then let it stand for stratification, and remove the lower - layer glycerol after stratification to obtain biodiesel; The preparation method of the immobilized lipase includes the following steps: 11) Add silane coupling agent KH - 560 into the mixed solution of ethanol and water, heat it to 50 °C, and obtain a coupling agent hydrolysis solution through stirring; 12) Add sepiolite into the coupling agent hydrolysis solution, continue stirring and reacting, and obtain coupling - agent - modified sepiolite through centrifugal separation, washing and drying; 13) Add carboxymethyl chitosan into deionized water and stir to dissolve it to obtain a carboxymethyl chitosan solution. Add the coupling - agent - modified sepiolite into the carboxymethyl chitosan solution, and the mass ratio of the coupling - agent - modified sepiolite to carboxymethyl chitosan is 1:(0.5 - 1.3). Heat it to 80 °C, stir and react for 2 h, and obtain primary sepiolite - carboxymethyl chitosan composite particles through filtration and separation; 14) Add glutaraldehyde into deionized water, stir evenly to obtain a glutaraldehyde solution. Add the primary sepiolite - carboxymethyl chitosan composite particles into the glutaraldehyde solution, heat and react, and obtain sepiolite - carboxymethyl chitosan composite particles through centrifugal separation, washing and drying; 15) Add aspergillus lipase into phosphate buffer solution to obtain an aspergillus lipase solution. Add the sepiolite - carboxymethyl chitosan composite particles into deionized water, stir to form a suspension, then add the aspergillus lipase solution into the suspension, stir evenly, let it stand, and obtain immobilized lipase through filtration and separation, washing and freeze - drying.

2. A method for accelerating the enzymatic esterification reaction by using an ultrasonic oscillation rod according to claim 1, characterized in that, In the step 1), the mass ratio of waste oil to methanol is 8:(1 - 2).

3. A method for accelerating the enzymatic esterification reaction by using an ultrasonic oscillation rod according to claim 1, characterized in that, In the step 1), the addition amount of the immobilized lipase is 1% - 5% of the mass of the waste oil.

4. A method for accelerating the enzymatic esterification reaction by using an ultrasonic oscillation rod according to claim 1, characterized in that, In the step 2), the oscillation frequency of the ultrasonic oscillator rod is 19 kHz - 20 kHz.

5. A method for accelerating the enzymatic esterification reaction by using an ultrasonic oscillation rod according to claim 1, characterized in that, In the step 2), the ultrasonic oscillation reaction time is 8 h - 10 h.

6. A method for accelerating the enzymatic esterification reaction by using an ultrasonic oscillation rod according to claim 1, characterized in that, In the step 14), the heating reaction temperature is 50 °C - 60 °C.

7. A method for accelerating the enzymatic esterification reaction by using an ultrasonic oscillating rod according to claim 1, characterized in that, In the step 14), the heating reaction time is 3 h - 8 h.

8. A method for accelerating the enzymatic esterification reaction by using an ultrasonic oscillation rod according to claim 1, characterized in that In the step 15), the standing time is 10 - 20 h.