Method for accelerating biological enzyme esterification reaction by using ultrasonic oscillation bar

Abstract

The invention relates to the technical field of biodiesel, and discloses a method for accelerating a biological enzyme esterification reaction by using an ultrasonic oscillation bar. Comprising the following steps: adding waste grease and methanol into a beaker, and then respectively adding deionized water and immobilized lipase; heating the beaker in a water bath kettle, inserting an ultrasonic oscillation rod into the beaker, and carrying out an ultrasonic oscillation reaction to obtain crude biodiesel; and carrying out precision filtration on the crude biodiesel, separating and recovering the immobilized lipase, then standing for layering, and removing glycerin at the lower layer after layering to obtain the biodiesel. According to the method, the reaction monomer is in full contact with the immobilized lipase through an ultrasonic oscillation method, so that the esterification reaction time is shortened, and the biodiesel synthesis efficiency is improved.

Description

Technical Field

[0001] The invention relates to the technical field of biodiesel, and in particular to a method for accelerating bioenzyme esterification reaction by utilizing an ultrasonic oscillating rod. Background Art

[0002] As the population grows, the demand for fuel is increasing, and the reserves of fossil fuels are decreasing. People are beginning 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 relieving the pressure of fossil fuels. Biodiesel is a green and safe fuel that can be made from edible and inedible vegetable oils, animal fats, waste cooking oils and many other oils of poor quality. Biodiesel is mainly long-chain fatty acid methyl esters or ethyl esters, such as higher fatty acid methyl esters, which can be made from animal and plant oils, waste frying oils, animal and plant waste oils, algae and cellulose, which are widely available, easy to obtain and rich in fatty acids, as raw materials, and short-chain alcohols such as methanol or ethanol are reacted under the action of acids, alkalis or biological enzymes. Biodiesel has good combustion performance and does not produce harmful gases such as sulfur dioxide when burned. It has the characteristics of being green, safe and renewable, and has gradually become a substitute for traditional petrochemical diesel.

[0003] At present, the production processes of biodiesel mainly include chemical method and bio-enzyme method. The chemical method generally uses inorganic acid or alkali as a catalyst, and has the disadvantages of complex process, high energy consumption, large alcohol consumption, and environmental pollution; the bio-enzyme method has the advantages of mild reaction conditions, easy separation of products, and environmental protection, and is considered to be a green process that is expected to replace the chemical method for producing biodiesel. If free lipase is used as a catalyst, it is difficult to separate, regenerate, and recycle the enzyme after the reaction. If immobilized lipase is used, the above-mentioned defects of free enzymes can be overcome, so that the enzyme molecules can be 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 many times, thereby reducing costs and showing good application prospects. However, compared with free lipase, immobilized lipase cannot fully contact with the reaction monomer, thereby affecting the rate of esterification reaction, and the esterification reaction time is long, which affects the efficiency of biodiesel production. Summary of the invention

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

[0005] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0006] A method for accelerating the esterification reaction of biological enzymes by using an ultrasonic oscillator, 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) placing the beaker in a water bath and heating it to 40-50° C., then inserting an ultrasonic oscillator into the beaker to perform ultrasonic oscillation reaction to obtain crude biodiesel;

[0009] 3) The crude biodiesel is finely filtered to separate and recover the immobilized lipase, and then allowed to stand for stratification. After stratification, the glycerol in the lower layer is removed to obtain biodiesel.

[0010] In the technical solution of the present invention, waste oil and methanol are used as reaction monomers to synthesize biodiesel under the catalytic action of immobilized lipase. The immobilized lipase molecules are protected while effectively exerting the catalytic action, are not easily affected by the environment, are easy to separate from the substrate and the product, and can be recycled for many times, thereby reducing costs. However, compared with free lipase, the immobilized lipase cannot fully contact with the reaction monomers, thereby affecting the rate of the esterification reaction. The esterification reaction time is relatively long, which affects the biodiesel production efficiency. The present invention uses an ultrasonic oscillator rod to allow the reaction monomers to enter the carrier of the immobilized lipase during the esterification reaction, so that the reaction monomers are fully in contact with the lipase in the carrier, thereby improving the esterification reaction rate and promoting the synthesis efficiency of biodiesel.

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

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

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

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

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

[0016] 11) Add silane coupling agent KH-560 to a mixed solution of ethanol and water, heat to 50° C., and stir to obtain a coupling agent hydrolyzate;

[0017] 12) adding sepiolite to the coupling agent hydrolyzate, continuing to stir the reaction, and performing centrifugal separation, washing and drying to obtain coupling agent-modified sepiolite;

[0018] 13) adding carboxymethyl chitosan into deionized water and stirring to dissolve to obtain a carboxymethyl chitosan solution, adding coupling agent-modified sepiolite into the carboxymethyl chitosan solution, heating to 80° C., stirring to react for 2 h, and filtering and separating to obtain primary sepiolite-carboxymethyl chitosan composite particles;

[0019] 14) adding glutaraldehyde into deionized water and stirring to obtain a glutaraldehyde solution, adding the primary sepiolite-carboxymethyl chitosan composite particles into the glutaraldehyde solution, heating for reaction, centrifugation, washing and drying to obtain the sepiolite-carboxymethyl chitosan composite particles;

[0020] 15) Adding Aspergillus lipase to phosphate buffer to obtain Aspergillus lipase solution, adding sepiolite-carboxymethyl chitosan composite particles to deionized water, stirring to form a suspension, then adding the Aspergillus lipase solution to the suspension, stirring evenly, standing, filtering, separating, washing and freeze-drying to obtain immobilized lipase.

[0021] In the technical solution of the present invention, as mentioned above, the reaction monomer is made to enter the carrier of the immobilized lipase under the action of an ultrasonic oscillator, so that the reaction monomer is fully in contact with the lipase in the carrier, thereby improving 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 biological enzyme for the esterification reaction, and sepiolite is used as the carrier of Aspergillus lipase. The porous adsorption effect of sepiolite is used to bind the Aspergillus lipase to the sepiolite. However, the problem is that the method of ultrasonic oscillation during the esterification reaction is easy to cause the Aspergillus lipase to separate from the sepiolite, and the lipase separated from the sepiolite is difficult to separate from the reaction product, which affects the recycling of the biological enzyme. In order to solve this problem, the present invention performs modification treatment on sepiolite, firstly grafts epoxy silane coupling agent on the surface and inside the pores of sepiolite, then immerses the sepiolite in a carboxymethyl chitosan solution, wherein amino groups on a part of carboxymethyl chitosan molecules in the solution react with epoxy groups grafted on the surface of sepiolite to undergo ring-opening reaction, thereby grafting carboxymethyl chitosan on the surface and inside the pores of sepiolite (bonding carboxymethyl chitosan molecules to sepiolite through chemical bonds to improve the binding force between the two), and another part of carboxymethyl chitosan that does not participate in the reaction is adsorbed inside the pores of sepiolite, and then the carboxymethyl chitosan molecules undergo cross-linking reaction under the cross-linking action of glutaraldehyde, and the cross-linked carboxymethyl chitosan modifies the pores of sepiolite, which is beneficial to the adsorption of sepiolite on Aspergillus lipase and improves the binding force between the two. In addition, due to the hydrogen bonding force between the hydroxyl groups on the carboxymethyl chitosan molecules and the amino groups on the Aspergillus lipase, the binding force between sepiolite and lipase is further improved. The above modification of sepiolite solves the problem of Aspergillus lipase falling off from sepiolite under the vibration of ultrasonic vibrator, and improves the recovery rate of biological enzyme. Preferably, in step 13), the mass ratio of coupling agent modified sepiolite to carboxymethyl chitosan is 1:(0.5-1.3).

[0022] In the technical solution of the present invention, in order to stably bind Aspergillus lipase to sepiolite and prevent Aspergillus lipase from detaching from sepiolite under ultrasonic oscillation, a sufficient amount of carboxymethyl chitosan must be grafted and cross-linked on sepiolite, so the mass ratio of coupling agent-modified sepiolite to carboxymethyl chitosan is controlled to be less than 1:0.5. The team of the present invention accidentally discovered that when the grafting and cross-linking of carboxymethyl chitosan are increased to a certain extent, that is, the mass ratio of coupling agent-modified sepiolite to carboxymethyl chitosan is less than 1:1.3, the Aspergillus lipase bound to sepiolite decreases significantly, causing the conversion rate of biodiesel to decrease. This may be because the excessive cross-linked carboxymethyl chitosan causes the microporous structure of sepiolite to be blocked, affecting the adsorption of sepiolite on Aspergillus lipase. Therefore, the present invention controls the mass ratio of 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 step 14), the heating reaction time is 3h-8h.

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

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

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

[0028] 2) During the esterification reaction, the reaction monomers are made to enter the carrier of the immobilized lipase under the action of an ultrasonic oscillator, so that the reaction monomers are fully in 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. Under the vibration of the ultrasonic vibrator, the Aspergillus lipase is not easy to fall off from the sepiolite, thereby improving the recovery rate of the biological enzyme. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction 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 generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should 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 available to those skilled in the art; unless otherwise specified, the methods used in the embodiments of the present invention are all methods known to 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 oscillator, comprising the following steps:

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

[0035] 2) placing the beaker in a water bath and heating it to 45° C., then inserting an ultrasonic oscillator into the beaker, the oscillation frequency of the ultrasonic oscillator being 20 kHz, and performing ultrasonic oscillation reaction for 9 hours to obtain crude biodiesel;

[0036] 3) The crude biodiesel is finely filtered to separate and recover the immobilized lipase, and then allowed to stand for stratification. After stratification, the glycerol in the lower layer is removed to obtain biodiesel.

[0037] The preparation method of immobilized lipase comprises 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 to 50° C., and stir to obtain a coupling agent hydrolyzate;

[0039] 12) adding 3 g of sepiolite to the coupling agent hydrolyzate, continuing to stir the reaction, and obtaining coupling agent-modified sepiolite through centrifugal separation, washing and drying;

[0040] 13) adding 1 g of carboxymethyl chitosan to 200 mL of deionized water and stirring to dissolve to obtain a carboxymethyl chitosan solution, adding coupling agent-modified sepiolite to the carboxymethyl chitosan solution, wherein the mass ratio of coupling agent-modified sepiolite to carboxymethyl chitosan is 1:1.2, heating to 80° C., stirring to react for 2 h, and filtering and separating to obtain primary sepiolite-carboxymethyl chitosan composite particles;

[0041] 14) adding 0.3 g of glutaraldehyde to 200 mL of deionized water and stirring to obtain a glutaraldehyde solution, adding the above-mentioned primary sepiolite-carboxymethyl chitosan composite particles to the glutaraldehyde solution, heating to 55° C., reacting for 7 hours, and centrifuging, washing and drying to obtain sepiolite-carboxymethyl chitosan composite particles;

[0042] 15) 0.5 g of Aspergillus lipase was added to 50 mL of phosphate buffer (pH = 7.5) to obtain an Aspergillus lipase solution, 2 g of sepiolite-carboxymethyl chitosan composite particles were added to 300 mL of deionized water, and stirred to form a suspension, and then the Aspergillus lipase solution was added to the suspension, stirred evenly, and allowed to stand for 18 hours. After filtration separation, washing and freeze-drying, immobilized lipase was obtained.

[0043] Example 2

[0044] A method for accelerating the esterification reaction of biological enzymes by using an ultrasonic oscillator, comprising the following steps:

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

[0046] 2) placing the beaker in a water bath and heating it to 45° C., then inserting an ultrasonic oscillator into the beaker, the oscillation frequency of the ultrasonic oscillator being 19 kHz, and performing ultrasonic oscillation reaction for 9 hours to obtain crude biodiesel;

[0047] 3) The crude biodiesel is finely filtered to separate and recover the immobilized lipase, and then allowed to stand for stratification. After stratification, the glycerol in the lower layer is removed to obtain biodiesel.

[0048] The preparation method of immobilized lipase comprises 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 to 50° C., and stir to obtain a coupling agent hydrolyzate;

[0050] 12) adding 3 g of sepiolite to the coupling agent hydrolyzate, continuing to stir the reaction, and obtaining coupling agent-modified sepiolite through centrifugal separation, washing and drying;

[0051] 13) adding 1 g of carboxymethyl chitosan to 200 mL of deionized water and stirring to dissolve to obtain a carboxymethyl chitosan solution, adding coupling agent-modified sepiolite to the carboxymethyl chitosan solution, wherein the mass ratio of coupling agent-modified sepiolite to carboxymethyl chitosan is 1:0.6, heating to 80° C., stirring to react for 2 h, and filtering and separating to obtain primary sepiolite-carboxymethyl chitosan composite particles;

[0052] 14) adding 0.3 g of glutaraldehyde to 200 mL of deionized water and stirring to obtain a glutaraldehyde solution, adding the above-mentioned primary sepiolite-carboxymethyl chitosan composite particles to the glutaraldehyde solution, heating to 55° C., reacting for 4 hours, and centrifuging, washing and drying to obtain sepiolite-carboxymethyl chitosan composite particles;

[0053] 15) 0.5 g of Aspergillus lipase was added to 50 mL of phosphate buffer (pH = 7.5) to obtain an Aspergillus lipase solution, 2 g of sepiolite-carboxymethyl chitosan composite particles were added to 300 mL of deionized water, and stirred to form a suspension, and then the Aspergillus lipase solution was added to the suspension, stirred evenly, and allowed to stand for 12 hours. After filtration separation, washing and freeze-drying, immobilized lipase was obtained.

[0054] Example 3

[0055] A method for accelerating the esterification reaction of biological enzymes by using an ultrasonic oscillator, comprising the following steps:

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

[0057] 2) placing the beaker in a water bath and heating it to 45° C., then inserting an ultrasonic oscillator into the beaker, the oscillation frequency of the ultrasonic oscillator being 20 kHz, and performing ultrasonic oscillation reaction for 9 hours to obtain crude biodiesel;

[0058] 3) The crude biodiesel is finely filtered to separate and recover the immobilized lipase, and then allowed to stand for stratification. After stratification, the glycerol in the lower layer is removed to obtain biodiesel.

[0059] The preparation method of immobilized lipase comprises 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 to 50° C., and stir to obtain a coupling agent hydrolyzate;

[0061] 12) adding 3 g of sepiolite to the coupling agent hydrolyzate, continuing to stir the reaction, and obtaining coupling agent-modified sepiolite through centrifugal separation, washing and drying;

[0062] 13) adding 1 g of carboxymethyl chitosan to 200 mL of deionized water and stirring to dissolve to obtain a carboxymethyl chitosan solution, adding coupling agent-modified sepiolite to the carboxymethyl chitosan solution, wherein the mass ratio of coupling agent-modified sepiolite to carboxymethyl chitosan is 1:1, heating to 80° C., stirring to react for 2 h, and filtering and separating to obtain primary eco-sepiolite-carboxymethyl chitosan composite particles;

[0063] 14) adding 0.3 g of glutaraldehyde to 200 mL of deionized water and stirring to obtain a glutaraldehyde solution, adding the above-mentioned primary sepiolite-carboxymethyl chitosan composite particles to the glutaraldehyde solution, heating to 55° C., reacting for 6 hours, and centrifuging, washing and drying to obtain sepiolite-carboxymethyl chitosan composite particles;

[0064] 15) 0.5 g of Aspergillus lipase was added to 50 mL of phosphate buffer (pH = 7.5) to obtain an Aspergillus lipase solution, 2 g of sepiolite-carboxymethyl chitosan composite particles were added to 300 mL of deionized water, and stirred to form a suspension, and then the Aspergillus lipase solution was added to the suspension, stirred evenly, and allowed to stand for 15 hours. After filtration separation, washing and freeze-drying, immobilized lipase was obtained.

[0065] Example 4

[0066] A method for accelerating the esterification reaction of biological enzymes by using an ultrasonic oscillator, comprising the following steps:

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

[0068] 2) placing the beaker in a water bath and heating it to 50° C., then inserting an ultrasonic oscillator into the beaker, the oscillation frequency of the ultrasonic oscillator being 20 kHz, and performing ultrasonic oscillation reaction for 10 hours to obtain crude biodiesel;

[0069] 3) The crude biodiesel is finely filtered to separate and recover the immobilized lipase, and then allowed to stand for stratification. After stratification, the glycerol in the lower layer is removed to obtain biodiesel.

[0070] The preparation method of immobilized lipase comprises 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 to 50° C., and stir to obtain a coupling agent hydrolyzate;

[0072] 12) adding 3 g of sepiolite to the coupling agent hydrolyzate, continuing to stir the reaction, and obtaining coupling agent-modified sepiolite through centrifugal separation, washing and drying;

[0073] 13) adding 1 g of carboxymethyl chitosan to 200 mL of deionized water and stirring to dissolve to obtain a carboxymethyl chitosan solution, adding coupling agent-modified sepiolite to the carboxymethyl chitosan solution, wherein the mass ratio of coupling agent-modified sepiolite to carboxymethyl chitosan is 1:1.3, heating to 80° C., stirring to react for 2 h, and filtering and separating to obtain primary sepiolite-carboxymethyl chitosan composite particles;

[0074] 14) adding 0.3 g of glutaraldehyde to 200 mL of deionized water and stirring to obtain a glutaraldehyde solution, adding the above-mentioned primary sepiolite-carboxymethyl chitosan composite particles to the glutaraldehyde solution, heating to 60° C., reacting for 8 h, and centrifuging, washing and drying to obtain sepiolite-carboxymethyl chitosan composite particles;

[0075] 15) 0.5 g of Aspergillus lipase was added to 50 mL of phosphate buffer (pH = 7.5) to obtain an Aspergillus lipase solution, 2 g of sepiolite-carboxymethyl chitosan composite particles were added to 300 mL of deionized water, and stirred to form a suspension, and then the Aspergillus lipase solution was added to the suspension, stirred evenly, and allowed to stand for 20 hours. After filtration separation, washing and freeze-drying, immobilized lipase was obtained.

[0076] Example 5

[0077] A method for accelerating the esterification reaction of biological enzymes by using an ultrasonic oscillator, comprising the following steps:

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

[0079] 2) placing the beaker in a water bath and heating it to 40° C., then inserting an ultrasonic oscillator into the beaker, the oscillation frequency of the ultrasonic oscillator being 19 kHz, and performing ultrasonic oscillation reaction for 8 hours to obtain crude biodiesel;

[0080] 3) The crude biodiesel is finely filtered to separate and recover the immobilized lipase, and then allowed to stand for stratification. After stratification, the glycerol in the lower layer is removed to obtain biodiesel.

[0081] The preparation method of immobilized lipase comprises 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 to 50° C., and stir to obtain a coupling agent hydrolyzate;

[0083] 12) adding 3 g of sepiolite to the coupling agent hydrolyzate, continuing to stir the reaction, and obtaining coupling agent-modified sepiolite through centrifugal separation, washing and drying;

[0084] 13) adding 1 g of carboxymethyl chitosan to 200 mL of deionized water and stirring to dissolve to obtain a carboxymethyl chitosan solution, adding coupling agent-modified sepiolite to the carboxymethyl chitosan solution, wherein the mass ratio of coupling agent-modified sepiolite to carboxymethyl chitosan is 1:0.5, heating to 80° C., stirring to react for 2 h, and filtering and separating to obtain primary eco-sepiolite-carboxymethyl chitosan composite particles;

[0085] 14) adding 0.3 g of glutaraldehyde to 200 mL of deionized water and stirring to obtain a glutaraldehyde solution, adding the above-mentioned primary sepiolite-carboxymethyl chitosan composite particles to the glutaraldehyde solution, heating to 50° C., reacting for 3 h, and centrifuging, washing and drying to obtain sepiolite-carboxymethyl chitosan composite particles;

[0086] 15) 0.5 g of Aspergillus lipase was added to 50 mL of phosphate buffer (pH = 7.5) to obtain an Aspergillus lipase solution, 2 g of sepiolite-carboxymethyl chitosan composite particles were added to 300 mL of deionized water, and stirred to form a suspension, and then the Aspergillus lipase solution was added to the suspension, stirred evenly, and allowed to stand for 10 hours. After filtration separation, washing and freeze-drying, immobilized lipase was obtained.

[0087] Comparative Example 1

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

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

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

[0091] Comparative Example 2

[0092] The difference between Comparative Example 2 and Example 1 is:

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

[0094] Replacing the sepiolite-carboxymethyl chitosan composite particles in step 15) with sepiolite;

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

[0096] Comparative Example 3

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

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

[0099] Replacing the coupling agent-modified sepiolite in step 13) with sepiolite;

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

[0101] Comparative Example 4

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

[0103] Step 14) is omitted during the preparation of immobilized lipase.

[0104] Replacing the sepiolite-carboxymethyl chitosan composite particles in step 15) with nascent sepiolite-carboxymethyl chitosan composite particles;

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

[0106] Comparative Example 5

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

[0108] Step 13) in the preparation process of immobilized lipase,

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

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

[0111] Comparative Example 6

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

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

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

[0115] Performance Testing

[0116] 1. Determination of biodiesel conversion rate: Take the glycerol solution of the lower clarified part of the biodiesel in Examples 1-5 and Comparative Examples 1 and 5, add 3mL, 0.05g / mL sodium hydroxide solution and 1mL, 0.05g copper sulfate solution in a test tube, then add the glycerol solution in the biodiesel diluted 100 times, ultrasonicate for 5 minutes, centrifuge at 4000r / min for 5 minutes, take the upper clear liquid, and perform colorimetry at 630nm with deionized water as blank. The conversion rate of biodiesel is calculated according to the following formula:

[0117] Conversion rate (%) = actual glycerol concentration / theoretical glycerol concentration x 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 solid lipase recovery and utilization effect:

[0120] The crude biodiesel of Examples 1-5 and Comparative Examples 2, 3, 4 and 6 was finely filtered to separate and recover the immobilized lipase.

[0121] The immobilized lipase separated and recovered in the embodiment and the comparative example is used to catalyze the esterification reaction to prepare biodiesel. The biodiesel preparation method comprises the following steps:

[0122] 40 g of waste oil and 7 g of methanol were added to a beaker, and then 2 mL of deionized water and immobilized lipase were added respectively; the beaker was placed in a water bath and heated to 45°C, and then an ultrasonic oscillator was inserted into the beaker with an oscillation frequency of 20 kHz, and ultrasonic oscillation reaction was performed for 9 hours to obtain crude biodiesel; the crude biodiesel was precisely filtered to remove the immobilized lipase, and then allowed to stand for stratification. After stratification, the glycerol in the lower layer was removed to obtain biodiesel, and the conversion rate of biodiesel was tested according to the above-mentioned biodiesel conversion rate determination method.

[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 preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for accelerating the esterification reaction of biological enzymes using an ultrasonic oscillator, characterized in that: The following steps are involved: 1) Add waste oil and methanol into a beaker, and then add deionized water and immobilized lipase respectively; 2) placing the beaker in a water bath and heating it to 40-50° C., then inserting an ultrasonic oscillator into the beaker to perform ultrasonic oscillation reaction to obtain crude biodiesel; 3) The crude biodiesel is finely filtered to separate and recover the immobilized lipase, and then allowed to stand for stratification. After stratification, the glycerol in the lower layer is removed to obtain biodiesel.

2. The method of accelerating the esterification reaction of biological enzymes by using an ultrasonic oscillating rod according to claim 1, characterized in that: In the step 1), the mass ratio of waste oil and fat to methanol is 8:(1-2).

3. The method of accelerating the esterification reaction of biological enzymes by using an ultrasonic oscillating rod according to claim 1, characterized in that: In the step 1), the amount of immobilized lipase added is 1%-5% of the mass of the waste oil.

4. The method of accelerating the esterification reaction of biological enzymes by using an ultrasonic oscillating rod according to claim 1, characterized in that: In the step 2), the oscillation frequency of the ultrasonic oscillation rod is 19kHz-20kHz.

5. The method of accelerating the esterification reaction of biological enzymes by using an ultrasonic oscillating rod according to claim 1, characterized in that: In the step 2), the ultrasonic oscillation reaction time is 8h-10h.

6. The method of accelerating the esterification reaction of biological enzymes by using an ultrasonic oscillating rod according to claim 1, characterized in that: In the step 1), the method for preparing the immobilized lipase comprises the following steps: 11) Add silane coupling agent KH-560 to a mixed solution of ethanol and water, heat to 50° C., and stir to obtain a coupling agent hydrolyzate; 12) adding sepiolite to the coupling agent hydrolyzate, continuing to stir the reaction, and performing centrifugal separation, washing and drying to obtain coupling agent-modified sepiolite; 13) adding carboxymethyl chitosan into deionized water and stirring to dissolve to obtain a carboxymethyl chitosan solution, adding coupling agent-modified sepiolite into the carboxymethyl chitosan solution, heating to 80° C., stirring to react for 2 h, and filtering and separating to obtain primary sepiolite-carboxymethyl chitosan composite particles; 14) adding glutaraldehyde into deionized water and stirring to obtain a glutaraldehyde solution, adding the primary sepiolite-carboxymethyl chitosan composite particles into the glutaraldehyde solution, heating for reaction, centrifugation, washing and drying to obtain the sepiolite-carboxymethyl chitosan composite particles; 15) Adding Aspergillus lipase to phosphate buffer to obtain Aspergillus lipase solution, adding sepiolite-carboxymethyl chitosan composite particles to deionized water, stirring to form a suspension, then adding the Aspergillus lipase solution to the suspension, stirring evenly, standing, filtering, separating, washing and freeze-drying to obtain immobilized lipase.

7. A method for accelerating the esterification reaction of biological enzymes by using an ultrasonic oscillating rod according to claim 6, characterized in that: In the step 13), the mass ratio of coupling agent-modified sepiolite to carboxymethyl chitosan is 1:(0.5-1.3).

8. The method of accelerating the esterification reaction of biological enzymes by using an ultrasonic oscillating rod according to claim 6, characterized in that: In the step 14), the heating reaction temperature is 50°C-60°C.

9. The method of accelerating the esterification reaction of biological enzymes by using an ultrasonic oscillating rod according to claim 6, characterized in that: In the step 14), the heating reaction time is 3h-8h.

10. The method of accelerating the esterification reaction of biological enzymes by using an ultrasonic oscillating rod according to claim 6, characterized in that: The standing time in step 15) is 10-20 hours.