CN114606222A - Method for preparing biodiesel by enzyme-loaded microspheres and Pickering emulsion enzyme method - Google Patents
Method for preparing biodiesel by enzyme-loaded microspheres and Pickering emulsion enzyme method Download PDFInfo
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- 239000004005 microsphere Substances 0.000 title claims abstract description 85
- 238000000034 method Methods 0.000 title claims abstract description 58
- 239000003225 biodiesel Substances 0.000 title claims abstract description 49
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- 239000002105 nanoparticle Substances 0.000 claims abstract description 77
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 66
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 66
- POULHZVOKOAJMA-UHFFFAOYSA-N dodecanoic acid Chemical compound CCCCCCCCCCCC(O)=O POULHZVOKOAJMA-UHFFFAOYSA-N 0.000 claims abstract description 30
- 238000006243 chemical reaction Methods 0.000 claims abstract description 29
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- 230000003197 catalytic effect Effects 0.000 claims abstract description 23
- 235000019421 lipase Nutrition 0.000 claims abstract description 23
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- 239000006185 dispersion Substances 0.000 claims description 21
- UYXTWWCETRIEDR-UHFFFAOYSA-N Tributyrin Chemical compound CCCC(=O)OCC(OC(=O)CCC)COC(=O)CCC UYXTWWCETRIEDR-UHFFFAOYSA-N 0.000 claims description 20
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- 239000004382 Amylase Substances 0.000 description 1
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- 239000002253 acid Substances 0.000 description 1
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- 239000010775 animal oil Substances 0.000 description 1
- 238000005815 base catalysis Methods 0.000 description 1
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- 150000002148 esters Chemical group 0.000 description 1
- 239000005431 greenhouse gas Substances 0.000 description 1
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- 238000009776 industrial production Methods 0.000 description 1
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- 235000019626 lipase activity Nutrition 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
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- 231100000956 nontoxicity Toxicity 0.000 description 1
- HMMGMWAXVFQUOA-UHFFFAOYSA-N octamethylcyclotetrasiloxane Chemical compound C[Si]1(C)O[Si](C)(C)O[Si](C)(C)O[Si](C)(C)O1 HMMGMWAXVFQUOA-UHFFFAOYSA-N 0.000 description 1
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
- C11C3/00—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom
- C11C3/003—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom by esterification of fatty acids with alcohols
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/649—Biodiesel, i.e. fatty acid alkyl esters
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Abstract
A method for preparing biodiesel by enzyme-loaded microspheres and a Pickering emulsion enzyme method is characterized in that the enzyme-loaded microspheres are prepared by a method of interface self-assembly assisted by an emulsion template by hydrophilic/hydrophobic silica nanoparticles and lipase with a mass ratio of 99:1 or 9: 1. The preparation method of the biodiesel comprises the steps of dissolving lauric acid in n-octane to serve as an oil phase, dispersing methanol in a phosphate buffer solution to serve as a water phase, adding enzyme-loaded microspheres into a mixed solution of the oil phase and the water phase, mixing the mixture through a homogenizer to form w/o or o/w type Pickering emulsion, and placing the Pickering emulsion with the stabilized enzyme-loaded microspheres on a rotary incubator or a shaking table to obtain the methyl laurate type biodiesel. The invention has the characteristics of high catalytic activity of the enzyme-loaded microspheres, good biocompatibility, recycling, mild reaction conditions, high quality of obtained products and the like.
Description
Technical Field
The invention relates to the technical field of biological heterogeneous catalysis based on immobilized enzyme, and particularly relates to a preparation method of enzyme-loaded microspheres and a method for preparing biodiesel by a Pickering emulsion enzyme method through stabilization of the enzyme-loaded microspheres.
Background
Environmental pollution and energy crisis caused by excessive consumption of non-renewable fossil energy have become major threats facing the global survival of living organisms. Under the guidance of 'double-carbon' planning in China, the development of non-renewable fossil energy substitutes becomes an important task for realizing green, low-carbon and sustainable development in China.
Biodiesel is defined as a mixture of mono-alkyl esters of long chain fatty acids derived from renewable lipid sources (e.g., vegetable oils, animal fats, or waste fats) that can be used directly in compression ignition engines, an excellent alternative to fossil energy. The present staged esterification/ester exchange reaction preparation has the characteristics of regeneration, clean combustion, biodegradability, no toxicity and the like, and the use of the biodiesel can effectively reduce the emission of greenhouse gases and environmental pollution. Chinese patent with publication No. CN101397504B (application No. CN200810218887.6) adopts homogeneous alkali (NaOH/KOH) as a catalyst to prepare biodiesel by catalyzing the transesterification reaction of waste animal and vegetable oil and methanol. However, the chemical catalysis methods (homogeneous acid and base catalysis) widely used at the present stage have the problems of difficult catalyst removal, more side reactions, low product quality, high requirement on equipment, high energy consumption, environmental pollution and the like. In contrast, the preparation of biodiesel by using esterification/transesterification reaction catalyzed by lipase is a more green and sustainable method, and because the lipase can catalyze and produce high-quality biodiesel with high catalytic efficiency and high selectivity under mild conditions, the method not only can effectively reduce environmental pollution and energy consumption brought by the biodiesel production process by a chemical catalysis method, but also can greatly reduce the steps of separation and purification of products and reduce the production cost.
However, the application of the enzymatic method in the actual production process of biodiesel is severely hampered by the following 2 factors: (1) the immiscibility of the enzyme with the reaction substrate results in a very limited reaction contact area and a very low reaction rate for the biphasic system; (2) the enzyme stability is poor, and the activity of lipase is greatly damaged by using short-chain alcohol (methanol/ethanol) as a substrate.
In order to solve the problem of immiscible two phases in the production of biodiesel, the traditional method of adding a large amount of organic reagents capable of dissolving two-phase substrates simultaneously and carrying out uninterrupted vigorous stirring in the reaction process has the problems of high energy consumption, environmental pollution, complex product separation and purification and the like, and the organic solvents and the vigorous stirring damage the activity of enzyme; a Pickering emulsion interface biocatalysis system based on immobilized enzyme provides a feasible scheme for solving the problem of immiscible phases in enzyme-method biodiesel biology. Pickering emulsion is an emulsion system stabilized by solid particles, one phase of which is dispersed in the form of micron-sized droplets in the other immiscible phase, and the solid particles stabilize the Pickering emulsion by irreversible adsorption at the interface of the two phases, greatly increasing the reaction area of the contact between the two immiscible phases, and thus increasing the reaction efficiency (Marc Pera-Titus, et al, AngewandTechemie-International Edition,2015,54(7): 2006-2021.). Further, the enzyme is fixed on an insoluble solid carrier by an immobilized enzyme technology, and the immobilized enzyme can be simultaneously used as an emulsifier for stabilizing the Pickering emulsion and a catalyst for catalytic reaction at an interface, so that efficient enzymatic catalytic reaction on a two-phase interface of the Pickering emulsion is realized. Yanjun Jiang et al (Yanjun Jiang, et al. Bioresource Technology,2014,153,278-83.) utilize stabilized Pickering emulsion of mesoporous silica adsorbing lipase to prepare biodiesel, but the physical adsorption method is easy to cause enzyme leakage during the production process to cause inactivation of immobilized enzyme; xiiaobo Liu et al (Xiiaobo Liu, et al. Green Chemistry,2021,23(2), 966-; the immobilized lipase of the Xiao He and the like (Xiao He, et al Langmuir,2021,37,2, 810-819.) effectively avoids enzyme leakage and enzyme activity loss caused by immobilization operation in the process of producing biodiesel by a Pickering emulsion enzyme method by immobilizing the lipase in a physical entrapment mode.
Disclosure of Invention
The invention aims to provide a preparation method of enzyme-loaded microspheres.
The purpose of the invention is realized by the following steps: a preparation method of enzyme-loaded microspheres is characterized in that the enzyme-loaded microspheres are prepared by hydrophilic/hydrophobic silica nanoparticles with different mass ratios and lipase through an emulsion template-assisted interface self-assembly method, and the surface wettability of the enzyme-loaded microspheres is adjusted by changing the mass ratio of the hydrophilic/hydrophobic silica nanoparticles in the self-assembly process;
the method comprises the following steps:
step 1: adding hydrophobic silica nanoparticles into 1mL of hydrophilic silica nanoparticle dispersion liquid (40 wt%) according to the mass ratio of hydrophilic/hydrophobic silica nanoparticles of 99:1 or 9:1, and uniformly mixing the dispersion liquid;
step 2: adding 0.2-1.8 mL of lipase solution and 0.2-1.8 mL of phosphate buffer (0.05M, pH 7.5) to the mixed solution obtained in step 1, and mixing the obtained 3mL of mixed solution thoroughly;
and step 3: adding 30-50 mL of n-butanol/tributyrin into the mixed solution obtained in the step 2, and emulsifying and homogenizing for 13min under the condition that the rotation speed of a homogenizer is 12000 r/m;
and 4, step 4: and (3) standing the emulsion obtained in the step (3) in a refrigerator at 4 ℃ for self-assembly for 12h, centrifuging at 4 ℃ for 5min at 6000rpm by using a refrigerated centrifuge, washing the precipitate, and freeze-drying to obtain the enzyme-loaded microspheres.
The enzyme-carrying microspheres can be recycled after being separated by centrifugation or filtration and the like, and after being recycled for 10 times, the enzyme-carrying microspheres have stable emulsifying performance and the catalytic activity is kept above 94 percent.
The invention also aims to provide application of the enzyme-loaded microspheres in preparation of biodiesel by using the Pickering emulsion through an enzyme method.
Still another object of the present invention is to provide a method for preparing biodiesel by a Pickering emulsion enzymatic method.
A further object of the invention is achieved by:
step 1: dissolving lauric acid in n-octane to prepare 15-55 wt.% lauric acid-n-octane solution as an oil phase; dispersing methanol in a phosphate buffer (pH 7.5) to prepare a 15-50 wt.% methanol aqueous solution as an aqueous phase;
step 2: under the conditions that the oil-water two-phase ratio is 1: 1-7: 1, the molar ratio of lauric acid to methanol is 1: 1-1: 8, and the addition amount of the enzyme-loaded microspheres is 1-2 wt% (based on the mass ratio of the oil phase to the water phase), forming a W/O or O/W Pickering emulsion by hand shaking (100 times per minute) or a homogenizer with 8000-20000 revolutions per minute for 2-8 min, wherein the diameter of an emulsion droplet is 150-650 mu m, and the emulsion droplet is kept stable in the catalytic reaction process within at least 12 h;
and step 3: the Pickering emulsion stabilized by the enzyme-loaded microspheres is placed on a rotary incubator or a shaking table, the temperature is kept at 15-25 ℃, and the reaction is carried out for 0-12 h at the rotating speed of 20-150 rpm, so that methyl laurate (one type of biodiesel) can be generated.
The enzyme-loaded microspheres are simultaneously used as an emulsifier and a biological interface catalyst to carry out catalytic reaction on an oil-water two-phase interface in the Pickering emulsion to efficiently prepare the biodiesel, the catalytic activity of the enzyme is improved to 7.52-8.18 times, and the conversion rate reaches 75-84%.
Compared with the prior art, the invention has the following characteristics and advantages:
compared with the prior art, the invention has the following advantages:
(1) according to the invention, the Pickering emulsion stabilized by the amylase microspheres is used for producing the biodiesel through an enzyme method, compared with the traditional chemical method, the catalytic condition is mild, the catalytic process is efficient, green and sustainable, the quality of the obtained product is high, and the separation and purification cost of the product can be effectively reduced;
(2) the method utilizes the Pickering emulsion with stable enzyme-loaded microspheres to break through the problem that the catalytic efficiency of a two-phase system in the biodiesel production by the enzyme method is low at the present stage, and can obviously improve the efficiency of the biodiesel production by the enzyme method;
(3) the preparation and modification method of the enzyme-loaded microsphere is flexible and simple, has good biocompatibility and high enzyme activity retention rate, and is easy for industrial production.
(4) The adsorption state of the enzyme-loaded microspheres at a two-phase interface can be changed by adjusting the mass ratio of hydrophilic/hydrophobic silica nanoparticles in the self-assembly process of the enzyme-loaded microspheres, so that the loaded enzyme is far away from the water phase where the short-chain alcohol is located, and the catalytic activity of the enzyme is effectively protected in the enzymatic production process of the biodiesel related to high-concentration short-chain alcohol;
(5) the enzyme-loaded microspheres have good reusability, good emulsifying property after 10 times of recycling, and catalytic activity of the enzyme-loaded microspheres is kept above 94%, so that the production cost of producing biodiesel by an enzyme method can be effectively reduced.
The invention changes the adsorption state of the immobilized enzyme at the interface of Pickering emulsion oil-water two-phase by regulating and controlling the surface wettability of the immobilized enzyme, so that the immobilized enzyme is more inclined to an oil phase and is far away from a water phase where short-chain alcohol is positioned, and the enzyme can be protected from the inactivation of the short-chain alcohol, thereby realizing long-time high-efficiency enzyme catalysis in the enzymatic production of biodiesel.
The method can obviously improve the efficiency of producing the biodiesel by the enzyme method and protect the enzyme from the inactivation of methanol in the catalytic process. The lipase-loaded microspheres are used for immobilizing lipase in a physical entrapment mode through a co-assembly process based on hydrophilic/hydrophobic nano particles and lipase; the wettability of the nano-particles can be adjusted by changing the mass ratio of the hydrophilic/hydrophobic nano-particles in the co-assembly process, the non-chemical modification method is more biocompatible, and the enzyme activity loss caused by chemical modification can be avoided. Furthermore, the adsorption state of the enzyme-loaded microspheres at the interface of the Pickering emulsion oil and the water phase is controlled by the method, so that the enzyme-loaded microspheres are more inclined to the oil phase and far away from the water phase where the short-chain alcohol is located, the enzyme is protected from the inactivation of the short-chain alcohol, and the catalytic efficiency and reusability of the enzyme are effectively improved. In conclusion, the application technology for preparing biodiesel based on the Pickering emulsion loaded with the enzyme microspheres provided by the invention is expected to solve the practical problems in the production process of the enzyme-method biodiesel at the present stage, so that the green, efficient and sustainable enzyme-method production of the biodiesel is realized.
Drawings
FIG. 1 is a schematic diagram of the principle of producing biodiesel by catalysis of Pickering emulsion stabilized by enzyme-loaded microspheres.
FIG. 2 is a schematic diagram showing comparison of adsorption states of enzyme-loaded microspheres prepared from hydrophilic/hydrophobic silica nanoparticles at a mass ratio of 99:1 and 9:1 at a two-phase interface.
FIG. 3 is an optical microscopic image of the Pickering emulsion stabilized by enzyme-loaded microspheres prepared from hydrophilic/hydrophobic silica nanoparticles at a mass ratio of 99:1 in example 1.
FIG. 4 is an optical microscopic image of the Pickering emulsion stabilized by enzyme-loaded microspheres prepared from hydrophilic/hydrophobic silica nanoparticles at a mass ratio of 9:1 in example 2.
FIG. 5 is a graph comparing the enzyme activity of free enzyme in the biphasic system of example 3 with the enzyme-loaded microspheroidal enzymes prepared with different hydrophilic/hydrophobic silica nanoparticle mass ratios in the Pickering emulsion system.
FIG. 6 is a graph showing the relationship between the yield and the reaction time in the catalytic production of biodiesel by the enzyme-loaded microsphere stabilized Pickering emulsion system in which the mass ratio of the biphasic system to the hydrophilic/hydrophobic silica nanoparticles is 99:1 in example 4.
FIG. 7 is a graph showing the relationship between the yield and the reaction time in the catalytic production of biodiesel by the enzyme-loaded microsphere stabilized Pickering emulsion system in which the mass ratio of the biphasic system to the hydrophilic/hydrophobic silica nanoparticles is 9:1 in example 5.
FIG. 8 is a graph comparing the yields of biodiesel in 10 cycles of reactions for enzyme-loaded microsphere-stabilized Pickering emulsions prepared according to different hydrophilic/hydrophobic silica nanoparticle mass ratios in example 6.
Detailed Description
The invention is described in detail below with reference to the accompanying drawings and specific embodiments so that the advantages and features of the invention may be more readily understood by those skilled in the art, and the scope of the invention may be clearly and clearly defined.
A method for preparing biodiesel by using Pickering emulsion comprises the following steps:
step 1: dissolving lauric acid in n-octane to prepare 15-55 wt.% lauric acid-n-octane solution as an oil phase; the methanol is dispersed in a phosphate buffer (pH 7.5) to prepare a 15 to 50 wt.% methanol aqueous solution as an aqueous phase.
Step 2: under the conditions that the oil-water phase ratio is 1: 1-7: 1, the molar ratio of lauric acid to methanol is 1: 1-1: 8, and the addition amount of the enzyme-loaded microspheres is 1-2 wt% (based on the mass ratio of the oil phase to the water phase), a W/O or O/W Pickering emulsion is formed by hand shaking (100 times per minute) or other emulsification modes (8000-20000 revolutions per minute) for 2-8 min, wherein the diameter of an emulsion droplet is 150-650 mu m, and the emulsion is stable in a catalytic reaction process within 12h at least.
And step 3: the Pickering emulsion stabilized by the enzyme-loaded microspheres is placed on a rotary incubator or a shaking table, the temperature is kept at 15-25 ℃, and the reaction is carried out for 0-12 h at the rotating speed of 20-150 rpm, so that methyl laurate (one type of biodiesel) can be generated.
The enzyme-loaded microsphere is prepared by an emulsion template-assisted interface self-assembly method of hydrophilic/hydrophobic silica nanoparticles and lipase with different mass ratios, and the surface wettability of the enzyme-loaded microsphere can be adjusted by changing the mass ratio of the hydrophilic/hydrophobic silica nanoparticles in the self-assembly process. The method comprises the following steps:
step 1: to 1mL of a hydrophilic silica nanoparticle dispersion (40 wt%) was added hydrophobic silica nanoparticles in a hydrophilic/hydrophobic silica nanoparticle mass ratio of 99:1 and 9:1, and the dispersions were uniformly mixed.
Step 2: to the mixture solution obtained in step 1, 0.2 to 1.8mL of lipase solution and 0.2 to 1.8mL of phosphate buffer (0.05M, pH 7.5) were added, and the resulting mixture solution (3 mL) was thoroughly mixed.
And step 3: and (3) adding 30-50 mL of n-butyl alcohol/tributyrin into the mixed solution obtained in the step (2), and emulsifying and homogenizing for 1-3 min under the condition that the rotation speed of a homogenizer is 12000 r/m.
And 4, step 4: and (3) standing the emulsion obtained in the step (3) in a refrigerator at 4 ℃ for self-assembly for 12h, and centrifuging at 4 ℃ for 5min at 6000rpm by using a refrigerated centrifuge. Washing the precipitate, and freeze-drying to obtain the enzyme-carrying microsphere.
The lipase activity is defined as: at room temperature, the enzyme amount required for catalyzing lauric acid to generate 1 mu mol of methyl laurate per minute is 1 enzyme activity unit, and U (U.mL) is used-1) Expressed by the following formula, wherein M (g) is the mass of dodecanoic acid, and M (g. mol)-1) Is tenMolar mass of diacid, Y (%) is the yield of methyl dodecanoate over time, VE(mL) is the amount of enzyme added and t (min) is the reaction time. Enzyme activity: u ═ M/M) × 106×(Y/100)/(VE×t)
Example 1
A method for preparing enzyme-loaded microspheres with a hydrophilic/hydrophobic silica nanoparticle mass ratio of 99:1 to stabilize Pickering emulsion comprises the following steps:
step 1: adding 6mg of hydrophobic silica nanoparticles (the mass ratio of hydrophilic/hydrophobic silica nanoparticles is 99:1) into 1mL of hydrophilic silica nanoparticle dispersion (40 wt%), and uniformly mixing the dispersion;
step 2: adding 1.4mL of lipase solution and 0.6mL of phosphate buffer (0.05M, pH 7.5) to the mixture obtained in step 1, and uniformly mixing;
and step 3: rapidly adding 30mL of n-butanol/tributyrin into the mixed solution obtained in the step 2, and emulsifying and homogenizing for 1min under the condition that the rotation speed of a homogenizer is 12000 rpm;
and 4, step 4: and (3) standing the emulsion obtained in the step (3) in a refrigerator at 4 ℃ for self-assembly for 12h, and centrifuging at 4 ℃ for 5min at 6000rpm by using a refrigerated centrifuge. Washing and freeze-drying the precipitate to obtain the enzyme-loaded microspheres with the mass ratio of the hydrophilic/hydrophobic silicon dioxide nanoparticles of 99: 1;
and 5: 2g of lauric acid was dissolved in 8mL of n-octane as an oil phase, and 1.614mL of methanol was dispersed in 2mL of a phosphate buffer (0.05M, pH 7.5) as an aqueous phase;
step 6: adding the hydrophilic/hydrophobic silica nanoparticles prepared in the step (4) into the two-phase reaction system in the step (5) in an adding amount of 2 wt% (based on the mass ratio of oil to water) of the enzyme-loaded microspheres, and shaking by hand (100 times per minute) for 2min to form a Pickering emulsion system from the separated two-phase system;
as shown in FIG. 3, under the condition of optimized oil-water ratio, the enzyme-loaded microspheres with the mass ratio of the hydrophilic/hydrophobic silica nanoparticles being 99:1 show good emulsifying performance, and the type of the formed emulsion is O/W type.
As shown in FIG. 3, the average diameter of Pickering emulsion formed by the enzyme-loaded microspheres with the hydrophilic/hydrophobic silica nanoparticle mass ratio of 99:1 is 477 μm.
Example 2
A method for preparing enzyme-loaded microspheres with a mass ratio of hydrophilic/hydrophobic silica nanoparticles of 9:1 to stabilize Pickering emulsion comprises the following steps:
step 1: adding 58mg of hydrophobic silica nanoparticles (the mass ratio of hydrophilic/hydrophobic silica nanoparticles is 99:1) to 1mL of hydrophilic silica nanoparticle dispersion (40 wt%), and uniformly mixing the dispersion;
and 2, step: adding 1.4mL of lipase solution and 0.6mL of phosphate buffer (0.05M, pH 7.5) to the mixture obtained in step 1, and uniformly mixing;
and step 3: rapidly adding 30mL of n-butyl alcohol/tributyrin into the mixed solution obtained in the step 2, and emulsifying and homogenizing for 1min under the condition that the rotation speed of a homogenizer is 12000 rpm;
and 4, step 4: and (3) standing the emulsion obtained in the step (2) in a refrigerator at 4 ℃ for self-assembly for 12h, and centrifuging at 4 ℃ for 5min at 6000rpm by using a refrigerated centrifuge. Washing and freeze-drying the precipitate to obtain the enzyme-loaded microspheres with the mass ratio of hydrophilic/hydrophobic silicon dioxide nanoparticles of 9: 1;
and 5: 2g of lauric acid was dissolved in 8mL of n-octane as an oil phase, and 1.614mL of methanol was dispersed in 2mL of a phosphate buffer (0.05M, pH 7.5) as an aqueous phase;
step 6: adding the hydrophilic/hydrophobic silica nanoparticles prepared in the step 4 into the two-phase reaction system in the step 5 by adding 2 wt% (based on the mass ratio of oil to water) of the enzyme-loaded microspheres, and shaking by hand (100 times per minute) for 2min to form a Pickering emulsion system;
as shown in FIG. 4, under the condition of optimized oil-water ratio, the enzyme-loaded microspheres with the mass ratio of the hydrophilic/hydrophobic silica nanoparticles being 9:1 show good emulsifying performance, and the formed emulsion is W/O.
As shown in FIG. 4, the average diameter of Pickering emulsion formed by the enzyme-loaded microspheres with the mass ratio of the hydrophilic/hydrophobic silica nanoparticles being 9:1 is 182 μm.
When the mass ratio of the hydrophilic/hydrophobic silicon dioxide is 9:1, the catalytic activity of the prepared Pickering emulsion catalytic system with stable enzyme-loaded microspheres is 8.18 times higher than that of a two-phase system; after 12 hours of reaction, the yield of the biodiesel can reach 84 percent; and maintains more than 94% of the initial catalytic activity after 10 times of recycling.
Example 3
A method for preparing biodiesel from Pickering emulsion stabilized by enzyme-loaded microspheres comprises the following steps:
step 1: adding hydrophobic silica nanoparticles into 1mL of hydrophilic silica nanoparticle dispersion liquid (40 wt%) according to the mass ratio of hydrophilic/hydrophobic silica nanoparticles of 99:1 and 9:1, and uniformly mixing the dispersion liquid;
step 2: adding 1.4mL of lipase solution and 0.6mL of phosphate buffer (0.05M, pH 7.5) to the mixture obtained in step 1, and thoroughly mixing the resulting mixture;
and step 3: rapidly adding 30mL of n-butanol/tributyrin into the mixed solution obtained in the step 2, and emulsifying and homogenizing for 1min under the condition that the rotation speed of a homogenizer is 12000 rpm;
and 4, step 4: and (3) standing the emulsion obtained in the step (3) in a refrigerator at 4 ℃ for self-assembly for 12h, and centrifuging at 4 ℃ for 5min at 6000rpm by using a refrigerated centrifuge. Washing the precipitate, and freeze-drying to obtain the enzyme-loaded microspheres with the mass ratio of the hydrophilic/hydrophobic silicon dioxide nanoparticles of 99:1 and 9: 1.
And 5: 2g of lauric acid was dissolved in 8mL of n-octane as an oil phase, and 1.614mL of methanol was dispersed in 2mL of a phosphate buffer (0.05M, pH 7.5) as an aqueous phase;
step 6: adding the enzyme-loaded microspheres prepared in the step 4 and having the mass ratio of the hydrophilic/hydrophobic silica nanoparticles of 99:1 and 9:1 into the two-phase reaction system in the step 5 in an adding amount of 2 wt.% (the mass ratio of the oil phase to the water phase), and shaking by hand (100 times per minute) for 2min to form a Pickering emulsion system from the separated two-phase system;
and 7: placing the Pickering emulsion reaction system in the step 5 on a rotary incubator, and reacting at the rotating speed of 40 rpm;
and 8: after 8h of reaction, 100 mu L of oil phase sample is extracted, after demulsification is carried out by centrifugation for 1min at 5000rpm, quantitative analysis is carried out on the content of methyl laurate by using gas chromatography, and the enzyme activity under different systems is calculated.
As shown in fig. 5, the enzyme activity of the two-phase free enzyme system is: 1.46 U.mL-1The enzyme activity of the Pickering emulsion system with stable enzyme-loaded microspheres and a hydrophilic/hydrophobic silica nanoparticle mass ratio of 99:1 is as follows: 10.98 U.mL-1The enzyme activity of the Pickering emulsion system with stable enzyme-loaded microspheres and the mass ratio of hydrophilic/hydrophobic silicon dioxide nanoparticles of 9:1 is 7.52 times that of a bidirectional system: 11.94 U.mL-18.18 times that of the biphasic system.
Example 4
A method for preparing biodiesel from an enzyme-loaded microsphere stable Pickering emulsion system prepared from hydrophilic/hydrophobic silica nanoparticles in a mass ratio of 99:1 comprises the following steps:
step 1: adding 6mg of hydrophobic silica nanoparticles (the mass ratio of hydrophilic/hydrophobic silica nanoparticles is 99:1) into 1mL of hydrophilic silica nanoparticle dispersion (40 wt%), and uniformly mixing the dispersion;
step 2: adding 1.4mL of lipase solution and 0.6mL of phosphate buffer (0.05M, pH 7.5) to the mixture obtained in step 1, and uniformly mixing;
and step 3: rapidly adding 30mL of n-butanol/tributyrin into the mixed solution obtained in the step 2, and emulsifying and homogenizing for 1min under the condition that the rotation speed of a homogenizer is 12000 rpm;
and 4, step 4: and (3) standing the emulsion obtained in the step (3) in a refrigerator at 4 ℃ for self-assembly for 12h, and centrifuging at 4 ℃ for 5min at 6000rpm by using a refrigerated centrifuge. Washing and freeze-drying the precipitate to obtain the enzyme-loaded microspheres with the mass ratio of the hydrophilic/hydrophobic silicon dioxide nanoparticles of 99: 1;
and 5: 2g of lauric acid was dissolved in 8mL of n-octane as an oil phase, and 1.614mL of methanol was dispersed in 2mL of a phosphate buffer (0.05M, pH 7.5) as an aqueous phase;
step 6: adding the hydrophilic/hydrophobic silica nanoparticles prepared in the step (4) into the two-phase reaction system in the step (5) in an adding amount of 2 wt% (based on the mass ratio of oil to water) of the enzyme-loaded microspheres, and shaking by hand (100 times per minute) for 2min to form a Pickering emulsion system from the separated two-phase system;
and 7: placing the Pickering emulsion reaction system in the step 6 on a rotary incubator, and reacting at the rotating speed of 40 rpm;
and 8: extracting 100 mu L of oil phase samples every 1h, centrifuging at 5000rpm for 1min to break emulsion, and quantitatively analyzing the content of methyl laurate by using gas chromatography.
As shown in fig. 6, the catalytic system for preparing the Pickering emulsion with stable enzyme-loaded microspheres and a hydrophilic/hydrophobic silica nanoparticle mass ratio of 99:1 shows higher catalytic efficiency than that of the two-phase free enzyme system; after 12 hours of reaction, the yield of the biodiesel is 79.75 percent.
Example 5
A method for preparing biodiesel from enzyme-loaded microsphere stable Pickering emulsion prepared from hydrophilic/hydrophobic silica nanoparticles in a mass ratio of 9:1 comprises the following steps:
step 1: adding 58mg of hydrophobic silica nanoparticles (mass ratio of hydrophilic/hydrophobic silica nanoparticles is 9:1) to 1mL of hydrophilic silica nanoparticle dispersion (40 wt%), and uniformly mixing the dispersion;
step 2: adding 1.4mL of lipase solution and 0.6mL of phosphate buffer (0.05M, pH 7.5) to the mixture obtained in step 1, and uniformly mixing;
and step 3: rapidly adding 30mL of n-butanol/tributyrin into the mixed solution obtained in the step 2, and emulsifying and homogenizing for 1min under the condition that the rotation speed of a homogenizer is 12000 rpm;
and 4, step 4: and (3) standing the emulsion obtained in the step (3) in a refrigerator at 4 ℃ for self-assembly for 12h, and centrifuging at 4 ℃ for 5min at 6000rpm by using a refrigerated centrifuge. Washing and freeze-drying the precipitate to obtain the enzyme-loaded microspheres with the mass ratio of hydrophilic/hydrophobic silicon dioxide nanoparticles of 9: 1;
and 5: 2g of lauric acid was dissolved in 8mL of n-octane as an oil phase, and 1.614mL of methanol was dispersed in 2mL of a phosphate buffer (0.05M, pH 7.5) as an aqueous phase;
step 6: adding the hydrophilic/hydrophobic silica nanoparticles prepared in the step 4 into the two-phase reaction system in the step 5 by adding 2 wt% (based on the mass ratio of oil to water) of the enzyme-loaded microspheres, and shaking by hand (100 times per minute) for 2min to form a Pickering emulsion system;
and 7: placing the Pickering emulsion reaction system in the step 6 on a rotary incubator, and reacting at the rotating speed of 40 rpm;
and 8: extracting 100 mu L of oil phase samples every 1h, centrifuging at 5000rpm for 1min to break emulsion, and quantitatively analyzing the content of methyl laurate by using gas chromatography.
As shown in fig. 7, the Pickering emulsion catalytic system with stable enzyme-loaded microspheres prepared from hydrophilic/hydrophobic silica nanoparticles at a mass ratio of 9:1 showed the highest catalytic efficiency; after 12 hours of reaction, the yield of the biodiesel is 84.27 percent.
Example 6
The reusability of the enzyme-loaded microspheres comprises the following steps:
step 1: adding hydrophobic silica nanoparticles into 1mL of hydrophilic silica nanoparticle dispersion liquid (40 wt%) according to the mass ratio of hydrophilic/hydrophobic silica nanoparticles of 99:1 and 9:1, and uniformly mixing the dispersion liquid;
step 2: adding 1.4mL of lipase solution and 0.6mL of phosphate buffer (0.05M, pH 7.5) to the mixture obtained in step 1, and thoroughly mixing the resulting mixture;
and step 3: rapidly adding 30mL of n-butanol/tributyrin into the mixed solution obtained in the step 2, and emulsifying and homogenizing for 1min under the condition that the rotation speed of a homogenizer is 12000 rpm;
and 4, step 4: and (3) standing the emulsion obtained in the step (3) in a refrigerator at 4 ℃ for self-assembly for 12h, and centrifuging at 4 ℃ for 5min at 6000rpm by using a refrigerated centrifuge. Washing the precipitate, and freeze-drying to obtain the enzyme-loaded microspheres with the mass ratio of the hydrophilic/hydrophobic silicon dioxide nanoparticles of 99:1 and 9: 1.
And 5: 2g of lauric acid was dissolved in 8mL of n-octane as an oil phase, and 1.614mL of methanol was dispersed in 2mL of a phosphate buffer (0.05M, pH 7.5) as an aqueous phase;
step 6: adding the enzyme-loaded microspheres prepared in the step 4 and having the mass ratio of the hydrophilic/hydrophobic silica nanoparticles of 99:1 and 9:1 into the two-phase reaction system in the step 5 in an adding amount of 2 wt.% (the mass ratio of the oil phase to the water phase), and shaking by hand (100 times per minute) for 2min to form a Pickering emulsion system from the separated two-phase system;
and 7: placing the Pickering emulsion reaction system in the step 5 on a rotary incubator, and reacting at the rotating speed of 40 rpm;
and 8: after 8h of reaction, centrifuging at 5000rpm for 5min to demulsify, extracting oil phase, and quantitatively analyzing the content of methyl laurate by using gas chromatography.
And step 9: recovering the water phase and the enzyme-carrying microspheres, supplementing the consumed methanol, adding the oil phase obtained in the step 5, and repeating the operations of the step 7 and the step 8 for 10 times.
As shown in fig. 8, in the 10-time recycling process, the catalytic activity of both of the enzyme-loaded microspheres was not significantly reduced, and the corresponding initial enzyme activity was maintained at more than 94%; after 10 cycles, the yield of the biodiesel is over 80 percent.
Example 7
The preparation method of the enzyme-carrying microsphere comprises the following steps:
step 1: adding hydrophobic silica nanoparticles into 1mL of hydrophilic silica nanoparticle dispersion liquid (40 wt%) according to the mass ratio of hydrophilic/hydrophobic silica nanoparticles of 99:1 and 9:1, and uniformly mixing the dispersion liquid; the particle size of the hydrophobic silicon dioxide nano particles is 12nm, and the hydrophobic silicon dioxide nano particles are modified by octamethylcyclotetrasiloxane. The particle size of the hydrophilic silicon dioxide nano particles is 22nm, and no modification is carried out.
Step 2: adding 0.2-1.8 mL of lipase solution and 0.2-1.8 mL of phosphate buffer (pH 7.5) into the hydrophilic/hydrophobic silica nanoparticle dispersion, and fully mixing the obtained 3mL of mixed solution;
and step 3: quickly adding 0-50 mL of n-butyl alcohol/tributyrin into the obtained mixed solution, and emulsifying and homogenizing for 1-3 min under the condition that the rotation speed of a homogenizer is 12000 r/min;
and 4, step 4: the obtained emulsion was placed in a refrigerator at 4 ℃ and allowed to stand for self-assembly for 12 hours, and centrifuged at 6000rpm for 5 minutes at 4 ℃ by a refrigerated centrifuge. Washing and freeze-drying the precipitate to obtain the enzyme-loaded microspheres.
As shown in figure 2, the enzyme-loaded microcapsules with the mass ratios of the hydrophilic/hydrophobic silica nanoparticles of 99:1 and 9:1 have different adsorption states at the emulsion interface due to different wettabilities (three-phase contact angles of 91 degrees and 121 degrees respectively), and the enzyme-loaded microspheres are more biased to an oil phase and far away from a water phase where methanol is located when the mass ratio is 9:1, so that the enzyme can be effectively protected from the inactivation of the methanol.
It is to be understood that the above description is not intended to limit the present invention, and the present invention is not limited to the above examples, and those skilled in the art may make modifications, alterations, additions or substitutions within the spirit and scope of the present invention.
Claims (5)
1. The preparation method of the enzyme-loaded microsphere is characterized in that the enzyme-loaded microsphere is prepared by an emulsion template-assisted interface self-assembly method of hydrophilic/hydrophobic silica nanoparticles and lipase with different mass ratios, and the surface wettability of the enzyme-loaded microsphere is adjusted by changing the mass ratio of the hydrophilic/hydrophobic silica nanoparticles in the self-assembly process;
the method comprises the following steps:
step 1: adding hydrophobic silica nanoparticles into 1mL of hydrophilic silica nanoparticle dispersion liquid (40 wt%) according to the mass ratio of hydrophilic/hydrophobic silica nanoparticles of 99:1 or 9:1, and uniformly mixing the dispersion liquid;
step 2: adding 0.2-1.8 mL of lipase solution and 0.2-1.8 mL of phosphate buffer (0.05M, pH 7.5) to the mixed solution obtained in the step 1, and fully mixing the obtained mixed solution;
and step 3: adding 30-50 mL of n-butanol/tributyrin into the mixed solution obtained in the step 2, and emulsifying and homogenizing for 1-3 min under the condition that the rotation speed of a homogenizer is 12000 r/min;
and 4, step 4: and (3) standing the emulsion obtained in the step (3) in a refrigerator at 4 ℃ for self-assembly for 12h, centrifuging at 4 ℃ for 5min at 6000rpm by using a refrigerated centrifuge, washing the precipitate, and freeze-drying to obtain the enzyme-loaded microspheres.
2. The preparation method of the enzyme-loaded microspheres according to claim 1, wherein the enzyme-loaded microspheres can be recycled after being separated by centrifugation or filtration, and after 10 times of circulation, the emulsification performance of the enzyme-loaded microspheres is stable and the catalytic activity is maintained at more than 94%.
3. Use of the enzyme-loaded microspheres of claim 1 or 2 in the enzymatic preparation of biodiesel from Pickering emulsion.
4. A method for preparing biodiesel by a Pickering emulsion enzymatic method is characterized by comprising the following steps:
step 1: dissolving lauric acid in n-octane to prepare 15-55 wt.% lauric acid-n-octane solution as an oil phase; dispersing methanol in a phosphate buffer (pH 7.5) to prepare a 15-50 wt.% methanol aqueous solution as an aqueous phase;
step 2: forming a W/O or O/W Pickering emulsion by hand shaking (100 times per minute) or a homogenizer with 8000-20000 revolutions per minute and 2-8 min under the conditions that the oil-water two-phase ratio is 1: 1-7: 1, the molar ratio of lauric acid to methanol is 1: 1-1: 8, and the addition amount of the enzyme-loaded microspheres is 1-2 wt% (based on the mass ratio of the oil phase to the water phase), wherein the droplet diameter of the emulsion is 150-650 mu m, and the emulsion is kept stable in a catalytic reaction process at least within 12 hours;
and step 3: the Pickering emulsion stabilized by the enzyme-loaded microspheres is placed on a rotary incubator or a shaking table, the temperature is kept at 15-25 ℃, and the reaction is carried out for 0-12 h at the rotating speed of 20-150 rpm, so that methyl laurate (one type of biodiesel) can be generated.
5. The method for preparing biodiesel through a Pickering emulsion enzymatic method according to claim 4, wherein the enzyme-loaded microspheres are simultaneously used as an emulsifier and a biological interface catalyst to perform catalytic reaction at an oil-water two-phase interface in the Pickering emulsion to efficiently prepare biodiesel, the catalytic activity of the enzyme is increased to 7.52-8.18 times, and the conversion rate reaches 75-84%.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101108730A (en) * | 2006-07-18 | 2008-01-23 | 德古萨股份公司 | Surface modified silicon dioxide |
| CN103374194A (en) * | 2012-04-16 | 2013-10-30 | 苏州萃智新技术开发有限公司 | Gas phase nano silicon dioxide master batches for plastic modification and preparation method thereof |
| CN105176944A (en) * | 2015-10-14 | 2015-12-23 | 天津现代职业技术学院 | Emulsion system immobilized lipase and method |
| JP6344878B1 (en) * | 2017-11-16 | 2018-06-20 | 旭化成ワッカーシリコーン株式会社 | Water dispersion, method for producing water dispersion, oil-in-water emulsion, and method for producing oil-in-water emulsion |
| US20180200689A1 (en) * | 2015-07-30 | 2018-07-19 | Dwi-Leibniz-Institut Für Interaktive Materialien E.V. | Method for the encapsulation of substances in silica-based capsules and the products obtained thereof |
| CN113249369A (en) * | 2021-03-26 | 2021-08-13 | 河北工业大学 | Immobilized lipase and preparation method and application thereof |
-
2022
- 2022-03-31 CN CN202210328694.6A patent/CN114606222A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101108730A (en) * | 2006-07-18 | 2008-01-23 | 德古萨股份公司 | Surface modified silicon dioxide |
| CN103374194A (en) * | 2012-04-16 | 2013-10-30 | 苏州萃智新技术开发有限公司 | Gas phase nano silicon dioxide master batches for plastic modification and preparation method thereof |
| US20180200689A1 (en) * | 2015-07-30 | 2018-07-19 | Dwi-Leibniz-Institut Für Interaktive Materialien E.V. | Method for the encapsulation of substances in silica-based capsules and the products obtained thereof |
| CN105176944A (en) * | 2015-10-14 | 2015-12-23 | 天津现代职业技术学院 | Emulsion system immobilized lipase and method |
| JP6344878B1 (en) * | 2017-11-16 | 2018-06-20 | 旭化成ワッカーシリコーン株式会社 | Water dispersion, method for producing water dispersion, oil-in-water emulsion, and method for producing oil-in-water emulsion |
| CN113249369A (en) * | 2021-03-26 | 2021-08-13 | 河北工业大学 | Immobilized lipase and preparation method and application thereof |
Non-Patent Citations (9)
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