CN113025607A - Interface immobilized enzyme based on amphiphilic mesoporous nano silicon spheres and preparation method thereof - Google Patents

Interface immobilized enzyme based on amphiphilic mesoporous nano silicon spheres and preparation method thereof Download PDF

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CN113025607A
CN113025607A CN202110311672.4A CN202110311672A CN113025607A CN 113025607 A CN113025607 A CN 113025607A CN 202110311672 A CN202110311672 A CN 202110311672A CN 113025607 A CN113025607 A CN 113025607A
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amphiphilic
mesoporous
enzyme
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silica spheres
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金青哲
董喆
金俊
吴港城
韦伟
王兴国
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Jiangnan University
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Abstract

The invention discloses an amphiphilic mesoporous nano-silicon sphere-based interface immobilized enzyme and a preparation method thereof, the amphiphilic mesoporous nano-silicon sphere interface immobilized enzyme comprises an enzyme and a carrier for immobilizing the enzyme, namely an amphiphilic mesoporous nano-silicon sphere, the amphiphilic mesoporous nano-silicon sphere comprises a hydrophobic mesoporous silicon dioxide sphere serving as a core and hydrophilic mesoporous silicon dioxide serving as a shell, the particle diameter is 100-400 nm, and the specific surface area is 300-800 m2The pore size of the mesopores is 3-30 nm, the water contact angle of the hydrophobic mesoporous silica spheres is 90-150 degrees, and the water contact angle of the hydrophilic mesoporous silica spheres is 10-70 degrees. The amphiphilic mesoporous nano silicon sphere interface immobilized enzyme achieves the aim of enzyme interface immobilization by forming pickering emulsion, wherein the enzyme is positioned on an interlayer of a core-shell structure and an oil-water interface, and the enzyme stability and the catalytic performance can be greatly improved.

Description

Interface immobilized enzyme based on amphiphilic mesoporous nano silicon spheres and preparation method thereof
Technical Field
The invention belongs to the technical field of enzyme immobilization, and particularly relates to an amphiphilic mesoporous nano silicon sphere interface immobilized enzyme and a preparation method thereof.
Background
The enzyme is a green and environment-friendly biocatalyst with high catalytic activity, good selectivity and strong specificity, and can be widely applied to various fields. In the using process, the problems of catalytic activity, stability, recovery, repeated use and the like of free enzyme restrict the application in practical production, in order to solve the problems, an immobilization technology is developed at the same time, the immobilization of the enzyme refers to a technology that natural free enzyme is limited in a certain space and can not move freely, and the catalytic activity and the stability of the enzyme are greatly improved. The carrier of the immobilized enzyme is one of the main factors affecting its performance. Commonly used carriers comprise porous silicon materials, macroporous resins, molecular sieves and the like, and usually, the carriers are directly and simply adsorbed and immobilized, so that the enzyme activity is reduced. Patent CN110655060A discloses a double-sided amphiphilic carrier, which has two opposite hydrophilic-hydrophobic interfaces, however, the carrier is in the form of lamellar, and under the catalysis condition of high-speed stirring or rapid oscillation, the stability of enzyme is not high, and it is easy to fall off, and the reuse rate of immobilized enzyme is reduced.
The catalytic performance of the enzyme is related to the active center state of the enzyme, and research shows that the active center of the lipase is covered by one or more alpha helical structures, which are called as 'covers', the 'covers' have the hydrophilic upper surface and the hydrophobic lower surface, and the 'covers' are opened on an oil-water interface to expose the active center, so that the lipase can exert the catalytic performance, which is the phenomenon of lipase 'interface activation'. Patent CN 105176944 a discloses a method for improving catalytic performance of enzyme in two-phase reaction by constructing a pickering emulsion system based on traditional immobilized enzyme, however, in the method, the enzyme is adsorbed and immobilized on a carrier, and then particles containing the enzyme are put into the system to form pickering emulsion.
Disclosure of Invention
The purpose of the invention is as follows: in order to overcome the defects in the prior art, the invention provides an amphiphilic mesoporous nano silicon sphere interface immobilized enzyme and a preparation method thereof, the aim of enzyme interface immobilization is achieved by forming Pickering emulsion, wherein the enzyme is positioned on an interlayer of a core-shell structure and an oil-water interface, and the enzyme stability and the catalytic performance can be greatly improved.
The amphiphilic mesoporous nano silicon sphere interface immobilized enzyme comprises an enzyme and a carrier amphiphilic mesoporous nano silicon sphere for immobilizing the enzyme, wherein the amphiphilic mesoporous nano silicon sphere comprises an inner hydrophobic mesoporous silicon dioxide sphere and an outer hydrophilic mesoporous silicon dioxide sphere, the particle diameter is 100-400 nm, and the specific surface area is 300-800 m2The pore size of the mesopores is 3-30 nm, the water contact angle of the hydrophobic mesoporous silica spheres is 90-150 degrees, and the water contact angle of the hydrophilic mesoporous silica spheres is 10-70 degrees. Emulsifying enzyme, organic solvent and amphiphilic mesoporous nano silicon spheres to form pickering emulsion.
According to the scheme, the amphiphilic mesoporous nano silicon spheres can be of a core-shell structure.
The water contact angle of the hydrophobic mesoporous silica spheres is 90-150 degrees, and the water contact angle of the hydrophilic mesoporous silica spheres is 10-70 degrees.
According to the scheme, the enzyme mainly comprises any one of lipase, phospholipase, pancreatic lipase or hydrolase and the like. From the class of enzymes, enzymes useful in the present invention include, but are not limited to, enzymes having an interfacial activation effect. According to the scheme, the organic solvent mainly comprises n-hexane, isooctane, n-heptane and the like.
The preparation method of the amphiphilic mesoporous nano silicon sphere interface immobilized enzyme mainly comprises the following steps:
1) preparation of hydrophobic mesoporous silica spheres: dispersing a surfactant into a reaction solvent, adding a catalyst, normal hexane and a silicon source, stirring for reaction, centrifuging after the reaction is finished, washing with ethanol for 3 times, and drying to obtain a solid, namely a silicon dioxide ball serving as a core; removing the surface template agent from the silica spheres to form mesoporous silica spheres, and performing surface modification on the mesoporous silica spheres by using a silane coupling agent in a toluene environment and using triethylamine as a catalyst to synthesize the hydrophobic mesoporous silica spheres.
2) Preparation of mesoporous silica with amphiphilic core-shell structure: dispersing hydrophobic mesoporous silica spheres in a reaction solvent, adding a catalyst, normal hexane and a silicon source, stirring for reaction, centrifuging after the reaction is finished, washing with ethanol for 3 times, drying, removing a surface template agent from the silica spheres to form the mesoporous silica spheres with the core-shell structure, performing surface hydrophilic modification on the mesoporous silica spheres by using a silane coupling agent in a toluene environment and using triethylamine as the catalyst to synthesize the amphiphilic mesoporous silica spheres with the core-shell structure, namely the amphiphilic mesoporous nano-silica spheres;
3) preparing an amphiphilic mesoporous nano silicon sphere interface immobilized enzyme: dispersing enzyme into a buffer solution to prepare an enzyme solution; emulsifying an organic solvent, amphiphilic mesoporous nano silicon spheres and an enzyme solution to form pickering emulsion, and simultaneously carrying out interface immobilization to obtain amphiphilic mesoporous nano silicon sphere interface immobilized enzyme;
according to the scheme, the surfactant in the step 1) and the step 2) is one or a mixture of a plurality of dodecyl trimethyl ammonium bromide, tetradecyl trimethyl ammonium bromide, hexadecyl trimethyl ammonium bromide and octadecyl trimethyl ammonium bromide according to any proportion. Preferably, the surfactant is cetyl trimethyl ammonium bromide.
According to the scheme, the silicon source in the step 1) and the step 2) is one or a mixture of more of ethyl orthosilicate, propyl orthosilicate, isopropyl orthosilicate and butyl orthosilicate in any proportion. Preferably, the silicon source is tetraethoxysilane.
According to the scheme, the catalyst in the step 1) and the step 2) is sodium hydroxide, potassium hydroxide or concentrated ammonia water. Preferably, the catalyst is concentrated ammonia.
According to the scheme, the silane coupling agent in the step 1) and the step 2) is n-octyltrimethoxysilane, n-octyltriethoxysilane, dodecyltrimethoxysilane, dodecyltriethoxysilane, hexadecyltrimethoxysilane, hexadecyltriethoxysilane, trimethoxy (3,3, 3-trifluoropropyl) silane and the like. Preferably, the silane coupling agent is n-octyltrimethoxysilane.
According to the scheme, in the step 1), the proportion of the surfactant to the n-hexane is 1g (10-30) mL; the concentration of the silicon source in the reaction solvent ranges from 0.5% to 3% (mL/mL).
According to the scheme, in the step 1), the reaction solvent consists of water, the volume of the reaction solvent is 150-200 mL, the catalyst is 28 wt% of concentrated ammonia water, and the addition amount of the catalyst is 3-5% of the volume of the reaction solvent.
According to the scheme, in the step 2), the reaction solvent is water, and the proportion of the water, the hydrophobic mesoporous silica spheres and the surfactant is (50-200) mL:1g (0.2-0.6) g, wherein the volume range of n-hexane is 10-50 mL; the concentration range of the silicon source in the reaction solvent is 0.5-3% (mL/mL), wherein the n-hexane and the silicon source are uniformly mixed and then are dropwise added into the reaction system, and the dropwise adding speed range is (0.5-3) mL/min.
According to the scheme, in the steps 1) and 2), the stirring reaction is carried out at the temperature of 20-40 ℃ for 12-30 h.
According to the scheme, in the step 1), the proportion of the silane coupling agent, the triethylamine, the toluene and the mesoporous silica is (0.5-2.5) mmol (20-100) mu L: 20ml of 1g, the reaction temperature is 100-140 ℃, and the reaction time is 12-30 h.
According to the scheme, the silane coupling agent in the step 2) is 3-aminopropyltrimethoxysilane, mercaptopropyltriethoxysilane and the like.
According to the scheme, silane coupling agents different from those in the step 1) are used for surface modification in the step 2), and the ratio of the silane coupling agents to triethylamine to toluene to mesoporous silica is (0.1-2.5) mmol, (20-100) mu L: 20ml of 1g, the reaction temperature is 100-140 ℃, and the reaction time is 12-30 h.
According to the scheme, in the step 3), the organic solvent is n-hexane, n-heptane, isooctane or acetone and the like.
According to the scheme, in the step 3), the mass ratio of the mesoporous silica with the amphiphilic core-shell structure, the enzyme and the organic solvent is (0.2-1) g (120-450) mg: (1-15) mL.
According to the scheme, in the step 3), the mixed liquid obtained in the step 3) is emulsified by utilizing contact probe ultrasound, a handheld homogenizer, a vortex instrument, a high-pressure homogenizer and the like to form a Pickering emulsion immobilization system with the particle size of 10-200 mu m; the enzyme immobilization temperature is 4-30 ℃, and the time is 0.1-3 h.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention firstly provides that the immobilized enzyme is prepared by taking the core-shell amphiphilic mesoporous nano silicon spheres as a carrier through interface adsorption, and a proper space is provided for the immobilization of the enzyme by combining the special structure of the core-shell interlayer, so that the stability and the enzyme activity of the enzyme can be greatly improved.
2. The amphiphilic mesoporous nano silicon spheres have a large number of silicon-oxygen groups, can modify carriers in a hydrophilic and hydrophobic manner, and provide a proper microenvironment for lipase, and in addition, the special structure of the hydrophilic surface of the shell and the hydrophobic surface of the inner core is beneficial to the opening of the structure of a lipase cover and the exposure of an enzyme active site;
3. according to the amphiphilic mesoporous nano silicon sphere interface immobilized enzyme disclosed by the invention, the pickering emulsion is formed for interface adsorption immobilization, and the enzyme is immobilized in the hydrophobic interlayer.
Drawings
Fig. 1 shows the core-shell amphiphilic mesoporous nano-silica spheres and the hydrophilic and hydrophobic contact angles obtained in example 1, wherein (a) is the mesoporous silica spheres as the core, (b) is the core-shell structure amphiphilic mesoporous silica, (c) is the water contact angle of the core-shell structure amphiphilic mesoporous silica of 126 °, and (d) is the water contact angle of the core-shell structure amphiphilic mesoporous silica of 34 °.
FIG. 2(a) shows the core-shell structure of the amphiphilic mesoporous silica having a cavity obtained in example 2, and (b) shows the amphiphilic mesoporous silica having a core-shell structure obtained in example 3.
FIG. 3 is a photograph under the microscope of the Pickering emulsion obtained in example 3.
Detailed Description
The following examples are given solely for the purpose of illustrating the invention in detail, to facilitate a better understanding of the invention, and are included within the scope of the invention, but are not intended to limit the invention.
Examples
The present invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the specific material ratios, process conditions and results thereof described in the examples are illustrative only and should not be taken as limiting the invention as detailed in the claims.
Example 1
An amphiphilic mesoporous nano silicon sphere interface immobilized enzyme and a preparation method thereof, comprising the following steps:
1) preparation of hydrophobic mesoporous silica spheres: dispersing 1g of hexadecyl trimethoxy ammonium bromide into 160mL of water, adding 3% of catalyst concentrated ammonia water, stirring for 20min, mixing 20mL of n-hexane with 0.5% of tetraethoxysilane (the concentration of a silicon source in the water), dropwise adding the mixture into the mixed solution at the speed of 0.5mL/min, stirring and reacting for 12h at the temperature of 30 ℃, centrifuging after the reaction is finished, washing with ethanol for 3 times, and drying to obtain a solid, namely a silicon dioxide ball serving as a core; and (3) putting the silica spheres in a muffle furnace, and calcining at 550 ℃ for 4h to remove the surface template agent to form mesoporous silica spheres, as shown in figure 1 (a). Dispersing 1g of mesoporous silica spheres into 20mL of toluene, respectively dropwise adding 20 mu L of triethylamine and 0.5mmol of n-octyltrimethoxysilane, reacting for 12h at 100 ℃ in a high-pressure reaction kettle, and synthesizing hydrophobic mesoporous silica spheres, wherein the water contact angle is 90 degrees as shown in figure 1 (c);
2) preparing an amphiphilic mesoporous silica sphere with a core-shell structure: dispersing 1g of hydrophobic mesoporous silica spheres and 0.5g of hexadecyl trimethyl ammonium bromide in 80mL of water, adding 1% of strong ammonia water, and mixing 20mL of n-hexane and 0.5% of tetraethoxysilane (siliconConcentration of the source in water), adding the mixture into the mixed solution drop by drop at a speed of 0.5mL/min, stirring and reacting for 12 hours at a temperature of 30 ℃, centrifuging after the reaction is finished, washing with ethanol for 3 times, drying, wherein the obtained solid is a silicon dioxide ball with a core-shell structure, putting the silicon dioxide ball into a muffle furnace, calcining at a temperature of 550 ℃ for 4 hours, and removing a surface template agent to form a mesoporous silicon dioxide ball with a core-shell structure, as shown in fig. 1 (b). Dispersing 1g of mesoporous silica spheres with a core-shell structure into 20mL of toluene, respectively dropwise adding 20 muL of triethylamine and 0.1mmol of 3-aminopropyltrimethoxysilane, reacting for 12h at 100 ℃ in a high-pressure reaction kettle, and synthesizing amphiphilic mesoporous silica spheres with a core-shell structure, wherein as shown in figure 1(d), the water contact angle of the outer hydrophilic mesoporous silica spheres is 34 degrees; the diameter of the obtained amphiphilic mesoporous nano silicon spheres is 100nm, and the specific surface area is 300m2The pore size of the mesopores is 3 nm.
3) Preparing an amphiphilic mesoporous nano silicon sphere interface immobilized enzyme: 150mg of Candida antarctica lipase was dispersed in 10ml of 20mmol of phosphate buffer solution having a pH of 7.0 to prepare an enzyme solution; emulsifying 2mL of n-hexane, 0.2g of amphiphilic mesoporous nano silicon spheres and 10mL of enzyme solution by using a handheld homogenizer to form pickering emulsion, simultaneously placing the pickering emulsion in a constant-temperature oscillator at 15 ℃ for interface immobilization, wherein the reaction time is 0.5h, after the reaction is finished, cleaning the buffer solution for 3 times, and freeze-drying after centrifugal separation to obtain the amphiphilic mesoporous nano silicon sphere interface immobilized enzyme.
The application comprises the following steps: synthesis of alpha-linolenic acid phytosterol ester: weighing 4.5g of phytosterol, weighing alpha-linolenic acid and 2.0g of amphiphilic mesoporous nano silicon sphere interface immobilized enzyme according to the molar ratio of the alpha-linolenic acid to the phytosterol of 5:1 (example 1), adding 100mL of n-hexane, placing in a constant-temperature water bath at 55 ℃ for reaction for 4h, and stirring at the rotating speed of 300 revolutions per minute; after the reaction is finished, the immobilized enzyme is removed by centrifugation, the solvent is removed by reduced pressure distillation, and the sterol esterification rate is 94.7 percent. The amphiphilic mesoporous nano silicon sphere interface immobilized enzyme is recovered after being cleaned by n-hexane for 3 times, the reaction is repeated for 10 times, and the sterol esterification rate is still more than 88%.
Example 2
An amphiphilic mesoporous nano silicon sphere interface immobilized enzyme and a preparation method thereof, comprising the following steps:
1) preparation of hydrophobic mesoporous silica spheres: dispersing 1g of dodecyl trimethyl ammonium bromide into 150mL of water, adding 5% of catalyst concentrated ammonia water, stirring for 20min, mixing 10mL of n-hexane with 3% of tetraethoxysilane (the concentration of a silicon source in the water), dropwise adding the mixture into the mixed solution at the speed of 0.5mL/min, stirring and reacting for 30h at the temperature of 20 ℃, centrifuging after the reaction is finished, washing with ethanol for 3 times, and drying to obtain solid, namely silicon dioxide spheres serving as cores; and (3) putting the silicon dioxide spheres in a muffle furnace, calcining for 4 hours at the temperature of 550 ℃ to remove the surface template agent, and forming the mesoporous silicon dioxide spheres. Dispersing 1g of mesoporous silica spheres into 20mL of toluene, respectively dropwise adding 100 mu L of triethylamine and 2.5mmol of dodecyl trimethoxy silane, reacting for 30h at 140 ℃ in a high-pressure reaction kettle, and synthesizing hydrophobic mesoporous silica spheres with a water contact angle of 120 degrees;
2) preparation of mesoporous silica with amphiphilic core-shell structure: dispersing 1g of hydrophobic mesoporous silica spheres and 0.6g of dodecyl trimethyl ammonium bromide in 200mL of water, adding 5% of concentrated ammonia water, mixing 50mL of n-hexane and 3% of tetraethoxysilane (the concentration of a silicon source in the water), dropwise adding the mixture into the mixed solution at the speed of 0.5mL/min, stirring and reacting at the temperature of 20 ℃ for 30 hours, centrifuging after the reaction is finished, washing with ethanol for 3 times, drying, putting the obtained solid, namely the silica with the core-shell structure into a muffle furnace, calcining at the temperature of 550 ℃ for 4 hours, and removing a surface template agent to form the mesoporous silica spheres with the core-shell structure. Dispersing 1g of mesoporous silica spheres with a core-shell structure into 20mL of toluene, respectively dropwise adding 100 mu L of triethylamine and 2.5mmol of mercaptopropyltriethoxysilane, reacting for 30h at 140 ℃ in a high-pressure reaction kettle, and synthesizing amphiphilic mesoporous silica spheres with a core-shell structure, as shown in FIG. 2 (a); the water contact angle of the outer hydrophilic mesoporous silica spheres is 30 degrees; the diameter of the obtained amphiphilic mesoporous nano silicon spheres is 200nm, and the specific surface area is 500m2The pore size of the mesopores is 5 nm.
3) Preparing an amphiphilic mesoporous nano silicon sphere interface immobilized enzyme: dispersing 120mg of Pseudomonas cepacia lipase into 10mL of phosphate buffer solution with the pH value of 7.0 and the concentration of 20mmol of the lipase to prepare an enzyme solution; carrying out ultrasonic emulsification on 1mL of n-hexane, 1g of amphiphilic mesoporous nano silicon spheres and 10mL of enzyme solution to form Pickering emulsion, meanwhile, placing the Pickering emulsion in a constant-temperature oscillator at 30 ℃ for interface immobilization, wherein the reaction time is 3h, after the reaction is finished, cleaning the buffer solution for 3 times, carrying out centrifugal separation, and carrying out freeze drying to obtain the amphiphilic mesoporous nano silicon sphere interface immobilized enzyme.
The application comprises the following steps: synthesis of human milk fat replacers: weighing 1g of tripalmitin, weighing oleic acid and 0.8g of amphiphilic mesoporous nano silicon sphere interface immobilized enzyme according to the molar ratio of the tripalmitin to the oleic acid of 1:6 (example 2), adding 0.6mL of deionized water, adding 30mL of n-hexane, placing in a constant temperature oscillator at 55 ℃ for oscillation for 2 hours at the oscillation rate of 200rpm, centrifuging to remove the immobilized enzyme after the reaction is finished, and evaporating the solvent under reduced pressure to obtain the esterification rate of 42.4%. After the immobilized enzyme is recovered and washed by normal hexane for 3 times, the esterification rate is still more than 40 percent after the reaction is repeated for 8 times.
Example 3
An amphiphilic mesoporous nano silicon sphere interface immobilized enzyme and a preparation method thereof, comprising the following steps:
1) preparation of hydrophobic mesoporous silica spheres: dispersing 1g of tetradecyl trimethyl ammonium bromide into 200mL of water, adding 3% of catalyst concentrated ammonia water, stirring for 20min, mixing 50mL of n-hexane and 1% of tetraethoxysilane (the concentration of a silicon source in the water), dropwise adding the mixture into the mixed solution at the speed of 0.5mL/min, stirring and reacting for 30h at the temperature of 40 ℃, centrifuging after the reaction is finished, washing with ethanol for 3 times, and drying to obtain a solid, namely a silicon dioxide ball serving as a core; and (3) putting the silicon dioxide spheres in a muffle furnace, calcining for 4 hours at the temperature of 550 ℃ to remove the surface template agent, and forming the mesoporous silicon dioxide spheres. Dispersing 1g of mesoporous silica spheres into 20mL of toluene, respectively dropwise adding 50 mu L of triethylamine and 1.5mmol of dodecyl triethoxysilane, reacting for 24h at 110 ℃ in a high-pressure reaction kettle, and synthesizing hydrophobic mesoporous silica spheres with a water contact angle of 130 degrees;
2) preparing the mesoporous silica spheres with the amphiphilic core-shell structure: 1g of hydrophobic mesoporous silica spheres and 0.2g of hydrophobic mesoporous silica spheresTetradecyl trimethyl ammonium bromide is dispersed in 50mL of water, 3% concentrated ammonia water is added, 30mL of n-hexane and 1% of tetraethoxysilane (the concentration of a silicon source in the water) are mixed, then the mixture is dropwise added into the mixed solution at the speed of 0.5mL/min, stirring reaction is carried out at the temperature of 35 ℃ for 12 hours, centrifugation is carried out after the reaction is finished, the obtained solid is silicon dioxide with a core-shell structure after washing for 3 times by ethanol and drying, the obtained solid is the silicon dioxide with the core-shell structure, the silicon dioxide spheres are placed into a muffle furnace, and the surface template agent is removed after calcining is carried out at the temperature of 550 ℃ for 4 hours, so that. Dispersing 1g of mesoporous silica spheres with a core-shell structure into 20mL of toluene, respectively dropwise adding 80 mu L of triethylamine and 1.5mmol of mercaptopropyltriethoxysilane, reacting for 15h at 110 ℃ in a high-pressure reaction kettle, and synthesizing amphiphilic mesoporous silica spheres with a core-shell structure, as shown in FIG. 2 (b); the water contact angle of the outer hydrophilic mesoporous silica spheres is 40 degrees; the diameter of the obtained amphiphilic mesoporous nano silicon spheres is 150nm, and the specific surface area is 450m2The pore size of the mesopores is 15 nm.
3) Preparing an amphiphilic mesoporous nano silicon sphere interface immobilized enzyme: dispersing 120mg of phospholipase into 10mL of phosphate buffer solution with the pH value of 7.0 and 20mmol of phospholipase to prepare enzyme solution; emulsifying 1mL of isooctane, 1g of amphiphilic mesoporous nano-silica spheres and 10mL of enzyme solution by a vortex apparatus to form Pickering emulsion, placing the Pickering emulsion in a constant temperature oscillator at 30 ℃ for interface immobilization as shown in figure 3, reacting for 3 hours, washing the buffer solution for 3 times after the reaction is finished, centrifuging, and freeze-drying to obtain the amphiphilic mesoporous nano-silica sphere interface immobilized enzyme.
The application comprises the following steps: weighing 3.0g of soybean lecithin, weighing ethyl linolenate according to the mass ratio of the ethyl linolenate to the soybean lecithin of 5:1, adding 3.0mL of deionized water, adding 30mL of n-hexane to completely dissolve the solution, adding 20mg of amphiphilic mesoporous nano silicon sphere interface immobilized enzyme (example 3) to synthesize functional phospholipid, placing the functional phospholipid in a constant-temperature oscillator at 55 ℃ for oscillation for 2 hours, wherein the oscillation rate is 200rpm, centrifugally removing the immobilized enzyme after the reaction is finished, and evaporating the solvent under reduced pressure to obtain the esterification rate of 90%. After the immobilized enzyme is recovered and washed by normal hexane for 3 times, the esterification rate is still more than 85 percent after the reaction is repeated for 10 times.
Example 4
An amphiphilic mesoporous nano silicon sphere interface immobilized enzyme and a preparation method thereof, comprising the following steps:
1) preparation of hydrophobic mesoporous silica spheres: dispersing 1g of octadecyl trimethyl ammonium bromide into 200mL of water, adding 4% of catalyst concentrated ammonia water, stirring for 20min, mixing 25mL of n-hexane and 4% of tetraethoxysilane (the concentration of a silicon source in the water), dropwise adding the mixture into the mixed solution at the speed of 0.5mL/min, stirring and reacting for 15h at the temperature of 30 ℃, centrifuging after the reaction is finished, washing with ethanol for 3 times, and drying to obtain a solid, namely a silicon dioxide ball serving as a core; and (3) putting the silicon dioxide spheres in a muffle furnace, calcining for 4 hours at the temperature of 550 ℃ to remove the surface template agent, and forming the mesoporous silicon dioxide spheres. Dispersing 1g of mesoporous silica spheres into 20mL of toluene, respectively dropwise adding 60 mu L of triethylamine and 2mmol of n-octyltrimethoxysilane, reacting for 24 hours at 110 ℃ in a high-pressure reaction kettle, and synthesizing hydrophobic mesoporous silica spheres with a water contact angle of 140 degrees;
2) preparing the mesoporous silica spheres with the amphiphilic core-shell structure: dispersing 1g of hydrophobic mesoporous silica spheres and 0.5g of octadecyl trimethyl ammonium bromide in 180mL of water, adding 4% of concentrated ammonia water, mixing 30mL of n-hexane and 4% of tetraethoxysilane (the concentration of a silicon source in the water), dropwise adding the mixture into the mixed solution at the speed of 0.5mL/min, stirring and reacting at the temperature of 35 ℃ for 12 hours, centrifuging after the reaction is finished, washing with ethanol for 3 times, drying, putting the obtained solid, namely the core-shell structured silica into a muffle furnace, calcining at the temperature of 550 ℃ for 4 hours, and removing a surface template agent to form the core-shell structured mesoporous silica spheres; dispersing 1g of mesoporous silica spheres with a core-shell structure into 20mL of toluene, respectively dropwise adding 80 mu L of triethylamine and 2.5mmol of mercaptopropyltriethoxysilane, reacting for 15h at 110 ℃ in a high-pressure reaction kettle, and synthesizing amphiphilic mesoporous silica spheres with a core-shell structure; the water contact angle of the outer hydrophilic mesoporous silica spheres is 60 degrees; the diameter of the obtained amphiphilic mesoporous nano silicon spheres is 300nm, and the specific surface area is 600m2The pore size of the mesopores is 20 nm.
3) Preparing an amphiphilic mesoporous nano silicon sphere interface immobilized enzyme: dispersing 120mg protease into 10mL, 20mmol and pH 7.0 phosphate buffer solution to prepare enzyme solution; emulsifying 1mL of isooctane, 1g of amphiphilic mesoporous nano-silica spheres and 10mL of enzyme solution by a high-pressure homogenizer to form pickering emulsion, simultaneously placing the pickering emulsion in a constant-temperature oscillator at 30 ℃ for interface immobilization, wherein the reaction time is 3h, after the reaction is finished, cleaning the buffer solution for 3 times, centrifugally separating, and freeze-drying to obtain the amphiphilic mesoporous nano-silica sphere interface immobilized enzyme.
The application comprises the following steps: catalytic hydrolysis of casein: dispersing 20mg of amphiphilic mesoporous nano silicon sphere interface immobilized enzyme (example 4) into 1mL of phosphate buffer solution (100mmol, pH 6.0), and preheating in a water bath at 40 ℃ for 10 min; then, 1mL of preheated casein protein solution (1g/100mL, prepared from phosphate buffer solution with pH 6.0) was added, the mixture was shaken in a thermostatic water bath at 40 ℃ for 10min, trichloroacetic acid solution (10%, 2mL) was immediately added to terminate the reaction, the immobilized enzyme was centrifuged, and the supernatant was measured for absorbance at 275nm with a spectrophotometer. One enzyme activity unit (U) is defined as the amount of enzyme required for 1g of immobilized enzyme to hydrolyze the substrate casein at 40 ℃ and pH 6.0 for 1min to produce 1. mu. mol L-tyrosine. The result shows that the enzyme activity of the amphiphilic mesoporous nano silicon sphere interface immobilized enzyme is 600U/g carrier, the enzyme activity is more than 500U/g after the immobilized enzyme is recovered, washed by n-hexane for 3 times and repeatedly reacted for 10 times.
Example 5
An amphiphilic mesoporous nano silicon sphere interface immobilized enzyme and a preparation method thereof, comprising the following steps:
1) preparation of hydrophobic mesoporous silica spheres: dispersing 1g of octadecyl trimethyl ammonium bromide into 160mL of water, adding 3% of catalyst concentrated ammonia water, stirring for 20min, mixing 20mL of n-hexane and 3% of n-butyl orthosilicate (the concentration of a silicon source in the water), dropwise adding the mixture into the mixed solution at the speed of 0.5mL/min, stirring and reacting for 15h at the temperature of 30 ℃, centrifuging after the reaction is finished, washing with ethanol for 3 times, and drying to obtain a solid, namely a silicon dioxide ball serving as a core; and (3) putting the silicon dioxide spheres in a muffle furnace, calcining for 4 hours at the temperature of 550 ℃ to remove the surface template agent, and forming the mesoporous silicon dioxide spheres. Dispersing 1g of mesoporous silica spheres into 20mL of toluene, respectively dropwise adding 80 mu L of triethylamine and 2.5mmol of trimethoxy (3,3, 3-trifluoropropyl) silane, reacting for 24h at 120 ℃ in a high-pressure reaction kettle, and synthesizing hydrophobic mesoporous silica spheres, wherein the water contact angle is 150 degrees;
2) preparing the mesoporous silica spheres with the amphiphilic core-shell structure: dispersing 1g of hydrophobic mesoporous silica spheres and 0.5g of octadecyl trimethyl ammonium bromide in 180mL of water, adding 2% of concentrated ammonia water, mixing 20mL of n-hexane and 3% of tetraethoxysilane (the concentration of a silicon source in the water), dropwise adding the mixture into the mixed solution at the speed of 0.5mL/min, stirring and reacting at the temperature of 20 ℃ for 12 hours, centrifuging after the reaction is finished, washing with ethanol for 3 times, drying, putting the obtained solid, namely the core-shell structured silica spheres into a muffle furnace, calcining at the temperature of 550 ℃ for 4 hours, and removing a surface template agent to form the core-shell structured mesoporous silica spheres. Dispersing 1g of mesoporous silica spheres with a core-shell structure into 20mL of toluene, respectively dropwise adding 100 mu L of triethylamine and 2.5mmol of mercaptopropyltriethoxysilane, reacting for 15h at 120 ℃ in a high-pressure reaction kettle, and synthesizing amphiphilic mesoporous silica spheres with a core-shell structure; the water contact angle of the outer hydrophilic mesoporous silica spheres is 70 degrees; the diameter of the obtained amphiphilic mesoporous nano silicon spheres is 360nm, and the specific surface area is 650m2The pore size of the mesopores is 30 nm.
3) Preparing an amphiphilic mesoporous nano silicon sphere interface immobilized enzyme: dispersing 120mg of Candida rugosa lipase into 10mL of phosphate buffer solution with the pH value of 7.0 and the concentration of 20mmol of the lipase to prepare enzyme solution; carrying out ultrasonic emulsification on 1mL of n-heptane, 1g of amphiphilic mesoporous nano silicon spheres and 10mL of enzyme solution to form Pickering emulsion, meanwhile, placing the Pickering emulsion in a constant-temperature oscillator at 30 ℃ for interface immobilization, wherein the reaction time is 3h, after the reaction is finished, cleaning the buffer solution for 3 times, carrying out centrifugal separation, and carrying out freeze drying to obtain the amphiphilic mesoporous nano silicon sphere interface immobilized enzyme.
The application comprises the following steps: 3.0g of fish oil, 1.5g of phosphoric acid buffer solution (0.1mol/L, pH 7) and 0.025g of amphiphilic mesoporous nano silicon sphere interface immobilized enzyme are put into a thermostatic water bath at 37 ℃ for reaction for 4 hours, and the stirring speed is 300 r/min. The mass fraction of glyceride in the oil phase of the hydrolysate obtained is 58.21%, and the mass fraction of free fatty acid is 41.79%. After the reaction, the immobilized enzyme is removed by centrifugation. After the immobilized enzyme is recovered and washed by n-hexane for 3 times, the reaction is repeated for 10 times, the mass fraction of glyceride is more than 50%, and the mass fraction of free fatty acid is more than 40%.
In conclusion, the amphiphilic mesoporous nano silicon sphere interface immobilized enzyme is prepared by interface adsorption, and combines the special structure of the core-shell interlayer, so that a proper space is provided for the immobilization of the enzyme, the damage and leakage of the enzyme caused by friction and shearing force in the reaction process can be effectively avoided, the stability of the enzyme can be greatly improved, in addition, the special structure of hydrophilic shell surface and hydrophobic core surface is beneficial to the opening of a lipase 'cover' structure and the exposure of an enzyme active site, so that a proper microenvironment is provided for the lipase, and the catalytic performance of the enzyme is improved. The amphiphilic mesoporous nano silicon sphere interface immobilized enzyme can effectively improve the immobilization stability and catalytic activity of the enzyme, has good reusability and has wide application prospect.
All the raw materials (such as specific raw materials of lipase) listed in the invention, the values of the upper limit and the lower limit and the interval of the raw materials of the invention, and the values of the upper limit and the lower limit and the interval of the process parameters (such as temperature, time and the like) can realize the invention, and the examples are not listed. The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, many modifications and changes can be made without departing from the inventive concept of the present invention, and these modifications and changes are within the protection scope of the present invention.

Claims (10)

1. An amphiphilic mesoporous nano silicon sphere is characterized in that: the amphiphilic mesoporous nano silicon spheres are of a core-shell structure and comprise inner hydrophobic mesoporous silicon dioxide spheres and outer hydrophilic mesoporous silicon dioxide spheres.
2. The amphiphilic mesoporous nano-silica sphere according to claim 1, characterized in that: particle diameter of100 to 400nm, and a specific surface area of 300 to 800m2The pore size of the mesopores is 3-30 nm.
3. The amphiphilic mesoporous nano-silica sphere according to claim 1, characterized in that: the water contact angle of the hydrophobic mesoporous silica spheres is 90-150 degrees, and the water contact angle of the hydrophilic mesoporous silica spheres is 10-70 degrees.
4. The preparation method of the amphiphilic mesoporous nano-silica spheres of claims 1-3, which is characterized in that: the method comprises the following steps:
1) preparation of hydrophobic mesoporous silica spheres: dispersing a surfactant into water, adding a catalyst, normal hexane and a silicon source, stirring for reaction, and centrifugally washing and drying to obtain silicon dioxide spheres; removing the surface template agent to form mesoporous silica spheres, and performing surface hydrophobic modification on the mesoporous silica spheres by using a silane coupling agent in a toluene environment and using triethylamine as a catalyst to synthesize hydrophobic mesoporous silica spheres;
2) preparation of amphiphilic mesoporous silica spheres: dispersing hydrophobic mesoporous silica spheres in a mixture of water and a surfactant, adding a catalyst, normal hexane and a silicon source, stirring for reaction, and centrifugally washing and drying to obtain core-shell structured silica spheres; removing the surface template agent to form mesoporous silica spheres with core-shell structures, and performing surface hydrophilic modification on the mesoporous silica spheres by using a silane coupling agent in a toluene environment and using triethylamine as a catalyst to synthesize the amphiphilic mesoporous silica spheres with core-shell structures, namely the amphiphilic mesoporous nano-silica spheres.
5. The preparation method of the amphiphilic mesoporous nano silicon spheres of claim 4, which is characterized in that: in the steps 1) and 2), the stirring reaction is carried out at the temperature of 20-40 ℃ for 12-30 h;
the surfactant is one or a mixture of a plurality of dodecyl trimethyl ammonium bromide, tetradecyl trimethyl ammonium bromide, hexadecyl trimethyl ammonium bromide and octadecyl trimethyl ammonium bromide in any proportion; in the step 1), the proportion of the surfactant to the n-hexane is 1g (10-30) mL;
the catalyst is any one of sodium hydroxide, potassium hydroxide and concentrated ammonia water, and the addition amount of the catalyst is 1-5% (v/v) of the water;
the silicon source is one or a mixture of more of ethyl orthosilicate, propyl orthosilicate, isopropyl orthosilicate and butyl orthosilicate in any proportion, the concentration of the silicon source in water is 0.5-3% (v/v), and after the n-hexane and the silicon source are uniformly mixed, the n-hexane and the silicon source are dripped into a reaction system at the speed of 0.5-3 mL/min;
in the step 2), the proportion of water, hydrophobic mesoporous silica spheres and surfactant is (50-200) mL: 1g: (0.2-0.6) g.
6. The preparation method of the amphiphilic mesoporous nano silicon spheres of claim 4, which is characterized in that: in the method for surface modification by using the silane coupling agent in the step 1), the ratio of the silane coupling agent to triethylamine to toluene to mesoporous silica spheres is (0.5-2.5) mmol: (20-100) μ L: 20ml:1g, the reaction temperature is 100-140 ℃, and the reaction time is 12-30 h;
the silane coupling agent in the step 1) is any one or a mixture of several of n-octyltrimethoxysilane, n-octyltriethoxysilane, dodecyltrimethoxysilane, dodecyltriethoxysilane, hexadecyltrimethoxysilane, hexadecyltriethoxysilane and trimethoxy (3,3, 3-trifluoropropyl) silane in any proportion;
the method for modifying the surface hydrophilicity in the step 2) comprises the following steps: carrying out surface modification by using a silane coupling agent different from the silane coupling agent in the step 1), wherein the ratio of the silane coupling agent to triethylamine to toluene to mesoporous silica spheres is (0.1-2.5) mmol: (20-100) μ L: 20ml:1g of a compound; the reaction temperature is 100-140 ℃, and the reaction time is 12-30 h;
the silane coupling agent in the step 2) is any one or a mixture of a plurality of 3-aminopropyl trimethoxy silane and mercaptopropyl triethoxy silane according to any proportion.
7. An amphiphilic mesoporous nano silicon sphere interface immobilized enzyme is characterized in that: comprising an enzyme and a carrier for immobilizing the enzyme, wherein the carrier is the amphiphilic mesoporous nano silicon sphere according to any one of claims 1 to 3, and the enzyme comprises any one of lipase, phospholipase, pancreatin and hydrolase.
8. The preparation method of the amphiphilic mesoporous nano silicon sphere interface immobilized enzyme according to claim 7, which is characterized in that: dispersing enzyme into a buffer solution to prepare an enzyme solution; emulsifying an organic solvent, amphiphilic mesoporous nano silicon spheres and an enzyme solution to form pickering emulsion, and carrying out interface immobilization to obtain amphiphilic mesoporous nano silicon sphere interface immobilized enzyme; the organic solvent is any one of n-hexane, n-heptane, isooctane and acetone.
9. The preparation method of the amphiphilic mesoporous nano silicon sphere interface immobilized enzyme according to claim 8, which is characterized in that: the emulsification methods include but are not limited to water bath ultrasound, contact probe ultrasound, hand-held homogenizers, vortexers, homogenizers.
10. The preparation method of the amphiphilic mesoporous nano silicon sphere interface immobilized enzyme according to claim 8, which is characterized in that: the mass ratio of the amphiphilic mesoporous silica spheres to the enzyme to the organic solvent is (0.2-1) g: (120-450) mg: (1-15) mL; forming a Pickering emulsion immobilization system with the particle size of 10-200 mu m; the enzyme immobilization temperature is 4-30 ℃, and the time is 0.1-3 h.
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