CN111621494A - Preparation method of magnetic immobilized lipase - Google Patents
Preparation method of magnetic immobilized lipase Download PDFInfo
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- CN111621494A CN111621494A CN202010646315.9A CN202010646315A CN111621494A CN 111621494 A CN111621494 A CN 111621494A CN 202010646315 A CN202010646315 A CN 202010646315A CN 111621494 A CN111621494 A CN 111621494A
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- methyltriethoxysilane
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
Abstract
The invention discloses a preparation method of magnetic immobilized lipase, which comprises the steps of hydrolyzing a mixture of ethyl orthosilicate and methyltriethoxysilane to obtain transparent sol at normal temperature by using dilute hydrochloric acid as a catalyst, wherein the mass ratio of the ethyl orthosilicate to the methyltriethoxysilane in the mixture is 1: 2-5: 1; cooling the transparent sol to room temperature, adding water while stirring, and then pumping out to remove a byproduct ethanol generated in the reaction system; adding magnetic nano Fe into the sol obtained after the treatment of the step S23O4And (3) adjusting the pH value of the system to 5-7 by using a dilute alkali solution under stirring, adding a lipase solution, stirring until gel is formed, coarsely crushing the gel, drying the gel in an oven at the temperature of 20-40 ℃ for 1-6 hours, finely grinding the dried gel, sieving the ground gel by using a sieve, and bagging the ground gel for later use. The activity of the finally obtained immobilized lipase product can reach more than 80% of that of free lipase, the immobilized lipase product can be repeatedly used for more than ten times to keep stable activity, and the preparation processSimple and environmentally friendly.
Description
Technical Field
The invention belongs to the technical field of new biological materials, and particularly relates to a preparation method of magnetic immobilized lipase.
Background
The lipase as a biocatalyst is an enzyme that can catalyze organic reactions such as ester hydrolysis, alcoholysis, lipid synthesis, or ester exchange at an oil-water interface. Compared with a chemical catalyst, the lipase has the advantages of mild reaction conditions, low energy consumption, high product conversion rate, less side reactions, environmental friendliness and the like, and is widely applied to the fields of agriculture, food, medicine, biodiesel and the like. Although lipases have wide application potential in industry, most lipases are very sensitive to the environment, have the problems of short life span, insolubility of free enzymes in organic phase, easy caking of reaction, difficult separation of products from enzymes, and high price, and the like, and greatly limit the application. Therefore, the enzyme is immobilized by an immobilization technique, so that the defect of free enzyme is overcome, and the problems can be effectively solved.
Patent CN104498472A discloses a magnetic immobilized cellobiase nano mesoporous material and a preparation method thereof, the method hydrolyzes TEOS with dilute acid at normal temperature to form transparent sol, extracts ethanol from the transparent sol, adds template solution, cellobiase and magnetic nano particles into the sol, adjusts the pH of the system to form gel, and dries the gel in silica gel to prepare the magnetic immobilized cellobiase nano mesoporous material. The method has simple preparation process, adopts the mesoporous silica carrier prepared by the non-surfactant to fix the biological enzyme, and has the characteristics of high efficiency, stability and reutilization.
However, the method disclosed in CN104498472A still has a problem in immobilizing lipase:
1. the method for preparing the gel cannot obtain immobilized enzyme with good lipophilicity;
2. the method for preparing the gel is usually carried out under the condition of pH 3-5 meta-acid, and the optimal activity of the lipase is influenced in the preparation process, so that unnecessary activity loss is caused;
3. the immobilized material needs to be embedded and dried by silica gel, which causes inconvenience to industrial production.
Disclosure of Invention
Aiming at the problems that in the prior art, lipophilicity is poor, the pH value of gel is too low to influence the lipase activity, the prepared immobilized carrier is insufficient in relative toughness and high in brittleness, the loss of grinding and screening at the later stage is large, the yield of the resultant product is low, the drying process is not beneficial to industrial production and the like, the application provides a preparation method of magnetic immobilized lipase to solve the existing problems.
In a first aspect, embodiments of the present application provide a method for preparing a magnetically immobilized lipase, comprising the steps of:
s1: hydrolyzing a mixture of ethyl orthosilicate and methyltriethoxysilane at normal temperature by using dilute hydrochloric acid as a catalyst to obtain transparent sol, wherein the mass ratio of the ethyl orthosilicate to the methyltriethoxysilane in the mixture is 1: 2-5: 1;
s2: cooling the transparent sol to room temperature, adding water while stirring, wherein the mass ratio of the water to the mixture of ethyl orthosilicate and methyltriethoxysilane is 1: 4-0.5: 1, and then removing a byproduct ethanol generated in a reaction system by pumping;
s3: adding magnetic nano Fe into the sol obtained after the treatment of the step S23O4Particles of magnetic nano Fe3O4The mass ratio of the particles to the mixture of the ethyl orthosilicate and the methyltriethoxysilane is 1: 32-6: 32, the pH of the system is adjusted to be 5-7 by using a dilute alkali solution under stirring, a lipase solution is added and stirred until gel is formed, and the mass ratio of the lipase to the mixture of the ethyl orthosilicate and the methyltriethoxysilane is 1:32-10: 32; and
s4: and (3) after the gel is coarsely crushed, placing the gel in an oven at the temperature of 20-40 ℃ for drying for 1-6 h, and finely grinding the dried gel, sieving and bagging the gel for later use.
In some embodiments, the mixture of tetraethoxysilane and methyltriethoxysilane is stirred vigorously for 5-30 min after adding dilute hydrochloric acid in step S1. Adding dilute hydrochloric acid as catalyst into the mixture of ethyl orthosilicate and methyltriethoxysilane, and stirring vigorously to make the hydrolysis reaction fully proceed.
In some embodiments, the mass ratio of the dilute hydrochloric acid to the mixture of tetraethoxysilane and methyltriethoxysilane in step S1 is 5:32 to 13: 32. The mass ratio of the dilute hydrochloric acid to the mixture is within the range, so that the hydrolysis reaction speed can be controlled, and the reaction is not too fast or too slow.
In some embodiments, the concentration of the dilute hydrochloric acid in step S1 is 0.1-0.25 mol/L. Controlling the catalyst concentration also ensures that the reaction proceeds at a suitable reaction rate.
In some embodiments, the ethanol is removed in step S2 by using a high vacuum system and heating in a water bath to 35-55 ℃ so that the content of ethanol in the reaction system is less than 6%. The existence of a large amount of ethanol in the system can influence the activity of the immobilized lipase, and the immobilized lipase can not be influenced after the ethanol is extracted by means of vacuum and the like to ensure that the ethanol content is less than 6 percent.
In some embodiments, the dilute alkaline solution in step S3 is NH3·H2O, NaOH solution or KOH solution.
In some embodiments, the NaOH solution is 0.4 to 0.8 mol/L.
In a second aspect, the present application also provides a magnetically immobilized lipase obtained by the preparation method of the first aspect.
The invention provides a preparation method of magnetic immobilized lipase, the activity of the product of the magnetic immobilized lipase prepared by the method can reach more than 80% of the activity of free lipase, and the product can be repeatedly used for more than ten times to keep stable activity. The immobilized material can be recycled through the internal magnetic nanoparticles, and the operability is strong; compared with free lipase, the lipase fixed by the method has wider application temperature, pH value and the like and wider environmental adaptation range. The invention performs the synthesis reaction at normal temperature and normal pressure, the system is basically close to neutral environment, the process is simple and environment-friendly, the condition is mild, and the method is suitable for industrialization. The magnetic immobilized enzyme prepared by the invention has better lipophilicity and is convenient for subsequent enzymolysis in oil. The invention also establishes that the lipase is immobilized under a proper pH value, the toughness of the material is increased by adjusting the process, the drying process is simplified, and the practical problems in industrialization are solved.
Drawings
The accompanying drawings are included to provide a further understanding of the embodiments and are incorporated in and constitute a part of this specification. The drawings illustrate embodiments and together with the description serve to explain the principles of the invention. Other embodiments and many of the intended advantages of embodiments will be readily appreciated as they become better understood by reference to the following detailed description. The elements of the drawings are not necessarily to scale relative to each other. Like reference numerals designate corresponding similar parts.
FIG. 1 is a process flow diagram of a method for preparing a magnetically immobilized lipase of the present invention;
FIG. 2 is a microscope photograph showing the lipophilic results of the gel obtained by the method of preparing a magnetically immobilized lipase of the present invention without adding MTES;
FIG. 3 is a microscope photograph showing lipophilic results of gels obtained by adding TEOS and MTES with the mass of 10g and 22g, respectively, in the preparation method of the magnetic immobilized lipase of the present invention;
FIG. 4 is a graph showing the comparison of the enzyme activity of the immobilized lipase prepared by adding TEOS and MTES with the mass of 10g and 22g, respectively, with that of the free lipase at different temperatures according to the preparation method of the magnetic immobilized lipase of the present invention;
FIG. 5 is a graph showing the recycling rates of immobilized lipases prepared by adding TEOS and MTES at mass of 10g and 22g, respectively, in the method for preparing a magnetic immobilized lipase of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the present invention will be described in further detail with reference to the accompanying drawings, and it is apparent that the described embodiments are only a part of the embodiments of the present invention, not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring now to FIG. 1, the present invention will be described in detail, and in one embodiment, as shown in FIG. 1, a method for preparing a magnetically immobilized lipase is provided, comprising the steps of:
s1: hydrolyzing a mixture of ethyl orthosilicate and methyltriethoxysilane at normal temperature by using dilute hydrochloric acid as a catalyst to obtain transparent sol, wherein the mass ratio of the ethyl orthosilicate to the methyltriethoxysilane in the mixture is 2: 1-5: 1;
s2: cooling the transparent sol to room temperature, adding water while stirring, wherein the mass ratio of the water to the mixture of ethyl orthosilicate and methyltriethoxysilane is 1: 4-0.5: 1, and then removing a byproduct ethanol generated in a reaction system by pumping;
s3: adding magnetic nano Fe into the sol obtained after the treatment of the step S23O4Particles of magnetic nano Fe3O4The mass ratio of the particles to the mixture of the ethyl orthosilicate and the methyltriethoxysilane is 1: 32-6: 32, the pH of the system is adjusted to be 5-7 by using a dilute alkali solution under stirring, a lipase solution is added and stirred until gel is formed, and the mass ratio of the lipase to the mixture of the ethyl orthosilicate and the methyltriethoxysilane is 1:32-10: 32; and
s4: and (3) after the gel is coarsely crushed, placing the gel in an oven at the temperature of 20-40 ℃ for drying for 1-6 h, and finely grinding the dried gel, sieving and bagging the gel for later use.
In a specific embodiment, in step S1, dilute hydrochloric acid is added to the mixture of tetraethoxysilane and methyltriethoxysilane, followed by vigorous stirring for 5-30 min. Wherein the ethyl orthosilicate is TEOS, and the methyltriethoxysilane is MTES. Adding dilute hydrochloric acid as catalyst into the mixture of ethyl orthosilicate and methyltriethoxysilane, and stirring vigorously to make the hydrolysis reaction fully proceed. The mass ratio of the dilute hydrochloric acid to the mixture of the ethyl orthosilicate and the methyltriethoxysilane is 5: 32-13: 32. The concentration of the dilute hydrochloric acid is 0.1-0.25 mol/L. The mass ratio of the dilute hydrochloric acid to the mixture can control the hydrolysis reaction speed within the range, the reaction cannot be too fast or too slow, and the reaction can be carried out at a proper reaction speed by controlling the concentration of the dilute hydrochloric acid.
In a specific embodiment, in step S2, a high vacuum system is used and the reaction system is heated in a water bath to 35 to 55 ℃ to remove ethanol by pumping, so that the content of ethanol in the reaction system is less than 6%. The existence of a large amount of ethanol in the system can influence the activity of the immobilized lipase, and the immobilized lipase can not be influenced after the ethanol is extracted by means of vacuum and the like to ensure that the ethanol content is less than 6 percent.
In a specific embodiment, the diluted alkaline solution in step S3 is NH3·H2O, NaOH solution or KOH solution. In a preferred embodiment, the NaOH solution is 0.4-0.8 mol/L.
The activities of the magnetic immobilized lipases prepared in the following examples were tested using the following methods.
The activity test method comprises the following steps: adopts a pNPP method, and the substrate is p-nitrophenyl palmitate. Mixing 9ml phosphate buffer (pH 8.0) containing 0.4% Triton X-100 with 1ml 8.89mM p-nitrophenylpalmitate-isopropanol solution, preheating for 5min, separating 2.9ml and 0.1ml lipase solution diluted appropriately, reacting at 37 deg.C for 15min, and measuring the absorbance at 420 nm. An equal amount of boiling inactivated enzyme solution was used as a blank control. The enzyme amount required for producing 1 mu mol p-nitrobenzoic acid by hydrolyzing the catalytic substrate within 1min is 1 enzyme activity unit (U).
Under proper conditions, the lipase can hydrolyze a substrate into a yellow substance p-nitrophenol, the absorption is at 405-420nm, and a standard curve is determined by measuring the content of the product p-nitrophenol: y is 0.08425+9.274X, R2 is 0.9952, where Y is the absorbance of the sample and X is the content of p-nitrophenol.
And (3) measuring the activity of the immobilized enzyme, namely weighing a certain amount of immobilized enzyme to replace the free enzyme to be added into a reaction system, measuring according to a free enzyme measuring method, and taking a blank carrier as a blank reference.
Wherein n is the dilution factor, t is the reaction time (min), V is the total reaction volume (mL), and 1000 is the ratio of mmol to μmol.
Example 1
According to the preparation method of the magnetic immobilized lipase, the steps S1 and S2 are sol precursor formation: weighing TEOS and MTES with the mass of 6g and 26g, 16g and 16g, 22g and 10g, 26g and 6g and 28g and 4g respectively, adding 7g of 0.1mol/L diluted hydrochloric acid solution under stirring, wherein the mass ratio of the diluted hydrochloric acid solution to the mixture of TEOS and MTES is 7:32, after stirring vigorously for 20min, hydrolyzing the mixture of TEOS and MTES with diluted acid at normal temperature to form a clear transparent sol precursor, and adding 20g of water under stirring. Extracting ethanol generated in the hydrolysis process in high vacuum, raising the temperature of the water bath to 37 ℃, and performing suction filtration until the content of the ethanol in the sol is less than 6%.
Proceeding to step S3 gel formation: adding magnetic nano Fe under stirring3O4Adjusting pH of the system to 5 with dilute alkali solution, adding lipase solution, and stirring to obtain gel. 5g of magnetic nano Fe is added3O4The mass ratio of the particles to the mixture of TEOS and MTES is 5:32, the dilute alkali solution is 0.4mol/L NaOH solution, and 1ml of lipase stock solution is added.
And finally, gel drying: after the gel was coarsely crushed, it was placed in an oven at 25 ℃ and dried for 3 hours. And finely grinding the dried gel, sieving with a 60-mesh sieve, and bagging for later use.
In the preparation process, TEOS and MTES are respectively hydrolyzed and condensed under the catalytic action of dilute hydrochloric acid to form sol, and then dilute alkali solution is used for adjusting the pH value of the system to form a gel material. The gel prepared by adjusting the mass ratio of TEOS to MTES can wrap the magnetic nano Fe3O4Particles and lipase, and can ensure that the lipase keeps good activity under the preparation process of the invention.
TABLE 1 Experimental results for different mass ratios of TEOS and MTES
As shown in figure 2, under the condition of not adding MTES, the gel material prepared by the method obviously forms an oil film in soybean oil, and forms a barrier to the later application in the field of biodiesel. As shown in fig. 3, in the case where MTES was added in example 1, TEOS and MTES were 10g and 22g, respectively, oil-free films were generated on the surface of the material, and dispersion was significant in soybean oil, so that gel materials having lipophilicity were prepared by the process of the present application. As shown in Table 1, the lipophilicity of the gel prepared by different mass ratios of TEOS and MTES is different from that of the gel prepared by different mass ratios of TEOS and MTES, and when the mass ratio of TEOS to MTES is less than 1:2, the lipophilicity of the prepared gel is good, but the gel is slowly formed and the elasticity is increased. When the mass ratio of TEOS to MTES is more than 5:1, the prepared gel has poor lipophilicity, easy cracking and high brittleness.
Example 2
On the basis of steps S1 and S2 of example 1, the formation of a gel proceeds to step S3: adding magnetic nano Fe under stirring3O4Adjusting system to different pH values of 4-8 with dilute alkali solution, adding lipase solution, and stirring to obtain gel. 5g of magnetic nano Fe is added3O4The mass ratio of the particles to the mixture of TEOS and MTES is 5:32, the diluted alkaline solution is 0.8mol/LKOH solution, and 3ml of lipase stock solution is added.
And finally, gel drying: after the gel was coarsely crushed, it was placed in an oven at 25 ℃ and dried for 3 hours. And finely grinding the dried gel, sieving with a 60-mesh sieve, and bagging for later use.
TABLE 2 Experimental results at different pH values in the gel step
As shown in Table 2, the pH value at the time of gelation can be increased to 5 to 7 in the range of 1:2 to 5:1 by mass of TEOS and MTES. When the pH value of the system is 5-7, the lipase has the optimal activity, so the optimal activity of the lipase is not influenced, unnecessary activity loss is caused, and the cost is reduced. In addition, the gel can be formed in a short time, and the gel is ensured to avoid the precipitation of magnetic particles and the full dispersion of enzyme substances in the system within a reasonable time. The pH value of the system in the gelling process can be enlarged from about 4 to about 7 by changing the mass ratio of TEOS to MTES, and the gelling can be completed within about 10min when the pH value is 5.6 when the mass ratio of TEOS to MTES is 10: 22. The results of the enzyme activity and the repeated recovery and utilization rate of the magnetic immobilized lipase prepared under the conditions are shown in fig. 4 and fig. 5, the activity of the product can reach more than 80% of the activity of free lipase, and the product can be repeatedly used for more than ten times to keep the activity stable; the immobilized material can be recycled through the internal magnetic nanoparticles, and the operability is strong; compared with free lipase, the lipase fixed by the method has higher temperature in use temperature, pH value and the like and wider environmental adaptation range. The invention performs the synthesis reaction at normal temperature and normal pressure, the system is basically close to neutral environment, the process is simple and environment-friendly, the condition is mild, and the method is suitable for industrialization.
In correspondence with the above-mentioned method for producing a magnetically immobilized lipase, a magnetically immobilized lipase obtained according to the above-mentioned production method is also proposed in examples of the present application. The activity of the product of the magnetic immobilized lipase prepared by the method can reach more than 80% of that of free lipase, and the product can be repeatedly used for more than ten times to keep stable activity. The immobilized material can be recycled through the internal magnetic nanoparticles, and the operability is strong; compared with free lipase, the lipase fixed by the method has wider application temperature, pH value and the like and wider environmental adaptation range. The invention performs the synthesis reaction at normal temperature and normal pressure, the system is basically close to neutral environment, the process is simple and environment-friendly, the condition is mild, and the method is suitable for industrialization. The invention also establishes that the lipase is immobilized under a proper pH value, the toughness of the material is increased by adjusting the process, the drying process is simplified, and the practical problems in industrialization are solved.
While the principles of the invention have been described in detail in connection with the preferred embodiments thereof, it will be understood by those skilled in the art that the foregoing embodiments are merely illustrative of exemplary implementations of the invention and are not limiting of the scope of the invention. The details of the embodiments are not to be interpreted as limiting the scope of the invention, and any obvious changes, such as equivalent alterations, simple substitutions and the like, based on the technical solution of the invention, can be interpreted without departing from the spirit and scope of the invention.
Claims (8)
1. A preparation method of magnetic immobilized lipase is characterized by comprising the following steps:
s1: hydrolyzing a mixture of ethyl orthosilicate and methyltriethoxysilane by taking dilute hydrochloric acid as a catalyst at normal temperature to obtain transparent sol, wherein the mass ratio of the ethyl orthosilicate to the methyltriethoxysilane in the mixture is 1: 2-5: 1;
s2: cooling the transparent sol to room temperature, adding water while stirring, wherein the mass ratio of the water to the mixture of the ethyl orthosilicate and the methyltriethoxysilane is 1: 4-0.5: 1, and then removing a byproduct ethanol generated in a reaction system by pumping;
s3: adding magnetic nano Fe into the sol obtained after the treatment of the step S23O4Particles of magnetic nano Fe3O4The mass ratio of the particles to the mixture of the ethyl orthosilicate and the methyltriethoxysilane is 1: 32-6: 32, the pH of a system is adjusted to be 5-7 by using a dilute alkali solution under stirring, a lipase solution is added and stirred until gel is formed, and the mass ratio of the lipase to the mixture of the ethyl orthosilicate and the methyltriethoxysilane is 1:32-10: 32; and
s4: and after the gel is coarsely crushed, placing the gel in an oven at the temperature of 20-40 ℃ for drying for 1-6 h, and finely grinding the dried gel, sieving and bagging the gel for later use.
2. The method of claim 1, wherein the diluted hydrochloric acid is added to the mixture of tetraethoxysilane and methyltriethoxysilane and the mixture is vigorously stirred for 5-30 min in step S1.
3. The method of claim 1, wherein the mass ratio of the dilute hydrochloric acid to the mixture of ethyl orthosilicate and methyltriethoxysilane in step S1 is 5: 32-13: 32.
4. The method of claim 1, wherein the concentration of the dilute hydrochloric acid in step S1 is 0.1-0.25 mol/L.
5. The method of claim 1, wherein the ethanol is removed by heating in a water bath at 35-55 ℃ under a high vacuum system in step S2, so that the ethanol content in the reaction system is less than 6%.
6. The method of claim 1, wherein the diluted alkaline solution in step S3 is NH3·H2O, NaOH solution or KOH solution.
7. The method of claim 6, wherein the NaOH solution is 0.4-0.8 mol/L.
8. A magnetically immobilized lipase obtained by the method according to any one of claims 1 to 7.
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