阅读说明：本技术 一种载酶干凝胶整体柱催化填料的制备方法 (Preparation method of enzyme-loaded xerogel monolithic column catalytic filler ) 是由 王洪海 岳文达 张淑玲 魏斯文 董凯 王中彪 于 2021-01-15 设计创作，主要内容包括：本发明为一种载酶干凝胶整体柱催化填料的制备方法。该方法通过溶胶-凝胶法包埋南极假丝酵母脂肪酶B,以有机硅为前驱体,经过水解、缩聚反应,利用圆柱型模具,常压干燥将其制备成圆柱型载酶干凝胶整体柱催化填料。本发明得到的载酶干凝胶整体柱催化填料中,干凝胶具有硅网络结构,酶包埋在其中,具有催化完整,表面无裂痕,机械强度高等优点。(The invention relates to a preparation method of an enzyme-loaded xerogel monolithic column catalytic filler. According to the method, candida antarctica lipase B is embedded by a sol-gel method, organic silicon is used as a precursor, and the cylindrical enzyme-loaded xerogel monolithic column catalytic filler is prepared by hydrolysis and polycondensation reactions and normal-pressure drying by using a cylindrical mold. In the monolithic column catalytic filler of the enzyme-loaded xerogel obtained by the invention, the xerogel has a silicon network structure, and the enzyme is embedded in the silicon network structure, so that the monolithic column catalytic filler has the advantages of complete catalysis, no crack on the surface, high mechanical strength and the like.)

1. A preparation method of a monolithic column catalytic filler of enzyme-supported xerogel is characterized by comprising the following steps:

(1) adding methyl orthosilicate and methyltrimethoxysilane into a solvent to obtain a solution A, putting the solution A into an ice-water bath, and standing for later use;

wherein the mass ratio of the methyl orthosilicate is as follows: methyltrimethoxysilane: the solvent is 1: 3.5-4: 5.6-6;

(2) mixing polyethylene glycol, sodium fluoride, enzyme solution and deionized water to obtain solution B; wherein, the mass ratio is that polyethylene glycol: sodium fluoride: enzyme solution: deionized water is 1: 3.2-3.6: 8.5-9.5: 14.5-15;

(3) mixing and stirring the solution A and the solution B in an ice-water bath to obtain enzyme-loaded wet gel; the volume ratio of the solution A to the solution B is 1.5-2.0: 1;

(4) injecting the enzyme-loaded wet gel obtained in the step (3) into a mold, covering a preservative film, and standing for 12-48 hours at room temperature;

(5) perforating the preservative film on one end of the die after standing at room temperature in the step (4), wherein the aperture ratio is 5-10%;

(6) putting the mold containing the wet gel loaded with the enzyme in the step (5) into an oven at the temperature of 25-40 ℃, and drying for 24-48 hours;

(7) and (4) taking the enzyme-loaded xerogel monolithic column obtained after drying in the step (6) out of the mold, and standing at room temperature for 24-48 hours to obtain the enzyme-loaded xerogel monolithic column catalytic filler biocatalyst for the transesterification reaction of ethyl acetate and n-butyl alcohol.

2. The process for preparing an enzyme-supported xerogel monolith catalytic filler according to claim 1, wherein the solvent in step (1) is methanol, ethanol or propanol, preferably methanol.

3. The process for preparing the enzyme-supported xerogel monolithic column catalytic filler according to claim 1, wherein the molecular weight of the polyethylene glycol in the step (2) is 400-600.

4. The process for preparing the enzyme-supported xerogel monolithic column catalytic filler according to claim 1, wherein the enzyme solution in step (2) is candida antarctica lipase B solution; the concentration is 3-10 wt%.

5. The method for preparing the enzyme-supported xerogel monolithic column catalytic packing as claimed in claim 1, wherein the mold is a plastic or glass round tube with an inner diameter of 1-1.5 cm, a thickness of 1-4 mm and a length of 1.5-2 cm.

6. Use of an enzyme-loaded xerogel monolithic catalytic filler prepared by the process of claim 1 for catalyzing the transesterification of ethyl acetate with n-butanol.

7. Use of an enzyme-loaded xerogel monolithic catalytic filler prepared according to claim 6 wherein the catalysis of the transesterification reaction of ethyl acetate with n-butanol comprises the steps of:

putting ethyl acetate and n-butyl alcohol into a kettle type reactor, heating a reaction solution to 65-75 ℃, putting an enzyme-loaded xerogel monolithic column catalytic filler into a load type stirring paddle, and reacting for 2-6 hours at a stirring speed of 200-300 revolutions per minute to obtain n-butyl acetate and ethanol;

wherein the mass ratio of ethyl acetate: n-butyl alcohol accounts for 2-5: 1; the mass ratio of n-butanol: the catalytic filler of the monolithic column of the enzyme-loaded xerogel is 2-10: 1.

8. Use of an enzyme-loaded xerogel monolithic catalytic filler prepared by the process of claim 7, characterized by the further steps of: and after the reaction is finished, taking out and separating out the enzyme-loaded xerogel monolithic column catalytic filler after the reaction, washing the enzyme-loaded xerogel monolithic column catalytic filler by using a phosphate buffer solution, drying the enzyme-loaded xerogel monolithic column catalytic filler for 2 to 4 hours at the temperature of 25 to 40 ℃, and regenerating to obtain the xerogel monolithic column catalytic filler.

Technical Field

The invention relates to a lipase-loaded CALB xerogel monolithic column catalytic filler biocatalyst with a silicon network structure and a preparation method and an application method thereof, in particular to an enzyme-loaded xerogel monolithic column catalytic filler biocatalyst for synthesizing n-butyl acetate by the transesterification reaction of ethyl acetate and n-butyl alcohol and a preparation method thereof.

Background

Butyl acetate is the most common solvent, can be used as an excellent organic solvent, is widely used in nitrocellulose varnish, is used as a solvent in the processing process of artificial leather, fabrics and plastics, is also used as an analytical reagent and an extraction agent in the perfume industry, is used for chromatographic analysis standard substances and solvents, is used for producing paints and coatings, and can also be used in the perfume industry. Therefore, the demand for the raw material butyl acetate is greatly increased, so that the research on the butyl acetate synthesis method becomes a hotspot of research.

The production process of n-butyl acetate includes two kinds of batch process and continuous process. Concentrated sulfuric acid is generally used as a catalyst in the synthesis process in industry, and the reaction method comprises two reaction methods, namely an esterification method and an ester exchange method. The batch method is to add acetic acid and butanol as reactants into the tower kettle, proportionally add concentrated sulfuric acid as a catalyst, and heat for esterification reaction. The excess sulfuric acid is removed through neutralization reaction, and the product n-butyl acetate is separated after washing and refining after dehydration. The method has the advantages of simple operation and the disadvantages that the used catalyst can damage the environment and severely corrode equipment, byproducts are generated in the reaction process, the yield is low, and the like, so the method is gradually eliminated by the market; currently, the research on the continuous method for preparing n-butyl acetate is more, but the catalyst in the continuous method is still strong acid, the requirement on equipment is higher, and the cost of the equipment is increased, so that the production of n-butyl acetate is not leap, and a new process method is urgently needed to change the situation.

The application of biocatalytic reactions in organic synthesis is becoming more and more widespread, because enzymes have high regioselectivity, stereoselectivity or enantioselectivity, and have become one of effective ways to obtain high value-added chemicals. However, in the case of actual industrial production, the enzyme has disadvantages such as high cost, low thermostability, and difficulty in recovery. The enzyme stability can be improved by immobilizing the enzyme, and the repeatability of the catalyst enzyme is increased.

The enzyme immobilization methods include adsorption, covalent bonding, entrapment, and crosslinking. The embedding method is the preferred immobilization method adopted in the development process of the enzyme catalytic reaction technology at present due to the simple operation process, mild operation conditions and difficult damage to the high-level structure and the active center conformation of the enzyme molecules. There are several examples of the integration of immobilized enzyme and catalytic reaction, for example, Tanztianwei et al (Biotechnology Advances,2010,28(5),628-34.) uses macroporous nonpolar resin NKA adsorption method to immobilize Candida lipase, the immobilization efficiency of resin NKA in n-heptane medium can reach 98.98%, and the conversion rate of catalyzing the transesterification reaction of soybean oil and methanol to synthesize biodiesel can reach 97.3%. Paiva et al (Biotechnology progress,2003,19(3):750-754.) show the first fully integrated setup of lipase-catalyzed transesterification in a laboratory-scale distillation column to maintain low temperatures of the heat-labile enzyme, applying a vacuum of kilopascals. Heating reactant ethyl butyrate and butanol at the bottom of the tower, and reacting in the inverted pear bulb by using immobilized lipase. The removal of the low-boiling alcohol improves the final yield of butyl butyrate. However, the degree of yield increase of the integrated process compared to the batch reaction is not shown. Furthermore, to a greater extent, the introduction of enzymes into the inverted pear bulbs leads to a lower accessibility of the substrate to the catalyst in the packed bed. Heils et al (Industrial & engineering chemistry research 2015,54(38), 9458-: dipping sol-gel methods and spray sol-gel methods. The dipping sol-gel method is that lipase is embedded in silica sol, the simple dipping sol-gel method is utilized to dry the gel on the surface of the filler at room temperature to form a catalytic coating, and the lipase catalyst embedded in the sol is coated on the surface of the filler to prepare the supported enzyme catalytic filler, however, a certain amount of catalyst is lost each time after the test of a repeatability test. Wierschem et al (Chemical engineering journal,2017,312, 106-. CN107115889A discloses an enzyme catalysis packing for reaction rectification and a coating method and application thereof. The enzyme-catalyzed filler is prepared by the following method, comprising the following steps: immersing the filler in the sol containing the biological enzyme for 20-30 seconds, and drying for 10-20 seconds after taking out; then immersing the substrate into the sol for 20-30 seconds again, taking out the substrate again, and drying the substrate for 10-20 seconds; repeating the immersion and drying for 8-15 times; finally, drying the filler until the weight is unchanged to obtain the enzyme catalytic filler attached with the biological enzyme coating; the enzyme-loaded filler obtained by the scheme has insufficient binding force between the enzyme and the filler, and the mass loss of the enzyme-loaded filler is large in the reaction process or the storage at room temperature.

As described above, the use of an immobilized enzyme heterogeneous catalyst in a catalytic reaction can improve the thermal stability and the reuse rate of the enzyme. However, the coated immobilized enzyme catalyst is easy to expose the active center of the enzyme, so that the enzyme is easy to inactivate, and in addition, the coating is easy to fall off, so that the reaction system is polluted, and the defects of complexity of the reaction system, high post-treatment difficulty, cost waste and the like are brought to the reaction process. Therefore, the development of a highly stable biocatalyst is of great importance.

Disclosure of Invention

The invention aims to provide a preparation method of an enzyme-loaded xerogel monolithic column catalytic filler aiming at the defects in the prior art. According to the method, candida antarctica lipase B is embedded by a sol-gel method, organic silicon is used as a precursor, and the cylindrical enzyme-loaded xerogel monolithic column catalytic filler is prepared by hydrolysis and polycondensation reactions and normal-pressure drying by using a cylindrical mold. In the monolithic column catalytic filler of the enzyme-loaded xerogel obtained by the invention, the xerogel has a silicon network structure, and the enzyme is embedded in the silicon network structure, so that the monolithic column catalytic filler has the advantages of complete catalysis, no crack on the surface, high mechanical strength and the like.

The technical solution of the invention is as follows:

a preparation method of an enzyme-supported xerogel monolithic column catalytic filler comprises the following steps:

(1) adding methyl orthosilicate and methyltrimethoxysilane into a solvent to obtain a solution A, putting the solution A into an ice-water bath, and standing for later use;

wherein the mass ratio of the methyl orthosilicate is as follows: methyltrimethoxysilane: the solvent is 1: 3.5-4: 5.6-6;

(2) mixing polyethylene glycol, sodium fluoride, enzyme solution and deionized water to obtain solution B; wherein, the mass ratio is that polyethylene glycol: sodium fluoride: enzyme solution: deionized water is 1: 3.2-3.6: 8.5-9.5: 14.5-15;

(3) mixing and stirring the solution A and the solution B in an ice-water bath to obtain enzyme-loaded wet gel; the volume ratio of the solution A to the solution B is 1.5-2.0: 1;

(4) injecting the enzyme-loaded wet gel obtained in the step (3) into a mold, covering a preservative film, and standing for 12-48 hours at room temperature;

(5) perforating the preservative film on one end of the mould after standing at room temperature in the step (4); the aperture ratio is 5 to 10 percent.

(6) And (3) putting the mold containing the enzyme-loaded wet gel in the step (5) into an oven at the temperature of 25-40 ℃, and drying for 24-48 hours.

(7) And (4) taking the enzyme-loaded xerogel monolithic column obtained after drying in the step (6) out of the mold, and standing at room temperature for 24-48 hours to obtain the enzyme-loaded xerogel monolithic column catalytic filler biocatalyst for the transesterification reaction of ethyl acetate and n-butyl alcohol.

The solvent in the step (1) is methanol, ethanol or propanol, preferably methanol.

The molecular weight of the polyethylene glycol in the step (2) is 400-600;

the enzyme solution in the step (2) is candida antarctica lipase B solution; the concentration is 3-10 wt%;

the mould is a plastic or glass round pipe, the inner diameter is 1-1.5 cm, the thickness is 1-4 mm, and the length is 1.5-2 cm.

The application of the enzyme-loaded xerogel monolithic column catalytic filler prepared by the method is used for catalyzing the ester exchange reaction of ethyl acetate and n-butyl alcohol.

The method for catalyzing the transesterification reaction of the ethyl acetate and the n-butanol comprises the following steps:

putting ethyl acetate and n-butyl alcohol into a kettle type reactor, heating a reaction solution to 65-75 ℃, putting an enzyme-loaded xerogel monolithic column catalytic filler into a load type stirring paddle, and reacting for 2-6 hours at a stirring speed of 200-300 revolutions per minute to obtain n-butyl acetate and ethanol;

wherein the mass ratio of ethyl acetate: n-butyl alcohol accounts for 2-5: 1; the mass ratio of n-butanol: the catalytic filler of the enzyme-loaded xerogel monolithic column is 2-10: 1;

also comprises the following steps: and after the reaction is finished, taking out and separating out the enzyme-loaded xerogel monolithic column catalytic filler after the reaction, washing the enzyme-loaded xerogel monolithic column catalytic filler by using a phosphate buffer solution, drying the enzyme-loaded xerogel monolithic column catalytic filler for 2 to 4 hours at the temperature of 25 to 40 ℃, and regenerating to obtain the xerogel monolithic column catalytic filler.

The invention has the substantive characteristics that:

in the prior art, a film is usually prepared by a sol-gel method, but the sol-gel method has the characteristics of serious shrinkage and large surface tension, and dried xerogel is easy to crack. As in the previous studies of the inventors, CN107115889a discloses an enzyme-catalyzed packing for reactive distillation and a coating method and application thereof. Coating enzyme sol-gel on the surface of a filler by adopting a sol-gel method, and drying to form an enzyme-loaded catalytic coating; CN 109837271A discloses that based on CN107115889A, enzyme-loaded sol-gel is sprayed on the surface of filler to make the coating uniform and stable. Since the 2 methods are all coating type biocatalysts, but the coating is unstable, the binding force with the surface of the filler is weak, the coating is easy to fall off, and the enzyme is easy to expose on the surface, expose active center and cause inactivation. The enzyme-loaded xerogel monolithic column catalytic filler is prepared by using a cylindrical mold through a casting method, is a catalytic filler, and solves the problems of unstable coating and enzyme inactivation.

The invention utilizes the cylindrical mould, and during the casting period, the special sealing treatment (sealing is carried out firstly, then certain air is exposed, and then the air is completely contacted) is carried out on the cylindrical mould, so that the volatilization of the solvent and the moisture can be controlled; the method mainly comprises 2 stages: gelation and aging; the first sealing is to make the sol react and gel, then exposing certain air is to control the solvent and water to slowly volatilize, which is beneficial to forming the silicon skeleton structure with high mechanical strength, and finally completely exposing the air is to remove the residual solvent and water, thereby preparing the enzyme-carrying xerogel monolithic column catalytic filler with high mechanical strength.

The invention has the beneficial effects that:

compared with the original method for synthesizing n-butyl acetate by using concentrated sulfuric acid as a catalyst, the enzyme-loaded xerogel monolithic column catalytic filler biocatalyst prepared by the invention has high activity and good selectivity; and the enzyme activity stability is high, the recovery is easy, and the enzyme can be repeatedly used. The production cost is reduced, and the harm to the environment is reduced.

1. Compared with the original method for synthesizing n-butyl acetate by using concentrated sulfuric acid as a catalyst, the enzyme-supported xerogel monolithic column catalytic filler biocatalyst prepared by the invention has high activity and high reaction rate (see example 10, the reaction time is 2.5 hours, and the conversion rate of n-butyl alcohol can reach 65.6%).

2. The catalytic filler of the enzyme-loaded xerogel monolithic column used in the invention can be recovered, has stable activity, can be repeatedly used, and reduces the production cost (see examples 4-9, the activity is maintained at 69.8% after being stored for more than 3 months at room temperature).

Drawings

FIG. 1 is a scanning electron microscope picture of the cross section of the enzyme-supported xerogel monolithic column catalytic filler obtained in example 1

FIG. 2 shows the catalytic packing of the enzyme-supported xerogel monolithic column prepared in example 1

Detailed Description

Example 1

(1) 0.58g of methyl orthosilicate and 2.08g of methyltrimethoxysilane are weighed out and mixed and dissolved in 3.39g of methanol.

(2) Putting the solution prepared in the step (1) into an ice water bath for later use.

(3) 0.14g of polyethylene glycol, 0.49g of NaF, 1.26g of an enzyme solution (Candida antarctica lipase B solution; concentration: 6 wt%) and 2.06g of deionized water were weighed, mixed and stirred uniformly.

(4) Slowly pouring the enzyme-containing mixed solution obtained in the step (3) into the mixed solution obtained in the step (1), and stirring in an ice-water bath for 3min to obtain the enzyme-loaded wet gel.

(5) Cutting a plastic circular tube with the inner diameter of 1cm and the thickness of 2mm into small sections with the length of 1.7cm, wherein the number of the small sections is 10, adhering one side of each small section after cutting on a plastic plate with the length of 5 multiplied by 6cm by glue, and drying the plastic plate in an oven at the temperature of 60 ℃ for 12 hours to obtain the cylindrical mold.

(6) And (4) transferring the enzyme-loaded wet gel obtained in the step (4) into the cylindrical mold obtained in the step (5), covering the mold with a preservative film, sealing, and standing at room temperature for 24 hours.

(7) After the standing process is finished, binding a plurality of small holes above the preservative film, wherein the hole ratio is 10%, enabling the small holes to be in contact with air, putting the die into a 40 ℃ oven for drying for 24 hours, after the drying process is finished, taking out the enzyme-loaded xerogel monolithic column from the die, and standing at room temperature for 24 hours to obtain the enzyme-loaded xerogel monolithic column catalytic filler.

The obtained enzyme-carrying xerogel monolithic column is characterized by a scanning electron microscope, the result is shown in figure 1, and the microsphere is SiO₂Particles in which enzyme molecules are embedded and interconnected to form a silicon network structure with a gap in the middle for a reaction channel.

Example 2

The difference between the embodiment and the embodiment 1 is that the opening rate of the pricked holes on the preservative film is replaced by 8 percent, the temperature of the mold put into the oven is replaced by 35 ℃, and other conditions are completely the same as those of the embodiment 1.

Example 3

The difference between the embodiment and the embodiment 1 is that the opening rate of the pricked holes on the preservative film is replaced by 6 percent, the temperature of the mold put into the oven is replaced by 25 ℃, and other conditions are completely the same as those of the embodiment 1.

Example 4

The enzyme activity and long-term stability of the prepared enzyme-carrying xerogel monolithic column catalytic filler in example 1 were tested as follows: 20mg of the enzyme-loaded xerogel powder was incubated in 3mL of phosphate buffer (50mM, pH 7.5) at 37 ℃ for 3min at 220rpm and 200. mu.L of p-nitrophenylpalmitate (5mg/mL) was added. Reacting 3mi in a constant-temperature water bath magnetic stirring tankAfter n, remove and add 1.5mL acetone quickly to stop the reaction. Finally, the reaction solution was detected with a spectrophotometer at 410 nm. All data experiments were tested in parallel 3 times. Specific enzyme activity (U.g)^-1): specific enzyme activity refers to the amount of enzyme required to catalyze the production of 1. mu. mol fatty acid from a substrate per minute.

Wherein C is the concentration of p-nitrophenol (calculated by substituting absorbance into standard) (mmol/ml); v is the volume (ml) of the reaction system; m is the amount (g) of immobilized enzyme; t is the action time (min).

Examples 5 to 9

The same test method as in example 4. The enzyme activity was measured every 2 weeks under the condition of storage at room temperature, except for the change time measurement. The results of the enzyme activity are shown in Table 1.

TABLE 1 Effect of storage time on enzymatic Activity of enzyme-Supported xerogel monolithic column catalytic Filler

	Enzyme activity (U/g)
		Example 4	476
Example 5	462
		Example 6	440
Example 7	378
		Example 8	356
Example 9	332

As can be seen from Table 1, after 5 cycles of tests, the enzyme-loaded xerogel monolithic catalytic filler can be stored at room temperature for more than 3 months, and compared with the initial enzyme activity of example 4, the enzyme-loaded xerogel monolithic catalytic filler still retains 69.8% of activity, and is completely suitable for practical operation.

Example 10

The prepared enzyme-carrying xerogel monolithic column catalytic filler is used for the ester exchange reaction of ethyl acetate and n-butyl alcohol.

Adding 50g of n-butyl alcohol and 119g of ethyl acetate into a glass reactor, heating the reactor to 70 ℃, then loading 5g of the catalytic filler of the enzyme-loaded xerogel monolithic column into a load type stirring paddle, placing the load type stirring paddle into the reactor for stirring reaction, sampling for the first time after 15 minutes from the beginning, sampling for every half hour at the later time until the balance is reached, analyzing by gas chromatography, and calculating the conversion rate without time period.

TABLE 2 Effect of time-free catalysis of transesterification reaction conversion of ethyl acetate and n-butanol on enzyme-loaded xerogel monolithic column

Example 11

The enzyme-supported xerogel monolithic catalytic filler is washed by phosphate buffer solution (pH 7.5) and then dried in an oven at 30 ℃ for 4 hours, and the obtained enzyme-supported xerogel monolithic catalytic filler is used for the transesterification reaction of ethyl acetate and n-butanol again.

Example 11 was repeated with regenerated catalyst packing and the experimental data obtained are shown in table 3:

TABLE 3 influence of the number of times of use of the catalytic packing of the enzyme-supported xerogel monolith on the conversion of n-butanol

Through the above embodiments, the preparation method is simple and has high repeatability; the enzyme-loaded xerogel monolithic column catalytic filler is a heterogeneous biocatalyst with high thermal stability, good activity and high selectivity, and the enzyme-loaded xerogel monolithic column catalytic filler prepared by the method is stored at room temperature for more than 3 months and also keeps 69.8 percent of activity. In addition, the method is applied to the ester exchange reaction of ethyl acetate and n-butanol, and the conversion rate reaches 65.6 percent in a batch reaction kettle; meanwhile, because of SiO₂The enzyme-loaded xerogel structure keeps stable after the reaction is finished, and inhibits the loss of active component enzyme, so that the biocatalyst keeps longer service life and can be repeatedly used. In addition, the enzyme-loaded xerogel monolithic column catalytic filler is used as a catalyst in the ester exchange reaction of ethyl acetate and n-butyl alcohol, and the defects of pollution, high post-treatment difficulty, high cost and the like of the catalyst used in the existing n-butyl acetate production process are overcome.

The invention is not the best known technology.