CN1908166A - Method of preparing high activity immobilization penicillin acylase hybridization carrier - Google Patents

Abstract

The invention discloses a method for preparing a highly active immobilized penicillin acylase hybrid carrier, comprising the following steps: uniformly mixing cyclohexane, polyethylene glycol octylphenyl ether and n-hexanol, adding ammonia water under stirring , be mixed with transparent and clear inverse microemulsion, add orthosilicate ethyl ester wherein after stirring 15min, add silylating agent after stirring 30min, the volume ratio _V of orthosilicate ethyl ester and silylating agent: V _{silylating reagent} = 0.5-9.5: 0.5-9.5, hydrolyzed and condensed for 22 hours, washed with acetone, and vacuum-dried at room temperature to obtain the high activity immobilized penicillin acylase hybrid carrier. The hybrid carrier prepared by the present invention is rich in organic groups, and the penicillin acylase can be covalently or adsorbed on the carrier by adopting a suitable activation loading method, thereby obtaining a higher immobilized enzyme than other carriers. activity, and has good operational stability and storage stability.

Description

A method for preparing highly active immobilized penicillin acylase hybrid carrier

technical field

The invention relates to a method for preparing a catalyst carrier, in particular to a method for preparing a highly active immobilized penicillin acylase hybrid carrier.

Background technique

Enzyme is a biological catalyst whose chemical essence is protein. Due to its high catalytic activity, strong selectivity, and mild reaction conditions, enzymes have been widely used in many fields such as biochemical industry, pharmaceutical chemical industry, food industry, and light industry. At present, thousands of enzymes have been discovered. Although water-soluble enzymes have many advantages that other catalysts cannot match, because water-soluble enzymes are produced according to the needs of organisms themselves, they are greatly limited in practical application. The main disadvantages are:

(1) Enzyme extraction and purification process is cumbersome and expensive;

(2) After the reaction, recycling and reuse has certain difficulties technically;

(3) Difficulty in product separation and purification, which increases production costs;

(4) It cannot be used in organic solvents, strong acids, strong alkalis or high temperatures, and its stability is poor, and some enzymes will lose their activity when used under mild conditions.

The immobilized enzyme produced after immobilizing the enzyme can overcome most of the above-mentioned shortcomings. The term "immobilized enzyme" was defined at the first Enzyme Engineering Conference held in Henniker, New Hampshire, USA in 1971 as: those that are confined to a specific area of a surface, retain their catalytic activity, and can be used repeatedly and continuously enzyme. Compared with water-soluble enzymes, immobilized enzymes have the following advantages:

(1) Repeated batch reaction and column packing continuous reaction can be carried out in a long period of time;

(2) It is very easy to separate the enzyme from the substrate and product;

(3) can improve the stability of enzyme;

(4) The reaction process can be strictly controlled;

(5) There is no enzyme residue in the product solution, which simplifies the purification process;

(6) Can improve product quality;

(7) The use efficiency of the enzyme is improved;

(8) There is basically no discharge of three wastes during the conversion process.

In view of the above advantages of immobilized enzymes and the development of immobilized enzyme technology in recent years, the large-scale industrial application of enzymes has been realized. Therefore, the study of enzyme immobilization has become an important direction of enzyme engineering research in recent years. So far, the preparation methods of immobilized enzymes can be roughly divided into the following four main methods: adsorption method, covalent binding method, cross-linking method and embedding method. The adsorption method is divided into physical adsorption method and ion adsorption method. The enzyme and the carrier are combined by ionic bonds and van der Waals force. Advantages such as less damage and less changes in the high-level structure of the enzyme. The covalent binding method is to bind the enzyme to the carrier with a covalent bond, first activate the related group on the surface of the carrier, and then couple with the related group in the enzyme molecule, or connect a bifunctional reagent on the surface of the carrier, and then When the enzyme is coupled, the immobilized enzyme prepared by this method is firmly combined with the carrier and is not easy to fall off, but the reaction conditions of this method are harsh, the operation is complicated, and the severe reaction conditions are likely to cause changes in the high-level structure of the enzyme protein and destroy part of the active center. It is often impossible to obtain immobilized enzymes with higher activity. The cross-linking method is to first adsorb the enzyme on an insoluble carrier, and then use a bifunctional or multifunctional reagent to make an intermolecular cross-link between the enzyme molecules to form a cross-linked network structure to immobilize the enzyme. This method also has a reaction Insufficient conditions such as severe conditions and loss of enzyme activity. The embedding method is to embed the enzyme in the microcapsule. The immobilized enzyme prepared by this method should not use macromolecular reactants (substrates), and the enzyme is easily inactivated. The reaction conditions must be cleverly designed. It is generally used to prepare immobilized cells. . The four methods have their own advantages and disadvantages. In industrial applications, in order to reduce the cost of immobilized enzymes, it is required that the prepared immobilized enzymes have good operational stability and can be used repeatedly. Based on the above four methods, it can be seen that the immobilized enzyme prepared by covalent method or cross-linking method has better operational stability, and the immobilized enzyme prepared by adsorption method has higher activity.

Penicillin acylase is a catalyst that catalyzes the cleavage of penicillin G potassium salt to produce 6-aminopenicillanic acid (6-APA). 6-APA is the key intermediate in the synthesis of semi-synthetic penicillins. Some side chains can be connected to the amino group of 6-APA to obtain highly efficient, broad-spectrum, and convenient semi-synthetic penicillins, such as ampicillin, trimethoprim, Amoxycillin and carbenicillin etc. Due to the wide clinical application of these semi-synthetic penicillins, the demand for 6-APA is increasing day by day. At present, the preparation methods of 6-APA mainly include chemical cleavage and enzymatic methods. Due to the problems of harsh reaction conditions, low yield, many side reactions and the discharge of three wastes in the chemical method, 6-APA is mainly produced by the enzymatic method in the world. However, if free enzymes are used to catalyze the hydrolysis reaction of penicillin, due to the weakness of free enzymes, they cannot compete with chemical methods economically. In recent years, with the development of enzyme immobilization technology, people can use immobilized penicillin acylase to hydrolyze penicillin G potassium salt to produce 6-APA. Because the immobilized penicillin acylase has the advantages of high stability, easy separation, and continuous operation, it is superior to chemical methods in terms of technology and economy. CN 1279287A invented a multi-component copolymer porous microbead as a carrier for immobilizing penicillin acylase, but the structure and properties of the polymer itself determine that it has the following weaknesses: control the pore structure of the polymer and the presence of functional groups on the surface The number and distribution are relatively difficult. Some functional groups are in the highly cross-linked network of polymers. Although the situation has improved after the solvent is swollen, it may still be difficult for the reactant molecules to approach, thereby reducing the surface of the carrier. Utilization rate; thermal stability, mechanical stability and solvent resistance of high polymer carrier are weak; it is difficult to dispose of high polymer carrier after waste. Aiming at the weakness of polymer carriers, some inorganic carriers have been developed and used, such as CN 1320688A, which uses MCM-41 as a carrier for immobilizing penicillin acylase, but the activity of the immobilized enzyme prepared is not high, and it is mainly combined by adsorption. Therefore, the operational stability of the immobilized enzyme is not good. In order to improve the stability of the immobilized enzyme, the surface of the inorganic carrier is usually modified and functional groups are grafted, but the carrier modified by this method has many disadvantages such as limited number of functional groups and uneven distribution. Therefore, in order to prepare an immobilized penicillin acylase with high activity and good stability, which can be used for industrialization, the following problems need to be solved: (1) the surface of the carrier should have an organic group that can covalently interact with the penicillin acylase, Ensure that the immobilized enzyme has good operational stability; (2) The specific surface area of the carrier should be large, so that more organic groups can be exposed on the surface; (3) The number and distribution of functional groups on the surface of the carrier should not only be (4) The carrier should have sufficient chemical stability and mechanical stability. During the chemical reaction (such as an enzyme-catalyzed reaction), the carrier surface group does not participate in the reaction, and the structure remains unchanged under the reaction conditions. (5) The particle size of the carrier should be appropriate to facilitate the separation of the product after the reaction.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for preparing a highly active immobilized penicillin acylase hybrid carrier. The hybrid carrier prepared by this method is rich in organic groups, adopts a suitable activation loading method, and successfully realizes the covalent immobilization and adsorption immobilization of penicillin acylase, which can obtain higher immobilization than other carriers. Enzyme activity, and has good operational stability and storage stability.

The invention provides a method for preparing a highly active immobilized penicillin acylase hybrid carrier, comprising the following steps: uniformly mixing cyclohexane, polyethylene glycol octylphenyl ether and n-hexanol, adding ammonia water under stirring , be mixed with transparent and clear inverse microemulsion, add orthosilicate ethyl ester wherein after stirring 15min, add silylating agent after stirring 30min, the volume ratio V of orthosilicate ethyl ester and silylating agent: V silylating reagent = 0.5-9.5: 0.5-9.5, after 22 hours of hydrolysis and condensation reaction, wash with acetone, and vacuum-dry at room temperature to obtain the high-activity immobilized penicillin acylase hybrid carrier. The silylating agent is selected from the group consisting of γ-aminopropyltriethoxysilane, γ-glycidyloxypropyl trimethyl (ethyl)oxysilane, γ-mercaptopropyl trimethyl (ethyl)oxysilane, phenyl trimethyl ( One of ethyl)oxysilane, vinyltrimethyl(ethyl)oxysilane or 4-trimethyl(ethyl)oxysilane butyronitrile. The carrier is directly adsorbed and immobilized with penicillin acylase, or the carrier is covalently bonded with glutaraldehyde preactivated carrier and penicillin acylase. Highly active penicillin acylase. For example: add a phosphate buffer solution with pH=8.0 to the carrier and mix it with diluted penicillin acylase solution, vibrate and fix it in a water bath shaker at 30°C for 24 hours, and then fully wash with deionized water to obtain the carrier Immobilized penicillin acylase. Add a phosphate buffer solution with pH=8.0 to the carrier, then add glutaraldehyde for activation for 2 h, and filter with suction; then add a phosphate buffer solution with a pH=8.0 to the carrier after suction filtration and mix it with the diluted penicillin acylase solution, Shake and fix in a water bath shaker at 30° C. for 24 hours, and then fully wash with deionized water to obtain carrier-immobilized penicillin acylase.

The carrier matrix prepared in the invention is inorganic silicon dioxide, the surface of which contains organic functional groups, and some silicon bonds connect the organic functional groups to form an organic-nonpolar hybrid carrier. The organic functional groups include aminopropyl, epoxy, mercaptopropyl, vinyl, phenyl and nitrile.

The carrier prepared by the invention can also be used as an immobilized carrier for trypsin, amylase, radish peroxidase, cytochrome C and lipase.

The invention provides a hybrid carrier with a unique structure that can be used for the immobilization of penicillin acylase and a preparation method. Using the rich organic groups of the carrier and adopting a suitable activation loading method, the acylation of penicillin is successfully realized The covalent immobilization and adsorption immobilization of enzymes can obtain higher activity than other immobilized enzymes, and it has good operation stability and storage stability. One of the advantages of the present invention is that the prepared carrier is an organic-nonpolar hybrid carrier in which silicon dioxide is used as a matrix, and aminopropyl groups are connected with some silicon bonds. The second advantage of the present invention is that the activity of the immobilized enzyme prepared by the carrier prepared by the present invention can be as high as 2214U/g. The third advantage of the present invention is that the immobilized penicillin acylase hybrid carrier is prepared in one step in the reverse microemulsion. The fourth advantage of the present invention is that when the configuration ratio of the inverse microemulsion, the ratio of ethyl orthosilicate and γ-aminopropyltriethoxysilane is changed within a certain range, the content of amino groups on the surface of the carrier can be affected. And specific surface area and other characteristics, thereby affecting the activity of immobilized enzymes. The fifth advantage of the present invention is that the surface of the prepared carrier has a certain amount of hydroxyl groups and aminopropyl groups, so it has strong hydrophilicity. The sixth advantage of the present invention is that the prepared carrier is a spherical particle with uniform size and an amorphous structure. Seventh advantage of the carrier of the present invention is that the prepared carrier has good thermal stability, acid resistance, solvent resistance and certain alkali resistance (except strong alkali). The eighth advantage of the present invention is that the surface of the prepared carrier is inert during the enzyme-catalyzed reaction. The ninth advantage of the present invention is that by changing the type of silylating reagent, a series of similar hybrid carriers can be synthesized, each containing different functional groups, such as epoxy, mercaptopropyl, vinyl, phenyl and nitrile groups.

Description of drawings

Figure 1 is the ²⁹ Si NMR characterization diagram of the carrier;

Figure 2 is a ¹³ C NMR characterization diagram of the carrier;

Fig. 3 is the SEM characterization figure of carrier;

Fig. 4 is a TEM characterization diagram of the carrier.

Detailed ways

The present invention is described in further detail below: a method for preparing a highly active immobilized penicillin acylase hybrid carrier, comprising the following steps: using an inverse microemulsion method to stir an organic phase, a surfactant and a cosurfactant Mix evenly under the pressure, add ammonia water, prepare a transparent and clear inverse microemulsion, add organic silicon source to it after stirring for 15 minutes, add silanization reagent after stirring for 30 minutes, wash with acetone after 22 hours of hydrolysis and condensation reaction, and vacuum dry at room temperature, namely The immobilized penicillin acylase hybrid carrier was obtained. Wherein the organic phase includes cyclohexane, the surfactant includes polyethylene glycol octyl phenyl ether, the co-surfactant includes n-hexanol, and the organic silicon source includes tetraethyl orthosilicate Fat, the silylating agent includes γ-aminopropyl triethoxysilane, γ-glycidyloxypropyl trimethyl (ethyl)oxysilane, γ-mercaptopropyl trimethyl (ethyl)oxysilane, phenyl Trimethyl(ethyl)oxysilane, vinyltrimethyl(ethyl)oxysilane or 4-trimethyl(ethyl)oxysilanebutyronitrile. The volume ratio of tetraethyl orthosilicate and the silylating agent V _{tetraethyl orthosilicate} : V _{silylating agent} = 0.5-9.5: 0.5-9.5. The carrier can be directly adsorbed and immobilized with penicillin acylase, or the carrier can be covalently bonded with glutaraldehyde preactivated carrier and penicillin acylase, that is, immobilized by covalent means, and the immobilized carrier can be obtained. Highly active penicillin acylase. For example: add a phosphate buffer solution of pH=8.0 to the carrier and mix it with diluted penicillin acylase solution, vibrate and fix it in a water bath shaker at 30°C for 24 hours, and then fully wash with deionized water to obtain the Carrier-immobilized penicillin acylase. It is also possible to add a phosphate buffer solution with a pH of 8.0 to the carrier, then add glutaraldehyde to activate it for 2 hours, and then filter with suction; add a phosphate buffer solution with a pH of 8.0 to the carrier after the suction filter and mix with the diluted penicillin acylase solution Mix, shake and fix in a water bath shaker at 30° C. for 24 hours, and wash thoroughly with deionized water to obtain carrier-immobilized penicillin acylase. The carrier matrix prepared in the invention is inorganic silicon dioxide, the surface of which contains organic functional groups, and some silicon bonds connect the organic functional groups to form an organic-nonpolar hybrid carrier. The organic functional groups include aminopropyl, epoxy, mercaptopropyl, vinyl, phenyl and nitrile. The carrier prepared by the invention can also be used as an immobilized carrier for trypsin, amylase, radish peroxidase, cytochrome C and lipase. The activity of the immobilized enzyme in the present invention is determined by alkali titration. The shape of the obtained carrier is a uniformly distributed spherical body, the particle size is about 100nm, it belongs to an amorphous structure, and there are a large number of amino groups on the surface of the carrier.

Example 1

The inverse microemulsion was prepared according to the ratio of 24mL cyclohexane: 9.4mL Triton X-100: 6mL n-hexanol: 12mL ammonia water, and the carrier was prepared under the condition that the volume of TEOS and APTES were both 6mL. Weigh 0.5g of this carrier to prepare immobilized enzymes by adsorption method and covalent method, and the activities of the immobilized enzymes obtained are 1046U/g and 1616U/g respectively. After repeating the operation three times, the activities retain 59% and 74%.

Example 2

According to the ratio of 24mL cyclohexane: 12mL Triton X-100: 12mL n-hexanol: 10mL ammonia water, the reverse phase microemulsion was prepared, and the carrier was prepared under the condition that the volume of TEOS was 4mL and the volume of APTES was 6mL. The amino group content of the obtained carrier was 5.52 mmol/g, the specific surface area is 7m ² /g. Use this carrier to covalently immobilize penicillin acylase, the activity of the obtained immobilized enzyme is 1478U/g, after repeated operations 5 times, the activity remains 94%, and after storage at 4°C for 48 days, the activity of the immobilized enzyme remains 89% .

Example 3

According to the ratio of 24mL cyclohexane: 12mL Triton X-100: 12mL n-hexanol: 10mL ammonia water, the reverse phase microemulsion was prepared, and the carrier was prepared under the condition that the volume of TEOS was 0.5mL and the volume of APTES was 9.5mL. Penicillin acylase was covalently immobilized, and the activity of the immobilized enzyme was 872U/g.

Example 4

According to the ratio of 24mL cyclohexane : 12mL Triton X-100 : 12mL n-hexanol : 10mL ammonia water, the reverse phase microemulsion was prepared, and the volume of TEOS was 9.5mL and the volume of γ-mercaptopropyltriethoxysilane was 0.5mL. The carrier is prepared under the conditions, and the carrier is used to covalently immobilize penicillin acylase, and the activity of the obtained immobilized enzyme is 623U/g.

Example 5

According to the ratio of 24mL cyclohexane: 12mL Triton X-100: 12mL n-hexanol: 10mL ammonia water, the reverse phase microemulsion was prepared, and the carrier was prepared under the condition that the volumes of TEOS and APTES were respectively 5mL. The amino group content of the obtained carrier was 4.84mmol/ g, and the specific surface area is 23m ² /g. Using this carrier to covalently immobilize penicillin acylase, the activity of the obtained immobilized enzyme is as high as 2214U/g, and the activity of the immobilized enzyme remains 96% after repeated operations for 5 times, and after storage at 4°C for 48 days, the activity of the immobilized enzyme remains 98% . The prepared carrier was characterized by NMR, SEM and TEM, and the results are shown in Figure 1, Figure 2, Figure 3, and Figure 4, respectively.

Example 6

According to the ratio of 24mL cyclohexane : 12mL Triton X-100 : 12mL n-hexanol : 10mL ammonia water, the volume of TEOS is 3.5mL and the volume of γ-glycidoxypropyl triethoxysilane is 6.5 The carrier was prepared under the conditions of mL, and the carrier was used to covalently immobilize penicillin acylase, and the activity of the obtained immobilized enzyme was 746U/g.

The above content is only a basic description of the concept of the present invention, and any equivalent transformation made according to the technical solution of the present invention shall fall within the scope of protection of the present invention.

Claims (2)

1. A method for preparing a highly active immobilized penicillin acylase hybrid carrier, comprising the following steps: mixing cyclohexane, polyethylene glycol octylphenyl ether and n-hexanol, adding ammonia under stirring, and preparing Form a transparent and clear inverse microemulsion, stir for 15 minutes, add tetraethyl orthosilicate therein, stir for 30 minutes and add silylating agent, the volume ratio of tetraethylorthosilicate and silylating agent V _{ethyl orthosilicate} : V _{silane Chemical reagent} = 0.5-9.5: 0.5-9.5, after hydrolysis and condensation reaction for 22 hours, wash with acetone, and vacuum-dry at room temperature to obtain the hybrid carrier for immobilized penicillin acylase with high activity. The silylating agent is selected from the group consisting of γ-aminopropyltriethoxysilane, γ-glycidyloxypropyl trimethyl (ethyl)oxysilane, γ-mercaptopropyl trimethyl (ethyl)oxysilane, phenyl trimethyl ( One of ethyl)oxysilane, vinyltrimethyl(ethyl)oxysilane or 4-trimethyl(ethyl)oxysilane butyronitrile. 2. The carrier prepared according to claim 1 can also be used as an immobilized carrier for trypsin, amylase, radish peroxidase, cytochrome C and lipase.

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