CN1908166B - A method for preparing highly active immobilized penicillin acylase hybrid carrier - Google Patents

Abstract

the invention discloses a hybridized carrier preparing method of high-activity solidified penicillin acylating enzyme, which comprises the following steps: blending cyclohexane, polyethylene glycol octylphenyl ether and positive hexyl alcohol evenly; adding ammonia; allocating transparent inverse-phase micro-emulsion; stirring for 15 min; adding silicic carbethoxy to stir for 30 min; adding siliconiting agent; setting the bulk rate of silicic carbethoxy and siliconiting agent at 0.5-9.5: 0.5-9.5; hydrolyzing; condensing for 22h; washing through acetone; drying in the vacuum at indoor temperature to obtain the product.

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. the

Background technique

Enzyme is a biological catalyst whose chemical essence is protein. Because of its high catalytic activity, strong selective specificity and mild reaction conditions, enzymes have been widely used in biochemical, pharmaceutical and chemical industries, food industry, light industry and other fields. 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 applications. The main disadvantages are:

(1) The extraction and purification are complicated and expensive;

(2) After the reaction, it is technically difficult to recycle and reuse;

(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 enzymes can overcome most of the above-mentioned shortcomings, so people are more and more interested in the application of immobilized enzymes. 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, the advantages of immobilized enzymes are as follows:

(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) Enzyme use efficiency is improved;

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

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, namely 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, van der Waals force, etc.; the binding force between the two is weak, so the enzyme is easy to lose, but this method has the advantage that the enzyme active center is not easy to be absorbed. 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. The specific operation is to 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 Coupling the enzyme, 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, destroying part of the active center, Therefore, it is often impossible to obtain immobilized enzymes with higher activity. The cross-linking method refers to the method of first adsorbing the enzyme on an insoluble carrier, and then using a bifunctional or multifunctional reagent to perform intermolecular cross-linking between the enzyme molecules to form a cross-linked network structure to immobilize the enzyme. There are deficiencies such as severe reaction conditions and loss of enzyme activity. The embedding method is to embed enzymes in microcapsules. The immobilized enzyme prepared by this method should not use macromolecular reactants (substrates), and the enzyme is easily inactivated, so the reaction conditions must be cleverly designed, and it is generally used to prepare immobilized cells. Each of the four methods has 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 repeatedly reacted in batches. Therefore, combining the above four methods, the immobilized enzyme prepared by the covalent method or the cross-linking method can meet the requirement of operational stability, while the immobilized enzyme prepared by the adsorption method has higher activity. the

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 three waste pollution 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 of penicillin, they cannot compete economically with chemical methods due to the weakness of free enzymes. In recent years, due to the development of immobilization technology, people use immobilized penicillin acylase to hydrolyze penicillin G potassium salt to produce 6-APA. Because immobilized penicillin acylase has high stability, easy separation, and continuous operation, it can compete with 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 quantity and its distribution are very difficult; some functional groups are in the highly cross-linked network of polymers. Although the situation has been improved after the solvent is swollen, it may still be difficult for the reactant molecules to approach, thus 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, people have developed and used some inorganic carriers, such as CN 1320688A, using 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 overcome: (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 high, 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 surface group of the carrier 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, and can obtain higher immobilized enzymes than other carriers. activity, and has good operational stability and storage stability. the

The technical scheme adopted in the present invention: 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, stirring Add ammonia water under the hood to prepare a transparent and clear inverse microemulsion. After stirring for 15 minutes, add ethyl orthosilicate thereto, and after stirring for 30 minutes, add silylating agent. The volume ratio of ethyl orthosilicate and silylating agent is V _{orthosilicic acid Ethyl ester} : V _{silylating reagent} = 0.5～9.5: 0.5～9.5, hydrolysis and condensation reaction for 22 hours, washing with acetone, and vacuum drying at room temperature to obtain the hybrid carrier of the highly active immobilized penicillin acylase; the silylating reagent is selected from From γ-aminopropyltriethoxysilane. The carrier is directly adsorbed and immobilized with penicillin acylase, or the carrier is preactivated with glutaraldehyde, and then covalently bonded with penicillin acylase, that is, immobilized by covalent means, which can be further immobilized by the carrier. 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 wash it thoroughly with deionized water to further 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

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

Beneficial effects of the present invention, the present invention provides a hybrid carrier with a unique structure for the immobilization of penicillin acylase, using the organic groups rich in the carrier, and adopting a suitable activation loading method, successfully realized The covalent immobilization and adsorption immobilization of penicillin acylase can obtain higher activity than the immobilized enzyme obtained from other carriers, and has good operation stability and storage stability. One of the advantages of the carrier of the present invention is that its structure is an organic-nonpolar hybrid carrier with silicon dioxide as the matrix and aminopropyl groups connected to some silicon bonds. The second advantage of the carrier of the present invention is that the activity of the prepared immobilized enzyme is 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 the configuration ratio of the inverse microemulsion, the ratio of ethyl orthosilicate and γ-aminopropyltriethoxysilane within a certain range can affect the content of amino groups on the surface of the carrier. 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 has a certain number of hydroxyl groups and aminopropyl groups, so it has strong hydrophilicity. The sixth advantage of the present invention is that the carrier is a spherical particle with uniform size and an amorphous structure. The seventh advantage of the carrier of the present invention is that it has good thermal stability, acid resistance, solvent resistance and certain alkali resistance (except strong alkali). The eighth advantage of the carrier of the present invention is that its surface 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. the

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 the TEM characterization figure of carrier;

Detailed ways

The present invention is described in further detail below by accompanying drawing: a kind of method for preparing highly active immobilized penicillin acylase hybrid carrier, comprises the following steps: adopts inverse phase microemulsion method to combine organic phase, surfactant and co-surfactant in Mix evenly under magnetic stirring, 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, hydrolysis and condensation reaction for 22 hours, wash with acetone, and vacuum dry at room temperature, The immobilized penicillin acylase hybrid carrier was obtained. Wherein said organic phase includes cyclohexane, said surfactant includes polyethylene glycol octyl phenyl ether, said co-surfactant includes n-hexanol, and said organosilicon 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 preactivated with glutaraldehyde, and then covalently bonded with penicillin acylase, that is, immobilized in a covalent manner, and can be further obtained. Carrier-immobilized 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. 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 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 activity of the immobilized enzyme in the present invention is determined by alkali titration. The shape of the obtained carrier is evenly distributed spherical, 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 volume of TEOS and APTES was 6mL to prepare the carrier. 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%. the

Example 2

According to the ratio of 24mL cyclohexane: 12mL Triton X-100: 12mL n-hexanol: 10mL ammonia water, the reverse phase microemulsion is prepared, the volume of TEOS is 4mL and the volume of APTES is 6mL. The carrier is prepared, and the amino group content of the obtained carrier is 5.52mmol/g , The specific surface area is 7m2/g. The carrier was used to covalently immobilize penicillin acylase, and the activity of the obtained immobilized enzyme was 1478U/g, and after repeated operations 5 times, the activity remained 94%, and after storage at 4°C for 48 days, the activity of the immobilized enzyme remained 89%. the

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, the volume of TEOS was 0.5mL and the volume of APTES was 9.5mL, and the carrier was prepared for covalent immobilization Penicillin acylase, the activity of the obtained immobilized enzyme is 872U/g. the

Example 4

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

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 volumes of TEOS and APTES were respectively 5mL to prepare the carrier. The amino group content of the obtained carrier was 4.84mmol/g, the ratio The surface area is 23m2/g. The carrier was used to covalently immobilize penicillin acylase, and the activity of the obtained immobilized enzyme was as high as 2214U/g, and 96% of the activity was retained after 5 repeated operations, and 98% of the activity of the immobilized enzyme was retained after being stored at 4°C for 48 days. Carriers prepared in Examples 1-5 were characterized by NMR, SEM and TEM respectively, and the results are shown in Fig. 1, Fig. 2, Fig. 3 and Fig. 4 respectively. the

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. the

Claims (1)

1. method for preparing high activity immobilization penicillin acylase hybridization carrier; comprise the following steps: hexanaphthene, Triton X-100 and n-hexyl alcohol are mixed; under agitation add ammoniacal liquor; be mixed with transparent clarifying reverse micro emulsion; stir behind the 15min to wherein adding tetraethoxy; add silylating reagent, the volume ratio V of tetraethoxy and silylating reagent after stirring 30min _{Positive silicic acid second Ester}: V _{Silylating reagent}=0.5～9.5: 0.5～9.5, hydrolysis-condensation reaction 22h uses washing with acetone, and room temperature vacuum-drying obtains described high activity immobilization penicillin acylase hybridization carrier; Described silylating reagent is a γ-An Bingjisanyiyangjiguiwan.

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