CN1995339A - Penicillin acylation enzyme-fixing carrier preparation method and carrying method - Google Patents

Abstract

The invention discloses a preparation method of immobilized penicillin acylase, which is characterized in that it comprises the following steps: (1) grinding a porous resin with side chain functional groups, screening, drying, activating and mixing with chitosan solution mixing and drying to obtain a solid; (2) adding the solid obtained in step (1) into a polar hydrophilic solvent, adding an auxiliary agent for modification reaction, suction filtering, washing, and drying to obtain a porous resin carrier; (3) The penicillin acylase is loaded on a porous resin carrier; the immobilized penicillin acylase is obtained. The invention improves the activity of the enzyme and maintains high stability through the biocompatibility of chitosan, and the high molecular polymer substrate has good mechanical properties, which makes the immobilized penicillin acylase of the present invention better meet Requirements for industrial production conditions of β-lactam antibiotics.

Description

Preparation and loading method of a carrier for immobilizing penicillin acylase

technical field

The invention relates to a preparation and loading method of a carrier for immobilizing penicillin acylase, which is mainly used in the industrial preparation and synthesis process of β-lactam antibiotics and their mother nuclei.

Background technique

In the early 1960s, the semi-synthetic industry of β-lactam antibiotics gradually emerged, which made 6-aminopenicillin alkanoic acid (6-APA), the mother nucleus of β-lactam, an important drug synthesis intermediate. Therefore, people first started to synthesize 6-APA by cleaving penicillin G by penicillin acylase (PGA). Subsequently, due to the unique selectivity of PGA, people studied the selective catalysis of PGA, using 6-APA or 7-amino deacetoxycephalosporanic acid (7-ADCA) as the mother nucleus of β-lactam, plus Appropriate side chains for the semisynthesis of β-lactam antibiotics.

For more than 40 years, PGA has become more and more stable through continuous screening and genetic recombination, and the production capacity of enzymes has also been continuously improved. Coupled with effective immobilization methods, the recovery of enzymes has become possible. This method The cost is also greatly reduced.

Now, through the catalysis of enzymes, the world can produce 20,000 tons of 6-APA a year, and the corresponding β-lactam antibiotics, such as cephalexin, amoxicillin and cefadroxil, have also been gradually realized. industrial enzymatic synthesis.

However, it is worth noting that an efficient PGA immobilization method is the key to enzymatic cleavage of penicillin G and β-lactam antibiotic semisynthesis. So much attention is paid to research in this field.

Many materials have been researched and developed for the immobilization of penicillin acylase, in general these materials can be divided into three categories:

1. High molecular polymer: mainly some polyacrylic acid and its derivatized porous resin materials, such as Eupergit C and Amberlite series resins.

2. Biocompatible materials: This category is mainly some natural or biocompatible materials, such as chitosan, dextran, sodium alginate, gelatin and so on.

3. Inorganic materials: These are mainly porous inorganic materials, such as molecular sieves, diatomaceous earth or silica gel, etc. But this type of material is usually modified by some chemical methods.

These three materials have been widely used in the immobilization of enzymes, not only the immobilization of penicillin acylase, but each of the three materials has advantages and disadvantages. Generally speaking, inorganic material carriers have better mechanical properties and higher specific surface area, while polymers have higher immobilization efficiency and better recovery and selectivity. Biomaterials, due to their better biocompatibility, will provide some help to immobilized enzymes, such as improving their chemical stability and reducing the inactivation of enzymes during immobilization, but often this type of carrier The mechanical properties are not very ideal, and it cannot be well adapted to the harsh reaction conditions in industry.

Contents of the invention

The invention provides a method for preparing and loading a carrier for immobilizing penicillin acylase, using a high molecular polymer with a certain active functional group as the base material, and then through the adsorption and modification of chitosan and some auxiliary agents, the The surface of the internal pores of the carrier is modified to make the surface of the internal pores of the polymer carrier more suitable for the adsorption and immobilization of enzymes to obtain higher stability and activity.

The preparation method of this carrier for immobilizing penicillin acylase comprises the steps:

(1) Grinding the porous resin with side chain functional groups, screening, drying, mixing with chitosan solution after activation, and drying to obtain solid;

(2) Add the solid obtained in step (1) into a polar hydrophilic solvent, adjust the pH value to 7-11 after the mechanical stirring speed is 100-800r/min for 10-100 minutes, and add additives for modification After reaction and modification reaction for 2-10 hours, filter with suction, wash and dry.

The porous resin is a granular solid porous resin with a particle diameter of 0.05-2.0 mm and a pore diameter of 2-100 nm.

The molecular skeleton of the porous resin is polypropylene, polystyrene, polyacrylic acid, polyethylene, polyacrylate, polyurethane, etc., and the side chain functional groups of the porous resin are hydroxyl, amino, carboxyl, sulfonic acid, and thiourea groups. , one or more of phosphonic acid groups or epoxy groups.

Described chitosan solution is that chitosan is dissolved in acetic acid or hydrochloric acid solution, and the mass percent concentration of chitosan is 0.5%～10.0%, and described chitosan is biological grade or industrial grade chitosan, The degree of deacetylation is 60% to 95%, and the molecular weight is 10,000 to 500,000. The mass ratio of the chitosan to the resin is between 1:1 and 1:50.

The chitosan solution can be added with a mass percent concentration of 1.0% to 10% hydrophilic substance, and the hydrophilic substance is polyethylene glycol (PEG) 2000, polyethylene glycol (PEG ) 5000, polyethylene glycol (PEG) 20000, dextran, dextran or polyamines.

The polar hydrophilic solvent described in step (2) is one or more of water, dimethylsulfoxide (DMSO), dimethylformamide (DMF), acetone, methanol, and ethanol.

The auxiliary agent described in the step (2) is formaldehyde, acetaldehyde, glutaraldehyde, epichlorohydrin, trimethoxychloromethylsilane, aminopropyltriethoxysilane or 2-chloroethylamine/hydrochloric acid One or several kinds of salts, the additive amount is 1.0%-10% of the weight of the modification reaction system.

The method for the carrier-loaded penicillin acylase prepared by the preparation method of the present invention comprises the following steps:

(a) Activating the carrier with an inorganic auxiliary agent, the inorganic auxiliary agent is an aqueous solution of ammonia water, sodium bicarbonate, sodium carbonate or sodium polyphosphate, the concentration is 0.1-4.0mol/L, and the activation temperature is 20-20 70°C, activation time 6-20 hours;

(b) Activate the carrier activated by the inorganic auxiliary agent with a bifunctional reagent, the bifunctional reagent is an aqueous solution of formaldehyde, glyoxal or glutaraldehyde, the mass percent concentration is 0.5% to 5.0%, and the activation temperature is 20 ～70℃, activation time 1～8 hours;

(c) The activated carrier is aged at a temperature of 20-70° C. for 12-48 hours, mixed with a free enzyme solution of penicillin acylase at a concentration of 50-2000 U/ml, and reacted at 5-40° C. for 12-60 hours. Add sodium borohydride to the solution, the amount of sodium borohydride per milliliter of free enzyme solution is 0.05-10 mg, react for 15-200 minutes, filter with suction, wash, and dry in vacuum at room temperature to obtain fine-grained or powdered penicillin acylase immobilized product .

The method of the carrier-loaded penicillin acylase is also applicable to the loading of some other types of enzymes, as long as the solution of the free penicillin acylase in step (c) is replaced with a solution of some other free enzymes, such as hydrolase , oxidoreductases, synthetases, etc. Preferred are aminoacylase, porcine pancreatic lipase, tryptophan synthase, urease.

In the step (c) of the method for loading penicillin acylase, the relationship between the amount of the carrier and the free enzyme solution can be determined as required, and generally 2-20 ml of the free enzyme solution is used per gram of the carrier.

To prepare the carrier of the present invention, a granular resin with macropores should be selected as the base material, for example, Amberlite IRC76C and Amberlite 1000Na from Rohm and Haas Company of the United States; common resins such as D101, D201, and D316, which are common types of ion exchange resins in China. This macroporous resin usually contains certain functional groups, such as hydroxyl, amino, carboxyl, sulfonic acid, thiourea, phosphonic acid, epoxy, and their derivatized groups. Chitosan is firstly adsorbed into the pores of the resin through porous adsorption, and then firmly connected with the resin through the action of different additives.

To illustrate with three examples:

One is the principle of covalent bonding of resins with hydroxyl groups as functional groups and chitosan. Since there are many hydroxyl groups on the resin, it is possible to use trimethoxy (or triethoxy) chloroalkylsilane and chitosan through these hydroxyl groups. The amino group of the sugar acts to achieve a modification purpose, and the chitosan is fixed and modified on the resin.

One is the principle of covalent bonding between resins with amino groups as functional groups and chitosan. Since there are many amino groups on the resin, these amino groups can be used to react with the amino groups of chitosan through the crosslinking effect of epichlorohydrin. , so as to achieve a modified purpose, chitosan is fixed and modified on the resin.

One is the principle of covalent bonding of resin with ester group as functional group and chitosan. Since there are many ester groups on the resin, dichloroethylamine (hydrochloride) can be added through these ester groups to interact with the shell. The ester group of the polycan reacts to achieve the purpose of modification, and the chitosan is fixed and modified on the resin.

In this way, chitosan-modified porous resin supports can be prepared by these different methods, and then the common covalent immobilization method is used to covalently immobilize the enzyme by utilizing the amino group of chitosan and the activation of bifunctional reagents.

In this carrier, it is not only necessary to utilize the high amino group density of chitosan to provide the basis and guarantee for covalent crosslinking, but also to utilize the abundant hydroxyl groups on the sugar ring of chitosan, only these hydroxyl groups can be used for immobilization. The enzyme provides an excellent hydrophilic microenvironment, so that the enzyme can maintain a high water activity, greatly reduce the enzyme inactivation rate, and maintain a high activity stability. Therefore, this is also one of the important purposes of using chitosan as a resin carrier modifier.

In summary, first, the porous macroporous resin provides the basis for the adsorption of chitosan and enzyme; second, the stable covalent modification of chitosan on the surface of the resin improves the stability of the entire carrier; Third, the rich hydroxyl groups of chitosan provide a very good microenvironment for enzymes; fourth, the variable resin backbone and functional groups and different covalent modification processes make the immobilized carrier diverse to adapt to different enzymes and use environments demand.

In this way, the high stability and activity of the enzyme itself is retained through the biocompatibility of chitosan, and because of the high molecular polymer as the substrate, it has good mechanical properties and chemical stability, making the whole immobilized The product can better adapt to harsh conditions in the industrial production of β-lactam antibiotics.

Description of drawings

Figure 1 is the cross-sectional SEM image of the resin before modification

Figure 2 is the cross-sectional SEM image of chitosan modified resin

Detailed ways

Example 1

5.0 g of polymethyl methacrylate spherical porous resin was ground and sieved to obtain resin particles with a particle size of 0.5 mm. Dissolve 1.0 g of chitosan in 50 ml of 2% acetic acid solution, and then add 0.5 g of PEG20000 to form a transparent and clear solution. Mix the resin and chitosan solution evenly, and dry at 40°C. Transfer the mixed solids into a round-bottomed flask, add 50ml of water, and stir mechanically at 200r/min. After 30 minutes, adjust the pH value to 10 with NaOH aqueous solution, add 0.5g of 2-chloroethylamine hydrochloride, and control the temperature at 40°C. After reacting for 5 hours, filter with suction, wash, and dry at 40°C.

Take 1.0 g of the prepared carrier in a Erlenmeyer flask, add 1 mol/L sodium bicarbonate solution, react at 50°C for 12 hours, separate, then add 10 ml of 2.0% glyoxal solution, shake on a shaking table, 200r/ min, the temperature is controlled at 40°C. Take it out after 6 hours, age at 30°C for 24 hours, add 400U/ml of free penicillin acylase solution, 5ml in total, react at 10°C for 24 hours, then add 15mg of NaBH ₄ to the system, treat for 30min, suction filter, wash, Dry under vacuum at room temperature. The product of immobilized penicillin acylase can be obtained by separation, the activity is about 600-800U/g.

Example 2

20.0 g of poly(p-styrene alkylhydroxyl) spherical porous resin was ground and sieved to obtain resin particles with a particle size of 0.5 mm. Dissolve 1.0 g of chitosan in 50 ml of 1% hydrochloric acid solution, and then add 0.5 g of PEG2000 to form a transparent and clear solution. Mix the resin with the chitosan solution and dry at 40°C. Transfer the mixed solid into a round bottom flask, add 50ml DMSO, after 30 minutes, mechanically stir at 500r/min, adjust the pH value to 9 with NaOH aqueous solution, add 5ml of trimethoxychloromethylsilane, control the temperature at 60°C, and react After 4 hours, filter with suction, wash, and dry at 40°C.

Take 1.0 g of the prepared carrier in an Erlenmeyer flask, add 1 mol/L ammonia water, react at 60°C for 15 hours, separate, then add 10 ml of 2.0% glutaraldehyde solution, vibrate on a shaker, 200r/min, temperature Controlled at 40°C. Take it out after 4 hours, age at 30°C for 36 hours, add 600U/ml of free penicillin acylase solution, 5ml in total, react at 20°C for 36 hours, then add NaBH ₄ 10mg to the system, treat for 60min, suction filter, wash, Dry under vacuum at room temperature. The product of immobilized penicillin acylase can be obtained by separation, the activity is about 500-700U/g.

Example 3

10.0 g of polyacrylic acid tertiary amino porous resin was ground and sieved to obtain resin particles with a particle size of 0.5 mm. Dissolve 1.0 g of chitosan in 50 ml of 1% hydrochloric acid solution, then add 0.5 g of glucose to form a transparent and clear solution. Mix the resin and chitosan solution evenly, and dry at 40°C. Transfer the mixed solid into a round bottom flask, add 25ml DMF and 25ml ethanol, after 30 minutes, mechanically stir at 500r/min, adjust the pH value to 11 with NaOH aqueous solution, add 5ml of epichlorohydrin, control the temperature at 90°C, and react After 8 hours, filter with suction, wash, and dry at 50°C.

Take 1.0g of the prepared carrier in a Erlenmeyer flask, add 1mol/L sodium polyphosphate solution, react at 30°C for 12 hours, separate, then add 10ml of 2.0% glyoxal solution, oscillate on a shaking table, 200r /min, the temperature is controlled at 40°C. Take it out after 6 hours, age at 30°C for 48 hours, add free penicillin acylase solution, add free penicillin acylase solution 800U/ml, a total of 5ml, react at 30°C for 50 hours, then add NaBH ₄ 20mg to the system to treat After 150 minutes, filter with suction, wash, and vacuum-dry at room temperature to obtain an immobilized penicillin acylase product with an activity of about 700-900 U/g.

Claims (10)

1. A carrier preparation method for immobilized penicillin acylase, characterized in that: comprising the steps: (1) Grinding the porous resin with side chain functional groups, screening, drying, mixing with chitosan solution after activation, and directly drying at 40-120° C. to obtain a solid; (2) Add the solid obtained in step (1) into a polar hydrophilic solvent, adjust the pH value to 7-11 after the mechanical stirring speed is 100-800r/min for 10-100 minutes, and add additives for modification After reaction and modification reaction for 2-10 hours, filter with suction, wash, and dry at 40-120°C. 2. The preparation method according to claim 1, characterized in that the porous resin is a granular solid porous resin with a particle diameter of 0.05-2.0 mm and a pore diameter of 2-100 nm. 3. The preparation method according to claim 1, characterized in that: the molecular skeleton of the porous resin is polypropylene, polystyrene, polyacrylic acid, polyvinyl chloride, polyacrylate, polyurethane, etc., with side chains The functional group is one or more of hydroxyl group, amino group, carboxyl group, sulfonic acid group, thiourea group, or epoxy group. 4. The preparation method according to claim 1, characterized in that: the chitosan solution is dissolved in 2% acetic acid or 1% hydrochloric acid solution, and the mass percent concentration of chitosan is 0.5 % to 10.0%, the chitosan is biological grade or industrial grade chitosan, the degree of deacetylation is 60% to 95%, and the molecular weight is 10,000 to 500,000. 5. The preparation method according to claim 4, characterized in that the mass ratio of chitosan to porous resin is 1:1-1:50. 6. The preparation method according to claim 4, characterized in that: said chitosan solution is added with a hydrophilic substance with a mass percentage concentration of 1.0% to 10%, and said hydrophilic substance is Macrogol 2000, macrogol 5000, macrogol 20000, dextran, dextran, or polyamines. 7. The preparation method according to claim 1, characterized in that: the polar hydrophilic solvent described in step (2) is water, dimethylsulfoxide, dimethylformamide, acetone, methanol, ethanol one or more of them. 8. The preparation method according to claim 1, characterized in that: the additives described in step (2) are formaldehyde, acetaldehyde, glutaraldehyde, epichlorohydrin, trimethoxychloromethylsilane, ammonia One or more of propyltriethoxysilane or 2-chloroethylamine hydrochloride, the additive amount is 1.0%-10% of the weight of the modification reaction system. 9. A carrier prepared by the preparation method as claimed in claim 1. 10. A carrier loading method according to claim 9, characterized in that it comprises the following steps: (a) Activating the carrier with an inorganic auxiliary agent, the inorganic auxiliary agent is an aqueous solution of ammonia water, sodium bicarbonate, sodium carbonate or sodium polyphosphate, the concentration is 0.1-4.0mol/L, and the activation temperature is 20-20 70°C, activation time 6-20 hours; (b) Activate the carrier activated by the inorganic auxiliary agent with a bifunctional reagent, the bifunctional reagent is an aqueous solution of formaldehyde, glyoxal or glutaraldehyde, the mass percent concentration is 0.5% to 5.0%, and the activation temperature is 20 ～70℃, activation time 1～8 hours; (c) The activated carrier is aged at a temperature of 20-70° C. for 12-48 hours, mixed with a free enzyme solution of penicillin acylase at a concentration of 50-2000 U/ml, and reacted at 5-40° C. for 12-60 hours. Add sodium borohydride to the solution, the amount of sodium borohydride per milliliter of free enzyme solution is 0.05-10 mg, react for 15-200 minutes, filter with suction, wash, and dry in vacuum at room temperature to obtain fine-grained or powdered penicillin acylase immobilized product .