KR20010100600A - Immobilized penicillin G acylase - Google Patents

Abstract

The present invention provides 6-amino peniclanic acid (6-APA), 7-aminodesacetooxycephalosporinic acid (7-ADCA), 7-aminocephalosporin, amoxicillin, cepha from penicillin G, cephalosporin C and the like. A novel immobilized penicillin acylase used to prepare beta-lactam antibiotics such as chlorine, cephalexin, and cepadroxyl, which is obtained by covalently immobilizing penicillin acylase with an immobilized carrier, wherein the immobilized carrier is 80 It is an acrylic resin composition to which an impact modifier having a particle size of ˜400 μm is added, which prevents the immobilization of the immobilized carrier from agitation, thereby ultimately increasing the number of reuse of enzymes. Manufacturing cost can be reduced.

Description

Immobilized penicillin G acylase

The present invention relates to a new immobilized penicillin acylase that significantly improves the number of times the enzyme is reused by increasing the mechanical strength of the penicillin acylase, an enzyme used to prepare betalactam antibiotics from penicillin.

Methods of immobilizing physiologically active substances such as enzymes on a carrier are very diverse and many studies have been made. If the type is largely divided, it can be divided into immobilization method by non-covalent bond such as adsorption and immobilization method by covalent bond, and the immobilization method by double covalent bond is applied to many industries. At this time, as the immobilization carrier, natural or synthetic organic or inorganic materials are used, and immobilization is performed by covalent bonding of a reactive functional group of an amino acid of a protein to an activated reactor of the carrier. Industrially, organic carriers such as polysaccharides, polystyrenes, polyacrylates, and the like are used, and these organic carriers are made to be reactive to form covalent bonds with proteins by general chemical methods.

Meanwhile, beta-lactam antibiotics are prepared by obtaining 6-amino peniclanic acid (6-APA) from the penicillin by enzyme-selective hydrolysis reaction, and then introducing appropriate side chains to synthesize desired beta-lactam antibiotics. It is becoming. Currently, the enzymes used to prepare 6-amino penicillane acid are penicillin amidase or penicillin acylase, which have been commercialized in a form that can be immobilized in a suitable form and reused several times.

U.S. Patent No. 5,782,260 discloses a method of preparing an enzyme having high enzymatic activity per unit volume by immobilizing some enzymes including penicillin acylase using an organosiloxane resin as an immobilization carrier, but referring to the mechanical properties of the enzyme. It is not known whether the carrier is damaged during long term use.

WO 97/04086 uses penicillin acylase prepared using a gel-forming substance such as gelatin and a polymer containing an amino group such as chitosan to increase the selectivity for the synthesis of side chains in the preparation of beta lactam antibiotics by enzymes. Although it is disclosed that there is no mention of stability in the long term use of the enzyme.

In addition, European Patent 0730035A1 discloses that by immobilizing penicillin acylase in an azlactone polymer, the ratio of hydrolysis of betalactam antibiotics, which are side chains and end products, can be lowered. none.

In general, in the reaction of the selective hydrolysis of penicillin or cephalosporin by enzymes to the production of beta-lactam antibiotics, the number of reuse of expensive penicillin acylase is known to be very important in the economics of the process. Mechanical and physical stability, such as breaking an immobilized carrier, has a great impact on the long-term stability of the enzyme.

In addition, after the enzymatic reaction, the enzyme and the product are mainly separated by using a mesh network of about 100 μm by the method of remaining the enzyme in the reactor and passing only the product. In this case, the broken enzyme fragments pass through the network, and then a new enzyme is added. The amount of enzyme lost must be replenished, and the end product is contaminated by the fragments of enzymes broken out of the reactor.

These problems are further exacerbated in the reaction to prepare semi-synthetic beta-lactam antibiotics from 6-aminophenicsilane acid. That is, in the production of beta-lactam antibiotics by enzymes, the proportion of 6-aminophenic silane acid, the side chains used, and the final product as solids in the reaction solution increases, resulting in a high viscosity of the reaction solution. The impact of the enzyme is greater than that of the penicillin hydrolysis reaction, and due to enzyme damage, the number of enzyme reuses is drastically reduced.

Thus, the present inventors while studying to improve the disadvantage that the immobilized carrier is easily damaged by physical impact such as stirring in the penicillin acyla immobilized enzyme as described above, using a polymethacrylate resin to which the impact modifier is added As a result, it was found that the impact strength of the immobilized enzyme can be improved, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a penicillin acylase that can increase the number of times of reuse of the enzyme by improving the degree of breakage of the carrier by stirring by using a resin containing an impact modifier as an enzyme immobilization carrier.

The immobilized penicillin acylase of the present invention for achieving the above object is obtained by immobilizing the penicillin acylase and the immobilized carrier through a covalent bond, wherein the immobilized carrier is acrylic-based to which an impact modifier having a particle size of 80 ~ 400㎛ is added It is characterized by being a resin composition.

1 is a graph showing a comparison of the activity of the immobilized penicillin acylase ( - ) prepared according to the present invention and the activity of PGA-450 (-), a conventional penicillin acylase of Roche (Roche).

The present invention will be described in more detail as follows.

The present invention provides 6-amino peniclanic acid (6-APA), 7-aminodesacetooxycephalosporinic acid (7-ADCA), 7-aminocephalosporin, amoxicillin, cepha from penicillin G, cephalosporin C and the like. A novel immobilized penicillin acylase used to prepare betalactam antibiotics such as chlor, cephalexin, cepadroxyl and the like.

In the present invention, in order to improve the disadvantage that the penicillin acylase immobilizing enzyme is easily damaged by the physical impact such as agitation, the immobilization carrier is added to the acrylic resin, specifically, polymethyl methacrylate resin as the immobilization carrier. use.

Specific immobilization carrier composition is composed of 100 parts by weight of the acrylic thermoplastic resin, 10 to 250 parts by weight of the impact modifier and 0.1 to 5.0 parts by weight of the processing additive, wherein the impact modifier has a particle size of 80 to 400㎛ and has a three-layer structure Use a specific one.

Such an impact modifier is filed by the present applicant as of May 1, 2000 (Patent Application No. 2000-23253), the schematic configuration is as follows;

An innermost layer made of methyl methacrylate or methyl methacrylate and an acrylic acid copolymer; An intermediate layer made of butyl acrylate copolymer or butadiene copolymer; And it has a three-layer structure of the outermost layer consisting of methyl methacrylate or methyl methacrylate and acrylic acid copolymer, wherein the molar ratio of methyl methacrylate of the innermost layer and the outermost layer is 1: 0.5 to 1:15, intermediate layer Prepared by including a UV stabilizer and a colorant in the polymerization.

The acrylic resin, ie, polymethyl methacrylate resin, to which the impact modifier is added, is transmitted from the outermost layer to the rubber layer from the outermost layer, and the energy is absorbed and diverged from the rubber layer to have an impact reinforcing effect.

In the polymerization of the impact modifier as described above, by appropriately changing the polymerization conditions, the size and physical properties can be adjusted to be suitable for the enzyme immobilization carrier.

The outermost layer of the impact modifier has a carboxyl group through copolymerization of a polymethacrylate resin with an acrylic acid monomer, and can form a covalent bond with an enzyme using such a carboxyl group.

There are many ways to covalently bond an enzyme with a carboxyl group. In general, a method using carbodiamide is used to activate a carboxyl group and then form a covalent bond with an enzyme.

The enzyme used in the present invention is an enzyme capable of catalyzing the acylation reaction of the beta lactam ring, and these enzymes have been known since 1961. Specifically, microorganisms that produce such catalytic activity as an enzyme of penicillin amidase or penicillin acylase (EC 3.5.5.11) system include Escherichia coli, Acetobacter pasteurianum , Xanthomonas citri ( Xanthomonas citri ), Kluyvera citrophila or Bacillus megaterium . Enzymes derived from double E. coli are commercially available.

To activate the carrier

N-cyclohexyl-N'-β [(N-methylmorpholine) -ethyl)]-carbodiimide para toluenesulfonate, N-ethyl-N '-(3-dimethylaminopropyl) carbodiimide hydrochloride and the like Carbodiimide compounds are used, in particular water soluble carbodiimide compounds are suitable for the present invention.

Penicillin acylase is added to the activated carrier in an aqueous solution of pH 4.5-8.5, particularly in a solution of 0.1 molar potassium phosphate buffer (pH 7.0). Immobilization bound to the carrier by a carbodiimide compound Enzyme is washed by known methods and stored at 0-4 ° C.

The penicillin acylase immobilized according to the present invention as a carrier has a catalytic activity of about 100 to 500 U per gram, which is similar to that of the conventional penicillin acylase. In terms of mechanical strength, the amount of the enzyme that is broken down under the same stirring conditions and reduced in size is reduced by 10-20% or more compared with the conventional immobilized enzyme, and as a result, the number of times of reuse of the enzyme is increased.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited by the Examples.

Definition and analysis method

Enzyme: The unit U of enzyme activity corresponds to the amount of enzyme required to hydrolyze 1 μmol of penicillin G potassium salt for 1 minute.

Reaction conditions-100 g / l penicillin G potassium salt, 0.05 M potassium phosphate buffer (pH 8.0)

HPLC analysis conditions: Column-Waters μBondapak C18 Column

Mobile phase-0.25 M potassium phosphate buffer, pH 5.0, 0.7% (v / v) acetonitrile

Flow rate-1 ml / min, measurement wavelength -230 nm

Particle size analysis: Fritsch analysette 22 particle size analyzer, non-sonic mode

Reference Example 1 Preparation of Impact Reinforcement Agent for Immobilized Carrier

75 kg of secondary distilled water was added to a 150 L reactor, and the internal temperature was heated to 80 ° C. under a nitrogen stream, followed by 12.77 kg of methyl methacrylate, 1.68 kg of ethyl acrylate, 73 g of allyl methacrylate, and 120 g of sodium diotylsulfosuccinate. 2.97 kg of the mixed solution was added to the reactor and stirred for 15 minutes. Then 720 g of a 1% potassium persulfate solution was added and stirred for 60 minutes. When the polymerization was almost complete, the remaining mixed solution was added dropwise to the reactor at a rate of 220 g per minute. After completion of the dropwise addition, the reaction proceeded for 60 minutes. 945 g of 1% potassium persulfate solution was added thereto, followed by stirring for 15 minutes. A mixed solution of 19.77 g of butyl acrylate, 4.49 kg of styrene, 480 g of allyl methacrylate, and 185 g of sodium dioctylsulfosuccinate was charged at a rate of 350 g per minute. Dropped in After completion of the dropwise addition, 250 g of a 1% potassium persulfate solution was added to the reactor, and polymerization was further performed for 15 minutes. When the conversion rate was 93% or more, 7.13 kg of methyl methacrylate, 0.37 kg of ethyl acrylate, and 0.87 kg of acrylic acid were added dropwise to the reactor at a rate of 150 g per minute, and then polymerization was completed for 50 minutes. .

The average particle diameter of the emulsion obtained after polymerization was 310 nm, and uniform particles with very low dispersion were obtained. The conversion rate was 95% based on the life cycle.

The emulsion thus obtained was obtained using a spray dryer to obtain the final product. The average particle size of the final product was 120-300 μm.

Example 1 Preparation of Immobilized Penicillin Acylase Using Impact Enhancer Carrier

100 mg of the final particles obtained in Reference Example 1 was suspended in 5 ml of 0.1 M potassium phosphate buffer (pH 7.0), and 103 mg of N-ethyl-N '-(3-dimethylaminopropyl) -carbodiimide hydrochloride was added thereto. It was dissolved in 2.5 ml of buffer (4 ° C) and added. After stirring for 10 minutes, 50U (sigma) of penicillin acylase was added thereto, and the mixture was left to stand at 0 to 4 ° C for 48 hours. The mixture was stirred twice for 6 hours.

The obtained immobilized enzyme was filtered off and washed three times with 0.1 M potassium phosphate buffer (pH 7.0) and three times with distilled water. 250 mg (wet) of immobilized penicillin acyla was obtained. The activity was measured by hydrolysis reaction of penicillin G potassium salt, and the activity per gram was 130 U / g wet enzyme.

Example 2 Preparation of 6-Aminophenicylanic Acid Using Immobilized Penicillin Acylase

0.15 BOU of penicillin G potassium salt was added to a 1.5 L reactor provided with a 100 μm mesh network at the bottom, followed by stirring at 28 ° C. after adding 1 L of pure water. 58 g of immobilized penicillin acylase obtained in Example 1 was added thereto, and the reaction was carried out. At this time, pH was maintained at 7.8-8.0 using 8% ammonia water. After 1 hour to 2 hours, if no decrease in pH occurred, the mixture was stirred for about 30 minutes and then the reaction solution was transferred to a new reactor. 0.5 L of methylene chloride was added to the transferred reaction solution and cooled to 0-4 ° C., and then the pH was lowered to 1.0-1.5 with concentrated hydrochloric acid. After stirring for 10 minutes, the mixture was allowed to stand for 30 minutes, and the methylene chloride layer was extracted to remove phenylacetic acid as a reaction byproduct. Thereafter, the pH of the reaction solution was adjusted to 4.0 to precipitate 6-aminophenic silane acid, which was filtered, washed and dried to obtain 53.5 g of 6-aminophenic silane acid (yield 88%).

Experimental Example 1 : Deactivation Trend of Long-Term Operation of Immobilized Penicillin Acylase

After preparing two reaction apparatuses as in Example 2, one side was immobilized penicillin acylase prepared in Example 1, the other was proceeded with the reaction using PGA-450 penicillin acylase from Roche, Germany, While repeating the reaction in the same manner as in Example 2, the time required for the reaction was confirmed.

The results are shown in FIG.

From the results of Figure 1, it can be seen that the immobilized penicillin acylase according to the present invention is superior in long-term stability compared to PGA-450 of the conventional Roche.

Experimental Example 2 : Mechanical Properties

1 g of the immobilized penicillin acylase prepared in Example 1 was placed in 50 ml of distilled water and stirred at 300 rpm for 24 hours using a screw-type stirrer.

The particle size distribution and average particle size before and after stirring were measured and the results are shown in Table 1 below.

Average particle size (㎛) <102㎛ distribution rate (%) Before stirring test 223 1.5 After stirring test 200 5.2

Comparative Experimental Example 1

Particle size analysis before and after stirring using Superzyme of Recordati, Italy, under the same conditions as in Example 2, and the results are shown in Table 2 below.

Average particle size (㎛) <102㎛ distribution rate (%) Before stirring test 223 1.6 After stirring test 168 24.4

Comparative Experimental Example 2

Particle size analysis before and after stirring using PGA-450, Roche, Germany, under the same conditions as in Example 2, and the results are shown in Table 3 below.

Average particle size (㎛) <102㎛ distribution rate (%) Before stirring test 191 3.1 After stirring test 152 20.5

From the results of Tables 1 to 3, the penicillin acylase using the immobilized carrier according to the present invention had a uniform particle size distribution and a small change in particle size before and after the agitation test, whereas the conventional immobilized penicillin acylase significantly reduced the average particle size. It can be seen. This means that the immobilized penicillin acylase breaks through stirring.

As described above in detail, an immobilized penicillin acylase obtained by covalently bonding with a penicsilane acylase using a polymethylmethacrylate resin containing a specific impact modifier according to the present invention as an immobilization carrier is a conventional immobilized penicillin acylase. Compared with the activity per unit weight, the mechanical strength is improved by about 10 to 20%, and the number of reuse can be increased in the production of beta-lactam antibiotics.

Claims (5)

In the immobilized penicillin acylase obtained by immobilizing a penicillin acylase and an immobilization carrier through a covalent bond, The immobilized carrier is an immobilized penicillin acylase, characterized in that the acrylic resin composition to which an impact modifier having a particle size of 80 ~ 400㎛ is added. The method of claim 1, wherein the acrylic resin composition comprises 100 parts by weight of an acrylic thermoplastic resin, 10 to 250 parts by weight of an impact modifier, and 0.1 to 5.0 parts by weight of a processing additive. The impact modifier of claim 1 or 2, wherein the impact modifier comprises: an innermost layer made of methyl methacrylate or a copolymer of methyl methacrylate and acrylic acid; An intermediate layer made of butyl acrylate copolymer or butadiene copolymer; And it has a three-layer structure of the outermost layer consisting of a methyl methacrylate or a copolymer of methyl methacrylate and acrylic acid, the molar ratio of methyl methacrylate of the innermost layer and the outermost layer is 1: 0.5 to 1:15, Immobilized phenylsilin acylase, characterized in that prepared by including an ultraviolet stabilizer and a colorant in the intermediate layer polymerization. The method of claim 1, wherein the penicillin acylase has an activity of 100 to 500 U per gram, E. coli, Acetobacter pasteurianum , Xanthomonas citri , Kluyvera citrophila Or immobilized penicillin acylase, characterized in that it is derived from Bacillus megaterium . A method for preparing a penicillin antibiotic or cephalosporin antibiotic using the immobilized penicillin acylase of claim 1.