KR100816495B1 - Separation and Purification of Cephalosporin C Acylase by Non-Cracking Cells - Google Patents

Abstract

The present invention relates to a method for isolating and purifying CPC acylases, and more particularly, to treat E. coli cells with buffers at pH 5.5 to 9.5 and 20 to 40 ° C .; Treating the cells with a surfactant and lysozyme to release CPC acylase (cephalosporin C acylase) present in the periplasm out of the cells; And desalination and concentration purification of the released CPC acylase using an ultrafiltration membrane. According to the present invention, the cephalosporin C acylase, an intracellular enzyme, is extracted without crushing the cells, and is easily separated and purified, thereby shortening the process and easily immobilized on an organic / inorganic complex immobilized carrier to prepare 7-aminocephalosporic acid This can be useful for.

Description

Separating and Purifying Method of Cephalosporin C Acylase by No Breaking of the Cell}

1 is glutaryl-7-aminocephalosporane in the step 1 using D-amino acid oxidase in the preparation of 7-aminocephalosporranic acid. After conversion to acid (Glutaryl-7-aminocephalosporanic acid), the glutaryl-7-aminocephalosporranic acid was converted to 2 using GL-7-ACA acylase in step 2. -A chemical formula showing a process for converting to aminocephalosporanic acid,

Figure 2 is a chemical formula showing a process for directly converting cephalosporin C to 7-aminocephalosporonic acid using cephalosporin C acylase.

The present invention provides an economical and efficient separation and purification of cephalosporin C acylase (hereinafter, abbreviated as CPC acylase) required for the direct preparation of 7-aminocephalosporranic acid from cephalosporin C by enzymatic reaction. It is about a method.

Cephalosporin C (abbreviated as CPC) is a β-lactam antibiotic that is commonly produced in fungi such as Acremonium Chrysogenium and inhibits cell wall synthesis by gram-negative bacteria. 7-aminocephalosporanic acid (7-ACA), which has antibiotic activity but is very weak and has been removed from the CPC by the amino adipoyl bran. ) Is used as a raw material for semisynthetic cephalosporin-based drugs.

As a method of producing 7-ACA from CPC, there are a chemical process and an enzyme process. The 7-ACA synthesis method by chemical process requires the use of silyl groups as both protecting groups for both amine and carboxyl groups of CPC, and requires multiple stages of cryogenic reactions. Organic solvents have a problem of polluting the environment and causing an increase in production costs. Enzymatic method is to prepare 7-ACA using D-amino acid oxidase, GL-7-ACA acylase, CPC acylase, etc. In recent years, these enzymes insert the transformed recombinant gene into E-coli It is produced by enemy.

Conventional enzyme production techniques such as D-amino acid oxidase, GL-7-ACA acylase and CPC acylase are described as follows.

The cells in the cultured E. coli cells were collected by centrifugation or membrane filtration, the collected cells were suspended in a buffer solution, and the suspension was physically crushed using a homogenizer or an ultrasonic sonicator. The supernatant is taken by centrifugation to remove the insoluble matter and cell debris. Ammonium sulfate is added to the crude supernatant solution at a concentration of 20-30%. After stirring for a certain time, the precipitate is removed by centrifugation again and ammonium sulfate is added to the supernatant so that the final concentration is 50-60%. do. After stirring for some time, the supernatant was discarded by centrifugation to recover the coenzyme precipitate. The recovered precipitate is suspended in a buffer solution to remove salt components by ultrafiltration (abbreviated as U / F) or dialysis.

The crude enzyme solution from which the salt is removed is purified through anion exchange resin, cation exchange resin, gel-filtration resin, and the ultrafiltration unit (U / F). Desalination and concentration by using acetone in the concentrate to precipitate the enzyme is used to dry or dried or freeze dried (freeze drying) the concentrate is used. In addition, in order to immobilize the enzyme, desalination and concentration of the purified enzyme is used by immobilization using a carrier in the concentrate.

As mentioned above, the production of enzymes requires several complex separation and purification processes.

Therefore, the present inventors tried to solve the problems of the conventional complex enzyme purification process as described above, instead of lysing the cells by using a homogenizer or an ultrasonic grinder to extract the enzyme, the lysozyme is a surfactant and cell wall degrading enzyme. The present invention was completed by developing a method of simply extracting the intracellular enzyme CPC acylase without disrupting the cells by controlling the pH and temperature using Lysozyme.

It is an object of the present invention to provide a method for isolating and purifying CPC acylase that separates and purifies the intracellular enzyme CPC acylase without disrupting the cells.

In order to achieve the above object, the present invention comprises the steps of treating E. coli cells with buffer at pH 5.5 ~ 9.5 and 20 ~ 40 ℃; Treating the cells with a surfactant and Lysozyme to release CPC aphalas (cephalosporin C acylase) present in Periplasm out of the cells; And desalting and concentrating the released CPC acylase using an ultrafiltration membrane, thereby providing a method for isolating and purifying CPC acylase by non- disruption of cells.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a method for extracting enzymes through pores generated by decomposing proteins and fat components of cell walls without disrupting cells by pH and temperature control using surfactants and lysozyme during CPC acylase production.

In the method for isolating and purifying the CPC acylase of the present invention, the CPC acylase is preferably present in E. coli intracellular Periplasm, and as the buffer (pH regulator), phosphoric acid is used. ), Acetic acid, citric acid, hydrochloride, sulfuric acid, sodium hydroxide, Trizma, potassium phosphate monobasic, potassium diphosphate It is preferably selected from the group consisting of (potassium phosphate dibasic), sodium phosphate monobasic and sodium phosphate dibasic.

In addition, in the method for separating and purifying the CPC acylase of the present invention, the surfactant is sodium dodecyl sulfate (Ammonium dodecyl sulfate), ammonium dodecyl sulfate (Cetyl) of formula (2) Cetyltrim-ethyl ammonium bromide, Cetyltrimethyl ammonium chloride, lysolecithin of formula 3, ether deoxylysolecithin of formula 4, of Sodium cholate, Ammonium cholate, Sodium taurodeoxy cholate of formula 6, Alkylpolyoxyethylene of formula 7, Alkylphenylpolyoxy of formula 8 ethylene (alkyl phenylpolyoxyethylene), p of formula (9) - teosi freeze-octylphenyl polyoxyethylene (p- tert-octylphenylpolyoxyethylene, Triton x -100), It is preferably selected from the group consisting of β-D-octylglucoside of Formula 10 and polyoxyethylene sorbitol ester of Formula 11, wherein the concentration of the surfactant is 0.1 to 3.0. It is more preferable that it is%.

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(x = y + v + w + z, number of oxyethylene units)

In addition, in the method for separating and purifying the CPC acylase of the present invention, the concentration of the lysozyme is preferably 0.01 to 0.5%.

Production of 7-ACA by the enzymatic method is performed in one step as shown in Figure 1 using D-amino acid oxidase (D-amino acid oxidase) CPC glutaryl-7-aminocephalosporanic acid (Glutaryl- 7-aminocephalosporanic acid (hereinafter abbreviated as GL-7-ACA) and convert GL-7-ACA to 7-ACA using GL-7-ACA acylase in two steps. There is a conversion method and a method of directly converting CPC to 7-ACA using CPC acylase as shown in FIG. 2. However, the two-stage enzymatic method of Figure 1 has a disadvantage in that a lot of by-products are generated by the hydrogen peroxide generated during the one-stage enzymatic reaction, the yield is reduced.

CPC acylase obtained by the separation and purification method of the present invention is not significantly different in terms of the purity of the enzyme in the activity of the enzyme according to the protein amount of the enzyme solution according to the prior art. The activity of CPC acylase is expressed as the amount of 7-ACA-producing enzyme, 30% sodidododecyl sulfate anionic in surfactant, 30% ammonium dodecyl sulfate, 30% cetyltrimethylammonium bromide cationic, 30% cetyltrimethylammonium chloride, nonionic, shows high activity upon treatment with 30% para-terial-octylphenylpolyoxyethylene (Triton x-100), 30% polyoxyethylene sorbitol ester (see Table 1). In addition, when used in combination with a surfactant and lyase, the activity of CPC acylase is significantly increased than when surfactant is used alone (see Table 2). In addition, when comparing the activity of CPC acylase according to the reaction pH, the activity of CPC acylase was observed between pH 7.5 and 8.5 when using Sodido dodecyl sulfate, Ammonium dodecyl sulfate, Cetyltrimethyl ammonium bromide, and Cetyltrimethyl ammonium chloride. When using para-tertiary-octylphenylpolyoxyethylene (Triton x-100) and polyoxyethylene sorbitol esters, the activity of CPC acylase remained high without a significant difference between pH 5.5 and 9.5 (see Table 3). ).

In addition, comparing the activity of CPC acylase according to the reaction temperature is maintained high without showing a large difference between 20 ~ 35 ℃ (see Table 4).

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are only illustrative of the present invention specifically, but the content of the present invention is not limited to the following examples.

Example 1 Isolation and Purification of CPC Acylase

After incubating E. coli BL21 (DE3) with CPC acylase in the periplasm of the cells, only cells were harvested by treatment with 0.1-5% CaCl ₂ and centrifugation. The collected cells were adjusted to pH 5.5-9.5 using buffer solution, and CPC acylase was extracted extracellularly at 20-40 ℃ using 0.1-1.0% surfactant and 0.01-0.5% lysozyme (Hylozyme). It was. The extracted CPC acylase was desalted and concentrated using ultra filtration membrane (Ultra filtration membrane, cut off MW 50,000 Da).

As a result, the enzyme activity prepared according to the present invention and the cells were crushed, and then the activity of the enzyme (specific activity) was measured according to the protein amount of the enzyme solution according to the prior art using a concentration step with ammonium sulfate and an ion exchange resin step. As 13.5 unit / mg protein and 13.3 unit / mg protein, respectively, there was no significant difference in the purity of the enzyme, and thus similar yields and purity were obtained without the complicated separation and purification processes as in the prior art.

Example 2 Measurement Process of CPC Acylase Activity

In a 100 ml beaker, add 50 ml of 75 mM CPC (31 g of pure CPC) in 0.2 M phosphate buffer and adjust the mark to 1 L. Then, add 10 ml of the activated CPC acylase supernatant isolated from Example 1, 37 The pH was maintained at 7.0-9.0 for 15 minutes with 0.1 M NaOH while stirring at < RTI ID = 0.0 > At this time, the amount of NaOH consumed was measured to determine the activity of CPC acylase. The activity units of CPC acylase (unit, u / ml) are expressed as the amount of enzyme that produces 1 μmole of 7-ACA per minute.

<Example 3> Comparison of activity (activity, unit / ml) of CPC acylase according to the type and amount of surfactant

Each surfactant was added to 100 ml of the CPC acylase culture solution separated in Example 1, stirred at 300 rpm at room temperature (20-25 ° C.) for 30 minutes, and the reaction solution was centrifuged to remove CPC acylase activity of the supernatant. (activity) was measured. The activity of CPC acylase according to the type and amount of surfactant is shown in Table 1 below.

Classification Surfactants Concentration (%) CPC acylase activity (u / ml)   Negative ion 30% Sodidododecyl Sulfate 0.1% 35 〃 1.0% 30 30% Ammonium Dodecyl Sulfate 0.1% 35 〃 1.0% 37   Cation 30% cetyltrimethylammonium bromide 0.1% 30 〃 1.0% 25 30% cetyltrimethylammonium chloride 0.1% 30 〃 1.0% 25     Amphoteric 30% Lysolecithin 0.1% 25 〃 1.0% 20 30% Etherdeoxylysolecithin 0.1% 25 〃 1.0% 15 30% Sodium Chelate 0.1% 15 〃 1.0% 20 30% Ammonium Cholate 0.1% 20 〃 1.0% 15 30% Sodipontaurodeoxycholate 0.1% 21 〃 1.0% 23    Nonionic Alkyl Polyoxyethylene 0.1% 20 〃 1.0% 15 Alkylphenyl Polyoxyethylene 0.1% 23 〃 1.0% 20 Para-tertiary-octylphenylpolyoxyethylene 0.1% 33 〃 1.0% 30 Beta-di-octylglucosite 0.1% 25 〃 1.0% 20 Polyoxyethylene Sorbitol Ester 0.1% 20 〃 1.0% 25

As a result, as shown in Table 1, anionic 30% sodidododecyl sulfate, 30% ammonium dodecyl sulfate, cationic 30% cetyltrimethylammonium bromide, 30% cetyltrimethyl ammonium chloride, As the temperature was high, 30% para-tertiary-octylphenylpolyoxyethylene (Triton x-100) and 30% polyoxyethylene sorbitol esters showed high activity.

Example 4 Comparison of CPC Acylase Activity According to Lysozyme Concentration When Using Surfactant and Lysozyme in Combination

In 100 ml of the CPC acylase culture isolated in Example 1, 30% sodium dodecyl sulfate, 30% ammonium dodecyl sulfate, 30% cetyltrimethyl ammonium queramide, 30% cetyltrimethyl ammonium chloride, 30% as a surfactant 1% of para-tertiary-octylphenylpolyoxyethylene and 30% polyoxyethylene sorbitol esters were added, and the lysozyme concentration was 0.01% to 0.5%, stirred at 300 rpm at room temperature for 30 minutes, and then the reaction solution was centrifuged. The activity of the supernatant was shown in Table 2 below.

 Surfactants Lysozyme Concentration (%) CPC acylase activity (u / ml)  30% Sodidododecyl Sulfate 1% 0.01 55 0.1 58 0.5 60  30% Ammonium dodecyl sulfate 1% 0.01 56 0.1 56 0.5 60  30% cetyltrimethylammonium bromide 1% 0.01 54 0.1 56 0.5 58  30% cetyltrimethylammonium chloride 1% 0.01 55 0.1 55 0.5 60  30% para-tertiary-octylphenylpolyoxyethylene 1% 0.01 60 0.1 63 0.5 65  30% polyoxyethylene sorbitol ester 1% 0.01 48 0.1 53 0.5 53

As a result, as shown in Table 2, by adding the lysozyme, the activity of the CPC acylase was significantly increased and the appropriate concentration was 0.01 ~ 0.5% than when using the surfactant of Example 3 alone.

Example 5 Comparison of CPC Acylase Activity According to Reaction pH

It was carried out under the same conditions as in Example 4. In 100 ml of the CPC acylase culture isolated in Example 1, 30% sodidododecyl sulfate, 30% ammonium dodecyl sulfate, 30% cetyltrimethylammonium bromide, 30% cetyltrimethyl ammonium chloride, 30% Para-tertiary-octylphenylpolyoxyethylene and 30% polyoxyethylene sorbitol ester were added 1%, respectively, and the addition concentration of lysozyme was fixed at 0.1%. The pH was adjusted between pH 5.5 and 7.5 using 0.1 M potassium monophosphate and 0.1 M potassium diphosphate. Between pH 8.5 and 9.5, 0.1 M tris (hydroxymethyl) amino methane and 0.1 The pH was adjusted using M hydrochloric acid and stirred at 300 rpm for 30 minutes at room temperature, and then the reaction solution was centrifuged to show the CPC acylase activity of the supernatant.

 Surfactants Lysozyme Concentration (%) PH used CPC acylase activity (u / ml)   30% Sodidododecyl Sulfate 1% 0.1 5.5 55 6.5 60 7.5 67 8.5 70 9.5 62   30% Ammonium dodecyl sulfate 1% 0.1 5.5 58 6.5 58 7.5 67 8.5 68 9.5 63   30% cetyltrimethylammonium bromide 1% 0.1 5.5 65 6.5 64 7.5 68 8.5 68 9.5 62   30% cetyltrimethylammonium chloride 1% 0.1 5.5 66 6.5 65 7.5 68 8.5 70 9.5 60   30% para-tertiary-octylphenylpolyoxyethylene 1% 0.1 5.5 73 6.5 75 7.5 78 8.5 78 9.5 73   30% polyoxyethylene sorbitol ester 1% 0.1 5.5 70 6.5 72 7.5 74 8.5 72 9.5 70

As a result, as shown in Table 3, the activity of CPC acylase was high between pH 7.5-8.5 when using sodicetdodecyl sulfate, ammonium dodecyl sulfate, cetyltrimethyl ammonium bromide, and cetyltrimethyl ammonium chloride. , Para-tertiary-octylphenylpolyoxyethylene (Triton x-100) and polyoxyethylene sorbitol esters remained high with no significant difference in CPC acylase activity between pH 5.5 and 9.5.

Example 6 Comparison of CPC Acylase Activity According to Reaction Temperature

It was carried out under the same conditions as in Example 5. To 100 ml of the CPC acylase culture isolated in Example 1, 30% para-terial-octylphenylpolyoxyethylene 1.0% and lysozyme 0.1% were added, and the reaction temperature was 20 ° C., 25 ° C. and 30 ° C. at a pH of 7.5. After stirring at 300 rpm for 30 minutes at 35 ° C and 40 ° C, the reaction solution was centrifuged to show the CPC acylase activity of the supernatant.

Surfactant Concentration (%) Lysozyme concentration (%) Reaction pH Reaction temperature (℃) CPC acylase activity (u / ml)   30% para-tertiary-octylphenylpolyoxyethylene 1% 0.1 7.5 20 75 25 72 30 72 35 70 40 60

As a result, as shown in Table 4 above, the reaction temperature was kept high without showing a large difference between 20 and 35 ° C.

As described above, according to the present invention, in the separation and purification of the intracellular enzyme cephalosporin C acylase, the separation step using ammonium sulfate, the ion exchange resin, the gel-resin, etc. can be omitted. Since the complex separation and purification process can be simplified, it can be easily immobilized on an organic / inorganic complex immobilization carrier to be useful for the production of 7-aminocephalosporanic acid. It has the effect of preventing occurrence.

Claims (5)

Iii) treating E. coli cells with buffer at pH 5.5-9.5 and 20-40 ° C .; Ii) treating the cells with a surfactant and Lysozyme to release CPC acease present in Periplasm out of the cells; And And v) desalting and concentrating the released CPC acylase using an ultrafiltration membrane. The method of claim 1, wherein the buffer solution is phosphoric acid, acetic acid, citric acid, hydrochloride, sulfuric acid, sodium hydroxide, Trizma , CPC acylase, characterized in that selected from the group consisting of potassium phosphate monobasic, potassium phosphate dibasic, sodium phosphate monobasic and sodium phosphate dibasic Separation and Purification Methods. The method of claim 1, wherein the surfactant is sodium dodecyl sulfate, ammonium dodecyl sulfate, cetyltrim-ethyl ammonium bromide, cetyltrimethyl ammonium chloride (Cetyltrimethyl ammonium chloride), lysolecithin, ether deoxylysolecithin, sodium cholate, ammonium cholate, sodium diurodeoxycholate (Sodium taurodeoxy cholate), alkylpolyoxyethylen polyoxyethylene, alkyl phenylpolyoxyethylene, p- tert-octylphenylpolyoxyethylene (Triton x-100), beta-di-octyl It is selected from the group consisting of glucoside (β-D-octylglucoside) and polyoxyethylene sorbitol ester Separation and purification method of CPC acylase. The method of claim 3, wherein the concentration of the surfactant is 0.1 ~ 3.0% CPC acylase separation and purification method. The method of claim 1, wherein the concentration of lysozyme is 0.01 ~ 0.5% CPC acylase separation and purification method.

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