DE3437224A1 - Process for the preparation of penicillin G acylase by fermentation - Google Patents

Abstract

The process is distinguished in that a microorganism which produces penicillin G is obtained by incorporating a plasmid with incorporated deoxyribonucleic acid (DNA) fragment from a strain of the genus Escherichia with genetic information for the synthesis of penicillin G acylase into a recipient microorganism of a strain of the genus Escherichia, the microorganism which produces penicillin G is fermented in a conventional culture medium in order to produce pure penicillin G acylase, and the acylase accumulated in the culture medium is isolated. The process therefore does not require an inducer such as phenylacetic acid and phenoxymethylacetic acid during the acylase synthesis.

Description

Process for the production of penicillin G acylase by

Fermentation The invention relates to a method of production of pennicillin G acylase by fermentation without induction procedure.

The prior art includes the use of penicillin G acylase (hereinafter referred to as PGA) as an enzyme for obtaining 6-aminopenicillanic acid (6-APA) from penicillin G. Penicillin G is easily decomposed by PGA and converted into 6-APA converted. This enzymatic process can be in higher yield without any unwanted side reactions are carried out under mild reaction conditions, and so the process has proven to be more economical than the compatible ones chemical process.

So far, many suggestions have been made to obtain PGA, to use certain microorganisms, e.g. B. Actinoolanes utahensis ATCC 14539, Arthrobacter tumescene ATCC 6947, Arthrobacter viscosus ATCC 15294, Bacillus megaterium ATCC 14945, Bacillus megaterium ATCC 14946, Bacillus megaterium B-400 FERN-p748, Erwinia stewartil ATCC 13531, Escherichia coli ATCC 9637, Escherichia coli ATCC 11105, Escherichia coli ATCC 13529, Escherichia coli NCIB 8743, Escherichia coli NCIB 8743A, Flavobacterium suaveolens ATCC 13533, Micrococcus candidus ATCC 8456, Micrococcus roseus ATCC 416, Micromonospora purpureochromogenes ATCC 13634, Nocardia sp. ATCC 13635, Proteus rettgeri ATCC 9250, Proteus rettgeri ATCC 31052, Pseudomonas aeruginosa NRRL 1014J, Pseudomonas fluorescens NRRL 1B10, Pseudomonas maltophila ATCC 17808, Serratia rubidacs ATCC 181, Streptomyces griseus KF0 3355, Streptomyces lavendlae ATCC 13664, Streptomyces lavendulae ATCC 13665, and Streptomyces lavendulae ATCC 21138.

The methods for obtaining PGA from such microorganisms require however, complex treatments of the microorganisms with an expensive inductor, such as phenylacetic acid, phenoxymethyl acetic acid and the like, during the fermentation steps and further complicated purification processes of the PGA thus obtained. Hence be the conventional methods that proceed from such microorganisms as below considered impractical from an economic point of view.

The aim of the invention is to provide a new process for the production of Penicillin G acylase, which is the most common method encountered Elaborate induction stage eliminated. The invention is also intended to provide a new method for extracting a penicillin G producing microorganism from a strain that has the genetic information of penioillin G acylase synthesis.

These and other objects can be achieved according to the invention by a method can be achieved in which a penicillin G producing microorganism is obtained in which a plasmid with a Deoxyribonucleic acid (DNA) -Fragment from a strain of the genus Escherichia with implanted genetic Information on the synthesis of penicillin G acylase in a recipient microorganism of the genus Escherichia, the penicillin G producing microorganism Fermented in a conventional culture medium to produce pure penicillin G acylase and the acylase accumulated in the culture medium is recovered.

In practicing the invention, the inventors have a strain of the genus Escherichia as a donor for deoxyribonucleic acid (DNA) selected, e.g. B. Escherichia coli ATCC 11105 capable of producing PGA, and then a DNA fragment with genetic information of the PGA synthesis from the strain obtained and the fragment inserted into a plasmid (as vector DNA). A preserved one recombinant plasmid is successfully introduced into a recipient microorganism of Escherichia introduced to produce the desired PGA.

According to the present invention, a strain having higher PGA productivity can be obtained if a microorganism that is resistant to tetracycline and opposed to Ampicillin is sensitive to being a recipient of such a recombinant plasmid is used.

It should be understood that the microorganism used in the present invention Gives PGA in much higher yield than any of the previously known PGA-producing Tribes.

The strain obtained according to the invention has been obtained from the Korean Federation of Culture Collection (KFCC) and was given the accession number Escherichia coli KFCC-84-3 dated March 28, 1984.

A chromosomal DNA is produced in the usual way from a PGA Standard strain, such as Escherichia coli ATCC 11105, is extracted and after a customary applied working method with a restriction endonuclease such as Eco RI and Pst I treated. Each plasmid extracted from a strain of Escherichia coli vom Relax type can be used as vector DNA.

For the introduction of the DNA fragment into the vector, customary Working methods are applied. The recombinant plasmid thus obtained (recombinant DNA) can be converted into a strain belonging to the genus Escherichia according to a conventional transformation technique to be built in. It should be understood that the recipient microorganism is the recombinant Plasmid must be one that is convenient for the selection and isolation of the desired Transformants can be used.

In the aspects of the invention, among the strains based on able to grow a prepared culture medium, only the transformants with higher PGA productivity can be obtained.

As such vector DNA, any relax-type plasmid which can be extracted from a strain of Escherichia coli, such as HB 101, according to the invention be used.

The methods for fermenting the PGA strain thus obtained are on well known in the art and similar to those for cultivating other PGA producers Microorganisms. Therefore, the composition of the culture medium can also easily vary conventional media. Such media can typically be sources for carbon, nitrogen and minerals. Such sources include Glucose, sucrose, starch hydrolyzate, molasses and corn steep liquor as a carbon source, Beef Extract, Baktopeptone and Bakto Yeast Extract as nitrogen sources and an inorganic one Salt as a source of minerals.

The fermentation can be carried out under aerobic conditions, and the pH and temperature of the culture medium used become desired ones Value set.

From the above it should be clear that it is possible according to the invention is, penicillin G acylase just by fermentation without adding any the acylase inducing materials, namely inducers, during fermentation to create.

The invention is further illustrated by the following examples, without limiting their spirit and scope.

Example 1 Escherichia coli ATCC 11105 became a PGA producing Strain, a new strain type with high PGA productivity according to the following levels obtain.

(1) Production of a chromosomal DNA with genetic information of PGA synthesis: 0 The starting strain ATCC 11105 was under at 37 C for three hours Shake in 1 1 L medium with 1% Bakto peptone, with 0.5% Bakto yeast extract, 0.1 % Glucose and 0.5% NaCl cultured.

The culture medium was adjusted to pH 7.2, and then were Bacterial cells collected in the exponential growth phase. A chromosomal DNA was extracted by a conventional phenol method to be purified to 5.3 mg Get DNA.

(2) Preparation of a vector DNA: At this stage, a DNA of Plasmid pBR322, a vector with both ampicillin and tetracycline resistance genes prepared as a marker as previously described for cloning the gene from PGA.

A strain of Escherichia coli HB 101 containing plasmid pBR322 was at 370C in 1 1 L medium at pH 5, containing 170 g / ml chloramphenicol, the Contains 10 g Bakto peptone, 5 g Bakto yeast extract and 10 g NaCl each per liter, incubated. By this procedure, the plasmid DNA was propagated and in the bacterial cells abundantly accumulated.

After 16 hours of incubation, the cells were harvested and then through Treat with a lysozyme and SDS lysed. The resulting lysate was one hour centrifuged at 30,000 g to give a supernatant. After this Concentrating the supernatant liquid, 580 pg of plasmid DNA was obtained by fractionation using cesium chloride-ethidium bromide equilibrium density gradient centrifugation obtain.

(3) Incorporation of the chromosomal DNA fragment into the vector DNA: 10 each g chromosomal DNA and vector DNA were treated with restriction endonuclease Eco RI and Pst I treated at 37 0C for one hour to cleave the DNA chains, and then 5 Heated to 65 ° C. for minutes.

The solutions of the cleaved chromosomal DNA and vector DNA were combined and the binding reaction of DNA fragments by T4 phage ligase in Subjected to the presence of ATP and dithiothreitol at iOOC for 24 hours. The reaction rate 0 The mixture was then heated to 65 ° C. for 5 minutes and twice the volume of ethanol was added. The resulting mixture was kept at -200C for 4 hours. After that, a failed recombinant DNA obtained.

(4) Genetic transformation with genetic information of high PGA productivity Hybrid plasmid containing: a strain of Escherichia coli HB101 was in 10 ml Medium cultured at 37 ° C. with shaking. Cells were in the exponential Harvested growth phase and suspended in a 0.1 M MgCl2 solution in an ice bath, whereby "efficient" cells with DNA uptake capacity were obtained.

For the suspension of the high-performance cells, that in the above Step (3) obtained DNA containing the hybrid plasmid DNA is given. After 30 minutes Holding the suspension in an ice bath, it was warmed to 42 ° C. for 2 minutes and could then stand in an ice bath for another 30 minutes. The cells the DNA thus incorporated was inoculated into an L medium, and the medium became Shaken for 2 hours at 37 0C, whereby the transformation reaction is complete have been done. The cells were collected, washed and then resuspended. A small portion of the cell suspension was placed on an agar plate containing lo g of Bakto-Pepton, 5 g Bakto yeast extract, 20 mg tetracycline and 2 g agar, each per liter, (the pH was adjusted to 7.2). Then the plate was at 370C incubated. After overnight incubation, all colonies were picked up, purified and isolated.

Each transformant thus obtained was found to be resistant to it Tetracycline and sensitive to ampicillin, which is evident in the Characteristics of the strain used as the recipient. This means, that only the cells that have the pBR322 plasmid with the built-in chromosomal fragments that cover gene specifying PGA synthesis as growing colonies were selected.

Among the clones thus obtained, only the most desirable one became High PGA productivity strain taken and deposited with KFCC under the accession number KFCC-84-3 deposited.

Example 2 In this example, experimental tests were carried out to determine the titer of the KFCC-84-3 strain obtained in Example 1, step (4).

A fermentation culture medium was prepared containing 10 g of Bakto -Trypton, 10 g yeast extract, 5 g beef extract and 10 g fructose, each per liter, and adjusted to pH 7.0 with KOH. 20 ml portions of the medium were poured into a Brought 500 ml flask inoculated with KFCC-84-3 strain and 24 hours cultivated with shaking at 37 ° C.

After the cultivation, the titer became that of the fermentation broth obtained PGA determined by a conventional method (Meyer, at. al., Plasmids of Medical, Environmental and Commercial Importances, p. 459 - 470 (1979), Elsevier / North-Holland Biomedical Press) - Test A. After 10 hours Culturing, 0.1% phenyl acetate was added to the collected PGA medium, and that Culturing was continued for another 14 hours to obtain fully cultured PGA. The titer of the purified PGA was determined by the same method as in the test A used - test B.

For comparison purposes, control tests were carried out using the parent strain ATCC 11105 under the same conditions as used for PGA.

The test results can be found in Table 1 below.

Table 1 Test A Test B Strain Titer of PGA Titer of PGA after induction ATCC 11105 720 E 2 540 E KFCC-84-3 17 280 E 15 252 E From the above table 1 results found that KFCC-84-3 compared a remarkable increase in PGA productivity to ATCC 11105, while productivity decreases considerably after induction.

Claims (4)

Claims 1. Process for the production of penicillin G acylase by fermentation without induction, characterized in that a penicillin G producing microorganism by incorporating a plasmid with incorporated deoxyribonucleic acid (DNA) fragment from a strain of the genus Escherichia with genetic information the synthesis of penicillin G acylase in a recipient microorganism of a strain of the genus Escherichia, the penicillin G producing microorganism Fermented in a conventional culture medium to produce pure penicillin G acylase and the acylase accumulated in the culture medium is recovered. 2. The method according to claim 1, characterized in that as penicillin G producing microorganism Escherichia coli KFCC-84-3 is used. 3. The method according to claim 1 or 2, characterized in that the Plasmid a deoxyribonucleic acid (DNA) fragment from Eco RI to Pst I sites of the strain of the genus Escherichia contains. 4. The method according to any one of the preceding claims, characterized in, that as a strain of the genus Escherichia Escherichia coli ATCC 11105 is used.