JPH0559705B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a chromosome containing an N-acylneuraminic acid aldolase gene derived from an N-acylneuraminic acid aldolase-producing bacterium belonging to the genus Escherichia.
The present invention relates to a new recombinant plasmid incorporating a DNA fragment, a microorganism transformed by introducing the plasmid, and a method for producing N-acylneuraminic acid aldolase from the microorganism. Conventional technology N-acylneuraminic acid aldolase (N-
acylneuraminate aldolase), also known as sialic acid aldolase, is classified as enzyme number EC4.1.3.3 by the International Union of Biochemistry Enzyme Committee, and its systematic name is N-acylneuraminate:pyruvate lyase. It is an enzyme called lyase). This enzyme produces sialic acid (N-acylneuraminic acid) as shown in the reaction formula below.
It is an enzyme that catalyzes the decomposition and synthesis reactions of Sialic acid = N-acylmannosamine + pyruvic acid When the present inventors culture known bacteria belonging to the genus Escherichia and several other genera in the presence of sialic acid, the above N-acylneuraminic acid He discovered that aldolase could be easily produced on an industrial scale and established the production technology for this enzyme (Tokuko Sho
56-54153, Patent No. 1111346). However, the established method described above requires the addition of sialic acid to the culture medium, and the sialic acid itself has the disadvantage that its preparation requires complicated operations and is expensive. Moreover, unless this sialic acid is added,
N-acylneuraminic acid aldolase was not produced or was produced only in very small amounts, and it was completely impossible to implement it industrially. That is, the microorganisms used in the above method cannot substantially exhibit the ability to produce N-acylneuraminic acid in the absence of sialic acid. Purpose of the Invention The present inventors have solved the biggest drawback of the above method, which is that it requires the use of sialic acid, and produced large quantities of N-acylneuraminic acid aldolase on an industrial scale without using the sialic acid. We conducted extensive research to develop the technology that would allow us to do so. As a result, a chromosomal DNA fragment containing the N-acylneuraminic acid aldolase gene was extracted from an N-acylneuraminic acid aldolase-producing bacterium belonging to the genus Escherichia among the microorganisms used in the above established method, and this was inserted into a vector to create a recombinant plasmid. They created a microorganism and succeeded in obtaining a transformed microorganism by introducing the plasmid, and also discovered that the microorganism can produce a significant amount of the desired enzyme by culturing in a sialic acid-free medium. The present invention was completed based on this new knowledge. Structure of the Invention That is, the present invention provides N-acylneuraminic acid aldolase-producing bacteria belonging to the genus Escherichia.
Recombinant plasmid PMK6 that can be replicated in Escherichia coli and is characterized by incorporating a Hind III-Eco RI fragment of chromosomal DNA containing the acylneuraminic acid aldolase gene and a DNA fragment approximately 1.2 kb in size.
The present invention also relates to a microorganism that is transformed by introducing the recombinant plasmid into the chromosomal DNA donor microorganism. According to the use of the above-mentioned recombinant plasmid of the present invention and the transformed strain into which it has been introduced, N-acylneuraminic acid aldolase inducers such as sialic acid and its analogs, which are difficult to prepare, are not added to the culture medium.
A large amount of the desired enzyme can be easily produced and collected on an industrial scale using a common culture medium for microorganisms. Hereinafter, the recombinant plasmid of the present invention and the method for producing a transformed strain introduced with the same will be described in detail. The plasmid of the present invention is produced by incorporating into a vector a chromosomal DNA fragment containing an N-acylneuraminic acid aldolase gene derived from an N-acylneuraminic acid aldolase-producing bacterium belonging to the genus Escherichia. The bacteria of the genus Escherichia used as the chromosomal DNA donor here is preferably one with high productivity of N-acylneuraminic acid aldolase, such as E. coli. There are no particular limitations as long as the bacteria are present, and any known variety of bacteria can be used. The preparation of chromosomal DNA from the above N-acylneuraminic acid aldolase-producing bacteria can be carried out using conventional methods, such as the method using phenol [Saito-Miura method,
Biochim. Biophys. Acta., 72 , 619 (1963)]. The prepared chromosomal DNA is then cut for ligation with the vector. This chromosomal DNA cleavage is carried out by a conventional method using a restriction endonuclease, but is not particularly limited to this method. For example, a method in which cleavage is performed by physically applying shearing force as long as the N-acylneuraminic acid aldolase gene is not cleaved It can also be done by When implementing a method of cutting chromosomal DNA using restriction endonucleases, when adopting reaction conditions that cause complete cleavage, use various restriction endonucleases that do not have a cleavage site in the target N-acylneuraminic acid aldolase gene. can be used, and all types of restriction endonucleases can be used if reaction conditions that cause only partial cleavage are employed. In particular, from the viewpoint of ease of ligation with a vector, it is preferable that the restriction endonuclease has a unique cleavage site in the vector used. As the vector DNA into which the thus cut chromosomal DNA fragments are inserted and ligated, any of a variety of commonly used vectors can be used, and Escherichia coli vectors are particularly preferred. Examples of the above vectors include ColE1 strain, pSC101 strain, pBR322 strain,
Examples include the pACY177 strain, the pCR1 strain, the R6K strain, and the lambda phage strain. The combination of the above chromosomal DNA and vector DNA is
Although this method is commonly used, for example, cutting the donor chromosome and vector with the same restriction endonuclease and then ligating them together using DNA ligase, this method is not limited to this method. However, any other method may be used. In this way, a recombinant plasmid of the present invention (a recombinant DNA molecule, ie, a conjugate of a donor chromosomal DNA fragment and a vector) is obtained. The present invention also provides microorganisms transformed by introducing the above-mentioned plasmids. Bacteria of the genus Escherichia, particularly the above-mentioned chromosomal DNA donor bacteria or N-acylneuraminic acid aldolase-deficient strains derived therefrom, are used as recipient bacteria to be transformed by introducing the recombinant plasmid of the present invention. Among these, N-acylneuraminic acid aldolase-deficient strains are particularly suitable for selecting transformants carrying the plasmid of the present invention. The method of introducing the plasmid of the present invention into a bacterium of the genus Escherichia as a host bacterium and causing transformation can be performed using a known method, for example, a typical transformation method using competent cells [Mol.Gen.Genet.
167, 251 (1979)] etc. The present invention is not particularly limited to this method, and any of various other known methods may be employed. Selection of a microorganism that possesses a donor chromosomal DNA fragment containing the target N-acylneuraminic acid aldolase gene from the transformed strain obtained by the above method and has the ability to produce the enzyme in the absence of an inducer such as sialic acid. Isolation can be easily carried out without using any special methods, using a medium that can selectively grow bacterial clones that have the desired genetic trait on the chromosome, the trait of the vector, or a combination of both. can. In this way, it is possible to obtain the desired transformed strain, which has antibiotic resistance, grows in a sialic acid minimal medium, and has the ability to produce a significant amount of N-acylneuraminic acid aldolase in a medium without the addition of inducers such as sialic acid. Until now, no bacteria of the genus Escherichia has been known to have such a significantly improved enzyme-producing ability, and of course the fact that known bacteria of the genus Escherichia can express such an enzyme-producing ability through genetic recombination methods, and the lack of sialic acid. There is also no known fact that a transformed strain having the ability to produce a significant amount of N-acylneuraminic acid aldolase can be bred using a supplemented medium. The present invention also provides a method for culturing the transformed strain obtained as described above and collecting N-acylneuraminic acid aldolase. The medium for culturing the above-mentioned transformed strain may be a synthetic medium, a semi-synthetic medium, or a natural medium that contains carbon sources, nitrogen sources, inorganic compounds, and other nutrients and is generally used for culturing bacteria. be able to. Examples of carbon sources used in each of the above-mentioned media include glucose, fructose, invert sugar, starch saccharification solution, carbohydrate solutions such as sorbitol and glycerol, and organic acids such as pyruvic acid, malic acid and succinic acid. Examples of the nitrogen source include ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium phosphate, ammonium hydroxide, ammonium tartrate, ammonium acetate, and urea. Examples of substances that can be used as both a carbon source and a nitrogen source include peptone, meat extract, and cornstarch liquor. Examples of inorganic compounds include monopotassium phosphate, dipotassium phosphate, monosodium phosphate, disodium phosphate, magnesium sulfate, magnesium chloride, potassium chloride, sodium chloride, ferrous sulfate, ferrous chloride, and ferrous sulfate. diiron, ferric chloride,
Examples include manganese sulfate and manganese chloride. Examples of other nutrients include yeast extract and vitamins. Cultivation can be carried out in either a liquid medium or a solid medium, but a liquid medium is generally more advantageous, and in particular, shaking culture or aerated agitation culture is advantageous in terms of mass production. The culture temperature is suitably 20-45°C, preferably 28-37°C. During cultivation, it is preferable to adjust the pH to 6 to 9 using a suitable neutralizing agent. By carrying out the above-mentioned culture for usually 10 to 50 hours, the activity of the target N-acylneuraminic acid aldolase reaches its maximum. Since this enzyme is generally an intracellular enzyme, when collecting and purifying this enzyme from a culture, the cells are collected from the above-mentioned culture solution by a method such as centrifugation, and the obtained cells are subjected to ultrasonic treatment. It is preferable to extract the enzyme by crushing by grinding using glass beads, French press, or the like. The extract can be processed by conventional methods such as ammonium sulfate salting out method, ion exchange chromatography, and gel filtration method to obtain purified N-acylneuraminic acid aldolase. Examples Hereinafter, the present invention will be explained in more detail with reference to Examples. Example 1 (1) N-acylneuraminic acid aldolase producing strain
Preparation of chromosomal DNA from E. coli K12C600 strain Escherichia coli (E. coli) K12C600 strain was cultured with shaking at 37° C. for about 3 hours in L medium 1 having the following composition, and cells in the logarithmic growth phase were collected. <L medium composition> Peptone 1g/dl Yeast extract 0.5g/dl Glucose 0.1g/dl NaCl 0.5g/dl PH adjusted to 7.2 The above bacterial cells were subjected to DNA extraction using the phenol method to obtain a final 3.8mg of chromosomal DNA. was extracted and purified. (2) Preparation of vector DNA and cutting with restriction enzymes DNA of plasmid pBR322 as vector
was prepared as follows. That is, first, Escherichia coli carrying pBR322 as a plasmid
Inoculate a type of K12 strain into GPM medium with the following composition,
After incubation at 37°C to mid-log phase, the final concentration
Add 100 μg/ml chloramphenicol,
It was further cultured overnight. <GPM composition> Glucose 10g Peptone 10g NH ₄ Cl 1g Na ₂ HPO ₄・12H ₂ O 15.2g KH ₂ PO ₄ 3g NaCl 3g Na ₂ SO ₄ 0.115g MgCl ₂・6H ₂ O 0.083g Yeast extract 1g Demineralized water Amount to be 1 By the above operation, a large amount of plasmid DNA was produced in the cells. Added chloramphenicol
Bacterial cells were collected at 16 hours, treated with lysozyme and SDS to lyse them, and ultracentrifuged at 30,000 xg for 1 hour to obtain a supernatant. From this, plasmid DNA is concentrated,
A final 700 μg of pBR322 plasmid DNA was fractionated by cesium chloride-ethidium bromide equilibrium density gradient centrifugation. (3) Introduction of chromosomal DNA fragment into vector Take 10μg of chromosomal DNA obtained in (1) above, apply restriction endonuclease Hind at 37°C for 30 minutes, and incubate for 60 minutes.
After reacting for 1 minute or 120 minutes, respectively, to partially cleave the DNA, the reaction was stopped by heat treatment at 65° C. for 10 minutes. On the other hand, vector pBR322 was completely cleaved using restriction endonuclease Hind, and then treated with alkaline phosphatase.
A DNA fragment of pBR322 was prepared. The above chromosomal DNA fragment and pBR322 DNA fragment 5μ
The DNA strands were ligated using _T4 phage-derived DNA ligase in the presence of ATP and dithiothreitol at 10°C for 16 hours. After heat treatment at 65℃ for 10 minutes, add 2 times the volume of ethanol to the reaction solution and add
DNA was precipitated and collected. (4) Transformation with recombinant plasmid DNA An N-acylneuraminic acid aldolase-deficient strain induced from Escherichia coli K12C600 strain by nitrosoguanidine mutation treatment was grown in L medium 10
After growing to the mid-logarithmic phase in
Competent cells (having the ability to take up DNA) were prepared by washing twice with 7.0).
Add 0.1 ml of the DNA solution obtained in (3) above to 0.4 ml of this competent cell suspension, hold at 0°C for 30 minutes, and immediately apply a heat pulse at 42°C for 2 minutes.
DNA was taken into cells. Next, this cell suspension was inoculated into L medium, statically cultured at 37°C for 2 hours to complete the transformation reaction, and the cells were harvested, washed, and resuspended on a minimal medium plate. and cultured at 37°C for 2 days. The above minimal medium plate contains 2g of sialic acid,
(NH ₄ ) 1 g of ₂ SO ₄ , 7 g of K ₂ HPO ₄ , KH ₂ PO ₄
2g of , 0.1g of MgSO ₄ 7H ₂ O, 20g of leucine
mg, 20 mg of threonine and 1 mg of thiamine in 1
Agar is dissolved in pure water and adjusted to pH 7.0.
Add 20g of ampicillin to the sterilized solid medium.
It was adjusted by adding 10 μg/ml. The colonies generated above were collected, and ampicillin resistance and intracellular N-acylneuraminic acid aldolase activity were examined.
pMK2 (about 14.2kb) was obtained. (5) Self-cloning of a plasmid carrying genetic information for N-acylneuraminic acid aldolase The transformed strain obtained in (4) above was cultured in 100 ml of L medium, and treated with chloramphenicol in the same manner as in (2) above. I did it. After collecting and washing the bacterial cells, the method of Birnboim and Doly [Nucleic Acids Research, 7 , 1513-1523]
(1979)], a solution containing a recombinant plasmid (hereinafter referred to as "pMK2") carrying genetic information for N-acylneuraminic acid aldolase was prepared. This solution was subjected to agarose gel electrophoresis (agarose 0.7%, 90V), the pMK2 band was cut out under ultraviolet irradiation, and this was placed in a dialysis tube.
Electrophoresis was performed again, and DNA was extracted from the gel. After removing ethidium bromide,
Add 2 times the volume of ethanol to precipitate and obtain
90μg of pMK2 in 5mM Tris-HCl buffer (PH7.5)
dissolved in Next, in the same manner as in (4) above, Escherichia coli K12C600 strain was endowed with DNA uptake ability, and then
The above pMK2 was incorporated. The bacterial strain thus obtained was cultured on a minimal medium plate containing 10 μg/ml of ampicillin, the resulting colonies were isolated, and the ampicillin resistance and intracellular N-acylneuraminic acid aldolase activity were examined to determine the transformed strain RC.
-K12C600/pMK2 was obtained. (6) Subcloning of N-acylneuraminic acid aldolase gene From the transformed strain obtained in (5) above, 10 μg of plasmid pMK2 was taken by the same method as in (5), and added to it by two restriction endonucleases, EcoRI and
Hind was simultaneously applied to carry out a partial cleavage reaction at 37°C for 1 hour, followed by heat treatment at 65°C for 10 minutes to stop the reaction. Restriction endonucleases EcoRI and Hind for vector pBR322 on the other hand
At the same time, the DNA fragment of pBR322 was prepared by completely cleaving the DNA fragment using alkaline phosphatase. The above pMK2 DNA fragment and pBR322 DNA fragment
A recombinant plasmid was prepared by mixing 5 μg of the DNA and performing a DNA strand ligation reaction at 1°C for 16 hours using _T4 phage-derived DNA ligase in the presence of ATP and dithiothreitol. did. Next, in the same manner as in (4) above, Escherichia coli K112C600 strain was made to have DNA uptake ability.
The recombinant plasmid prepared above was incorporated. The bacterial cells obtained in this way were treated with ampicillin 10 μg/
The resulting colonies were isolated and examined for ampicillin resistance and intracellular N-acylneuraminic acid aldolase activity to obtain a transformed strain RC-K12C600/pMK6. This transformed strain possesses a recombinant plasmid into which a chromosomal DNA fragment containing the N-acylneuraminic acid aldolase gene of the present invention has been integrated, and this plasmid is hereinafter referred to as "pMK6". The restriction enzyme cleavage map of pMK6 is shown in FIG. This is derived from pBR322, Pst, Pvu,
DNA fragments (Hind III-Eco RI fragments,
4331bp and a chromosomal DNA fragment derived from N-acylneuraminic acid aldolase-producing bacteria (Hind III-
Eco RI fragment, approximately 1.2 kb, contains Eco RI and Hind
It is linked at the cleavage site and has a size of approximately 5.5 kb. The transformed strain carrying the above-mentioned plasmid pMK6 has been deposited with the Institute of Microbiology, Agency of Industrial Science and Technology under the name Escherichia coli K12C600/pMK6, and its deposit number is FEIB Deposit No. 7797.
This is the number. (6) Production of N-acylneuraminic acid aldolase using the transformed strain of the present invention The transformed strain obtained in (5) above was compared with the DNA donor strain Escherichia coli K12C600 and its sialic acid-added medium and The productivity of N-acylneuraminic acid aldolase in each of the sialic acid-free medium (yeast extract medium) was examined. As culture media for each microorganism, media with the following compositions were used. <Sialic acid-added medium composition> Sialic acid 5g (NH ₄ ) ₂ SO ₄ 1g K ₂ HPO ₄ 7g KH ₂ PO ₄ 2g MgSO ₄・7H ₂ O 0.1g Yeast extract 0.5g Pure water Amount to make the whole 1 (PH6 .0) <Yeast extract medium composition> Yeast extract 20g Succinic acid 10g Pure water Amount set to 1 (PH6.0) Each of the above-mentioned media was inoculated with each microorganism, and cultured with shaking at 30°C for 24 hours. After that, the bacterial cells were collected from the culture medium by centrifugation, and the cells were added to 25mM phosphate buffer (PH).
7.5) Suspend in 100 ml, crush the cells by ultrasonication, extract the enzyme inside the bacterial cells, separate the sediment and supernatant by centrifugation, and use the crude enzyme solution as the extract (supernatant). Obtained. The activity of the obtained enzyme solution was determined by the method of Barnett et al. [JEGBarnett, DLCorina, and G. Rasool,
Biochemical Journal, 125 , 275 (1971)]. N-acylneuraminic acid aldolase activity is
It is measured by the above method, and one unit of the enzyme activity refers to the activity of decomposing 1 micromole of N-acylneuraminic acid per minute at a reaction temperature of 37°C. The results obtained are shown in Table 1 below. [Table] From Table 1 above, it is clear that according to the present invention, a significant amount of N-acylneuraminic acid aldolase can be obtained in a sialic acid-free medium. The above-mentioned crude enzyme solution can be made into a crude enzyme powder by salting out with ammonium sulfate, centrifuging, dialysis, and freeze-drying according to a conventional method. Further, the enzyme powder can be purified by means such as ion exchange chromatography and gel filtration to obtain a further purified enzyme preparation.

[Brief explanation of the drawing]

FIG. 1 shows a restriction enzyme cleavage map of the recombinant plasmid pMK6 of the present invention.

Claims (1)

[Scope of Claims] 1. Chromosomal DNA containing an N-acylneuraminic acid aldolase gene derived from an N-acylneuraminic acid aldolase-producing bacterium belonging to the genus Escherichia.
Hind III-Eco RI fragment, approximately 1.2 kb in size.
A recombinant plasmid pMK6 that incorporates a DNA fragment and is capable of replicating in E. coli. 2. Chromosomal DNA containing the N-acylneuraminic acid aldolase gene derived from N-acylneuraminic acid aldolase-producing bacteria belonging to the genus Escherichia.
Hind III-Eco RI fragment, approximately 1.2 kb in size.
Recombinant plasmid that incorporates a DNA fragment
A transformed microorganism characterized in that pMK6 is introduced into the above-mentioned chromosomal DNA donor bacterium.