JPH0638751A - Production of n-acylneuraminic acid aldolase - Google Patents

Abstract

PURPOSE:To massively and readily obtain an N-acylneuraminic acid aldolase in an ordinary medium free from sialic acid in an industrial scale by employing the N-acylneuraminic acid aldolase-producing bacterium of genus Escherichia transformed with a new recombined plasmid. CONSTITUTION:A recombined plasmid pMK6 which has been recombined with a DNA fragment having a size of 1.2 kb and which is the Hind-III-EcoRI fragment of a chromosomal DNA containing an N-acylneuraminic acid aldolase gene originated from an N-acylneuraminic acid aldolase-producing bacterium belonging to the genus Escherichia is first introduced into the chromosomal DNA-donating bacterium to produce the transformant [e.g. Escherichia.coli K 12C600/pMK6(FERM P-833). The transformant is cultured, and the objective enzyme is then collected from the cultured product. The culture is preferably performed at 28-37 deg.C at a pH of 6-9 for 10-50hr by a vibration-culturing method or by an aeration-stirring method.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to N derived from an N-acylneuraminic acid aldolase-producing bacterium belonging to the genus Escherichia.
-A method for producing N-acylneuraminic acid aldolase from a microorganism transformed by introducing a new recombinant plasmid having a chromosomal DNA fragment containing the acylneuraminic acid aldolase gene.

[0002]

2. Description of the Related Art N-acylneuraminate aldolase (N-acylneuraminate aldolase), which is also known as sialic acid alginase, has an enzyme number of E. C. 4.1.3.3. N-acylneuraminic acid: pyrivic acid lyase (N-
It is an enzyme called acylneuraminate (pyruvate lyase). The present enzyme is an enzyme that catalyzes the decomposition and synthesis reaction of sialic acid (N-acylneuraminic acid) as shown in the following reaction formula.

Sialic acid = N-acylmannosamine + pyruvic acid When the above-mentioned bacteria belonging to the genus Escherichia and several other genera are cultured in the presence of sialic acid, the above-mentioned N It was found that an acylneuraminic acid aldolase can be easily produced on an industrial scale, and a production technique for the enzyme was established (Japanese Patent Publication No. 56-54153, Patent No. 11).
11346). However, in the above-mentioned established method, the addition of sialic acid to the medium is indispensable, and the sialic acid itself has a disadvantage that it requires a complicated operation and is expensive. Moreover, unless this sialic acid was added, N-acylneuraminic acid aldolase was not produced or was produced only in an extremely small amount, and could not be industrially implemented at all. That is, the microorganism used in the above-mentioned method itself cannot substantially express the N-acylneuraminic acid-producing ability in the absence of sialic acid.

[0004]

The object of the present invention is to eliminate the essential drawback of the above-mentioned method, which is the essential use of sialic acid, and to produce a large amount of N-acylneuraminic acid aldolase on an industrial scale without using the sialic acid. Earnestly researched to develop the technology that can manufacture As a result, a chromosomal DNA fragment containing the N-acylneuraminic acid aldolase gene is extracted from an N-acylneuraminic acid aldolase-producing bacterium belonging to the genus Escherichia among the microorganisms used in the above-mentioned method, and the chromosomal DNA fragment is incorporated into a vector to construct a recombinant plasmid , And succeeded in obtaining a transformed microorganism by introducing the plasmid, and discovered the fact that the microorganism can produce a desired amount of the target enzyme by culturing in a sialic acid-free medium. The present invention has been completed based on this new finding.

[0005]

That is, the present invention relates to N-acylneuraminate aldolase-producing N-derived bacteria belonging to the genus Escherichia.
Chromosome D containing the acylneuraminic acid aldolase gene
A recombinant strain pMK6, which is a HindIII-EcoRI fragment of NA and has a DNA fragment of about 1.2 kb in size, is introduced into a donor strain of the chromosomal DNA, and the resulting transformant is cultured and cultured. N-acylneuraminic acid aldolase is collected from the product.
It relates to a method for producing an acylneuraminic acid aldolase.

According to the use of the above recombinant plasmid of the present invention and the transformant into which it is introduced, the inducer of N-acylneuraminic acid aldolase such as sialic acid and its analogs, which is troublesome to prepare, is not added to the medium. In both cases, a large amount of the target enzyme can be easily produced and collected on an industrial scale by using an ordinary microorganism culture medium.

The method for producing the above recombinant plasmid and the transformant into which it is introduced will be described in detail below.

The plasmid used in the present invention is produced by incorporating a chromosomal DNA fragment containing the N-acylneuraminic acid aldolase gene derived from the N-acylneuraminic acid aldolase-producing bacterium belonging to the genus Escherichia into a vector. The bacterium belonging to the genus Escherichia used as the chromosomal DNA donor is preferably one having high productivity of N-acylneuraminic acid aldolase, such as E. coli, but has N-acylneuraminic acid aldolase-producing ability. There is no particular limitation so long as it has any of various known bacteria.

The chromosomal DNA is prepared from the N-acylneuraminic acid aldolase-producing bacterium by a conventional method, for example, a method using phenol [Saito-Miura method, Biochim.
Biophys. Acta., 72 , 619 (1963)] and the like.

The chromosomal DNA prepared is then cut to ligate it with the vector. This chromosomal DNA is cleaved by a method using an ordinary restriction endonuclease, but it is not particularly limited to this method, and unless the N-acylneuraminic acid aldolase gene is cleaved, for example, a method in which a shearing force is physically applied to cleave the chromosomal DNA. You can also do Chromosomal DNA using restriction endonucleases
In carrying out the method for cleaving the enzyme, various reaction endonucleases having no cleavage site in the target N-acylneuraminic acid aldolase gene can be used when the reaction conditions that cause the complete cleavage are adopted. When using reaction conditions that only cause cleavage,
All types of restriction endonucleases can be used. Particularly, as the restriction endonuclease, a vector having a unique cleavage site in the vector to be used is preferable from the viewpoint of easy ligation with the vector.

As the vector DNA into which the chromosomal DNA fragment thus cleaved is inserted and ligated, various commonly used ones can be used, and the Escherichia coli vector is particularly preferable. Examples of the above vector include, for example, Col E1 strain, pSC101 strain, pBR322 strain, pACYC177 strain,
Examples thereof include pCR1 strain, R6K strain, and lambda phage strain.

The above-mentioned chromosomal DNA and vector DNA are ligated by a commonly used method, for example, the donor chromosome and the vector are cleaved with the same restriction endonuclease, and then ligated with DNA ligase. However, the method is not limited to this method, and any other method may be used. Thus, the desired recombinant plasmid (recombinant DNA molecule, that is, a conjugate of the donor chromosomal DNA fragment and the vector) is obtained.

The present invention also provides a microorganism transformed by introducing the above plasmid. As a recipient bacterium as a host transformed with the recombinant plasmid, a bacterium belonging to the genus Escherichia, particularly the above-mentioned chromosome D
NA-donating bacteria or N-acylneuraminic acid aldolase-deficient strains derived therefrom are used. Of these, the N-acylneuraminic acid aldolase-deficient strain is particularly suitable for selecting a transformant strain carrying the target plasmid.

A method for introducing a target plasmid into a bacterium belonging to the genus Escherichiah as a host bacterium to carry out transformation is a known method, for example, a transformation method using competent cells [Mol. Gen. Genet., 167 , 251 (19
79)] etc. The present invention is not particularly limited to this method, and any other known various methods can be adopted.

From the transformant obtained by the above method, a target chromosomal DNA fragment containing the target N-acylneuraminic acid aldolase gene is retained, and the enzyme has the ability to produce the enzyme in the absence of an inducer such as sialic acid. Selective isolation of microorganisms, without employing any special method, using a medium capable of selectively growing clones of a desired trait on the chromosome or the trait of the vector, or a bacterium having both of these traits. Easy to implement.

Thus, an objective transformant strain that has antibiotic resistance, grows in a sialic acid minimum medium, and has a significant amount of N-acylneuraminic acid aldolase-producing ability in a medium without addition of an inducer such as sialic acid is obtained. it can.

No bacterium belonging to the genus Escherichia having such a markedly improved enzyme-producing ability has hitherto been known, and of course, a known bacterium belonging to the genus Escherichia can express such an enzyme-producing ability by a gene recombination method. The fact that a transformant having a large amount of N-acylneuraminic acid aldolase-producing ability is bred in a sialic acid-free medium is not known.

The present invention provides a method for culturing the transformant obtained as described above to collect N-acylneuraminic acid aldolase.

The medium for culturing the above-mentioned transformant contains a carbon source, a nitrogen source, an inorganic compound and other nutrients,
Any of synthetic medium, semi-synthetic medium and natural medium generally used for culturing bacteria can be used.
Examples of the carbon source used in each of the above media include glucose, fructose, invert sugar, saccharified starch solution, sugar 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, urea and the like. Peptone, meat extract, corn steep liquor and the like can be used as the carbon source and the nitrogen source. Examples of the inorganic compound include monopotassium phosphate, dipotassium phosphate, monosodium phosphate, disodium phosphate, magnesium sulfate, magnesium chloride, potassium chloride, sodium chloride, ferrous sulfate, ferrous chloride and ferric sulfate. Examples include ferric iron, ferric chloride, manganese sulfate, manganese chloride and the like. Examples of other nutrients include yeast extract and vitamins.

The culture can be carried out in either a liquid medium or a solid medium, but the liquid medium is generally advantageous, and shaking culture or aeration-agitation culture is particularly advantageous in terms of mass production. The culture temperature is 20 to 45 ° C., preferably 2
The temperature is preferably 8 to 37 ° C. During the culturing, it is preferable to adjust the pH to 6 to 9 using an appropriate neutralizing agent. The desired N-acylneuraminic acid aldolase activity is maximized by carrying out the above culture for 10 to 50 hours. Since this enzyme is generally an intracellular enzyme, when collecting and purifying the enzyme from the culture, the bacterial cells are collected from the culture solution by a method such as centrifugation, and the obtained bacterial cells are subjected to ultrasonic treatment. It is preferable that the enzyme is extracted by crushing by grinding treatment using glass beads, French press treatment, or the like. The extract is purified by treatment with ammonium sulfate salting-out method, ion-exchange chromatography, gel filtration method and the like.
It can be an acylneuraminic acid aldolase.

[0021]

EXAMPLES The present invention will be described in more detail below with reference to examples.

Example 1 (1) N-acylneuraminic acid aldolase producing strain E.
Preparation of Chromosomal DNA from E. coli K12C600 Strain Escherichia coli (E.
coli) K12C600 strain was shake-cultured at 37 ° C. for about 3 hours, and the bacterial cells in the logarithmic growth phase were collected.

<L medium composition> Peptone 1 g / dl yeast extract 0.5 g / dl glucose 0.1 g / dl NaCl 0.5 g / dl pH adjusted to 7.2 DNA extraction operation by the phenol method was performed on the above cells. The final 3.8 mg of chromosomal DNA was extracted and purified.

(2) Preparation of vector DNA and digestion with restriction enzymes DNA of plasmid pBR322 as a vector was prepared as follows. That is, first, one kind of Escherichia coli K12 strain having pBR322 as a plasmid was inoculated into a GPM medium having the following composition, cultivated at 37 ° C. until mid-log phase, and then chloramphenicol at a final concentration of 100 μg / ml was added. , And further cultured overnight. <GPM composition> Glucose 10 g Peptone 10 g NH ₄ Cl 1 g Na ₂ HPO ₄ · 12H ₂ O 15.2 g KH ₂ PO ₄ 3 g NaCl 3 g Na ₂ SO ₄ 0.115 g MgCl ₂ · 6H ₂ O 0.083 g Yeast extract 1 g Desorption Amount of salt water to be 1 liter A large amount of plasmid DNA was produced intracellularly by the above operation. At 16 hours after the addition of chloramphenicol, the cells were collected and treated with lysozyme / SDS to lyse the cells.
The supernatant was obtained by ultracentrifugation at 000 × g for 1 hour. From this, the plasmid DNA was concentrated and subjected to cesium chloride-ethidium bromide equilibrium density gradient centrifugation to give a final 7
00 μg of pBR322 plasmid DNA was fractionated.

(3) Introduction of Chromosomal DNA Fragment into Vector 10 μg of the chromosomal DNA obtained in (1) above was taken, and restriction endonuclease Hind III was added at 37 ° C. for 30 minutes.
After reacting for 0 minutes or 120 minutes respectively to partially cut the DNA, the reaction was terminated by heat treatment at 65 ° C. for 10 minutes. On the other hand, the vector pBR322 was completely cleaved with a restriction endonuclease HindIII and then treated with alkaline phosphatase to prepare a DNA fragment of pBR322.

The above-mentioned chromosomal DNA fragment and D of pBR322
5 μg of NA fragment was mixed, and DNA strand ligation reaction was carried out in the presence of ATP and dithiothreitol using T ₄ phage-derived DNA ligase at 10 ° C. for 16 hours. After heat treatment at 65 ° C for 10 minutes, add 2 to the reaction solution.
A double volume of ethanol was added to precipitate and collect DNA after the ligation reaction.

(4) Transformation with Recombinant Plasmid DNA A N-acylneuraminic acid aldolase-deficient strain derived from Escherichia coli K12C600 strain by nitrosoguanidine mutation treatment was grown in L medium (10 ml) until mid-logarithmic growth. Competent (capable of DNA uptake) cells were prepared by washing twice with Tris buffer (50 mM, pH 7.0) containing 50 mM calcium chloride. To 0.4 ml of this competent cell suspension, 0.1 ml of the DNA solution obtained in the above (3) was added, and the mixture was kept at 0 ° C for 30 minutes and then immediately heated at 42 ° C for 2 minutes to give DNA. Incorporated into cells.

Next, this cell suspension was inoculated into L medium and 3
After performing stationary culture at 7 ° C for 2 hours to complete the transformation reaction, the cells were collected, washed, and the resuspension was spread on a minimal medium plate and cultured at 37 ° C for 2 days.

The above minimum medium plate contained 2 g of sialic acid, 1 g of (NH ₄ ) ₂ SO ₄ , and 7 g of K ₂ HPO ₄ .
g, KH ₂ PO ₄ 2 g, MgSO ₄ .7H ₂ O 0.
1 g, 20 mg of leucine, 20 mg of threonine and 1 mg of thiamine were dissolved in 1 liter of pure water, pH was adjusted to 7.0, and 20 g of agar was added to the sterilized solid medium so that ampicillin was adjusted to 10 μg / ml. It was prepared by adding to.

The colonies generated as described above were picked up, ampicillin resistance and intracellular N-acylneuraminic acid aldolase activity were examined, and the transformant RC-H1 / pMK was examined.
2 (about 14.2 kb) was obtained.

(5) Self-cloning of a plasmid that carries the genetic information of N-acylneuraminic acid aldolase The transformant obtained in (4) above was cultured in 100 ml of L medium and treated in the same manner as in (2) above. Call processing was performed. After collecting and washing the cells, the method of Birnboim and Doly (Nucleie Acids Re
search, 7 , 1513-1523 (1979)], a solution containing a recombinant plasmid (hereinafter referred to as “pMK2”) carrying the genetic information of N-acylneuraminic acid aldolase was prepared.

This solution was subjected to agarose gel electrophoresis (agarose 0.7%, 90 V) to cut out the pMK2 band under ultraviolet irradiation, and this was put in a dialysis tube,
Electrophoresis was performed again to extract DNA from the gel. After removing ethidium bromide, 90 μg of pMK2 obtained by adding 2 volumes of ethanol for precipitation
Was dissolved in 5 mM Tris-HCl buffer (pH 7.5).

Then, in the same manner as in the above (4), the Escherichia coli K12C600 strain was made to have the DNA-incorporating ability, and then the above-mentioned pMK2 was incorporated. The strain thus obtained is cultivated in a minimal medium plate containing 10 μg / ml of ampicillin, the resulting colonies are separated, the resistance to ampicillin and the N-acylneuraminic acid aldolase activity in the cells are examined, and the transformant RC-K12C600 is obtained.
/ PMK2 was obtained.

(6) Subcloning of N-acylneuraminic acid aldolase gene From the transformant obtained in (5) above, 10 μg of plasmid pMK2 was taken by the same method as in (5) above, and two kinds of restriction endonucleases were added to this. EcoRI and Hind
Partial cleavage reaction was performed at 37 ° C. for 1 hour by simultaneously acting III.

A DNA fragment of pBR322 was prepared by preliminarily heating at 65 ° C. for 10 hours, completely digesting it with restriction endonucleases EcoRI and HindIII, and then treating with alkaline phosphatase.

The above-mentioned pMK2 DNA fragment and pBR322
5 μg of the DNA fragment of Example 1 was mixed, and DNA strand ligation reaction was carried out at 10 ° C. for 16 hours in the presence of ATP and dithiothreitol using a DNA ligase derived from T ₄ phage to give a recombinant plasmid. Was prepared.

Then, the Escherichia coli K12C600 strain was made to have a DNA-incorporating ability in the same manner as in (4) above, and then the recombinant plasmid prepared above was incorporated.

Ampicillin 10 was used as the bacterial cells thus obtained.
Cultivate on a minimal medium plate containing μg / ml, separate the resulting colonies, and confirm ampicillin resistance and intracellular N-
The acylneuraminic acid aldolase activity was examined to obtain a transformant RC-K12C600 / pMK6.

This transformant strain carries a recombinant plasmid incorporating a chromosomal DNA fragment containing the desired N-acylneuraminic acid aldolase gene, and the plasmid is hereinafter referred to as "pMK6".

The restriction map of pMK6 is shown in FIG. This is derived from pBR322 and is PstI, Pst
A DNA fragment (HindIII-EcoRI) having a cleavage site which is cleaved with vuII, SalI and BamHI, respectively.
Fragment, 4321 bp) and a chromosomal DNA fragment (Hind III) derived from an N-acylneuraminic acid aldolase-producing bacterium.
-EcoRI fragment (about 1.2 kb) is ligated at the cleavage sites of EcoRI and HindIII, and is about 5.5.
It has a size of kb.

The transformant strain carrying the above plasmid pMK6 has been deposited at the Institute of Microbial Science and Technology of the Institute of Industrial Science and Technology under the name of Escherichia coli K12C600 / pMK6, and the deposit number is Micromachine Research Institute No. 7797.
No. (Microtechnical Institute Article 833).

(7) Production of N-acylneuraminic acid aldolase by the transformant The transformant obtained in (6) above was compared with the Escherichia coli K12C600 strain which is a DNA donor.
The N-acylneuraminic acid aldolase productivity in each of the sialic acid-added medium and the sialic acid-free medium (yeast extract medium) was examined.

As the culture medium for each microorganism, the medium having the following composition was used.

<Sialic acid-added medium composition> Sialic acid 5 g (NH _{4) 2} SO ₄ 1 g K ₂ HPO ₄ 7 g KH ₂ PO ₄ 2 g MgSO ₄ / 7H ₂ O 0.1 g Yeast extract 0.5 g Pure water 1 l Amount (pH 6.0) <Yeast extract medium composition> Yeast extract 20 g Succinic acid 10 g Pure water Amount to make the total amount 1 l (pH 6.0) Each of the above-mentioned medium was inoculated with each microorganism and shake-cultured at 30 ° C. for 24 hours. Was done. Thereafter, the cells were collected from the culture solution by a centrifugation method, and the cells were mixed with 25 mM phosphate buffer (pH 7.5) 10
After suspending in 0 ml and sonicating, the cells were disrupted, the enzyme in the bacterial cells was extracted, the precipitate and the supernatant were separated by centrifugation, and a crude enzyme solution was obtained as an extract (supernatant). .

The activity of the resulting enzyme solution was determined by the method of Burnett et al. [JEG Barnett, DL Corina, and G. Rasool, Bioch.
emical Journal, 125 , 275 (1971)].

The N-acylneuraminic acid aldolase activity is measured by the above-mentioned method. One unit of the enzyme activity means that 1 micromol of N-acetylneuraminic acid is decomposed in 1 minute at a reaction temperature of 37 ° C. Activity.

The results obtained are shown in Table 1 below.

[0048]

[Table 1]

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 crude enzyme solution can be made into a crude enzyme powder by salting out with ammonium sulfate according to a conventional method, centrifuging, dialysis, and freeze-drying. Further, the enzyme powder can be purified by a means such as ion exchange chromatography or gel filtration to obtain a further purified enzyme preparation.

[Brief description of drawings]

FIG. 1 shows a restriction map of the recombinant plasmid pMK6.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display area C12R 1:19) (C12N 1/21 C12R 1:19) (C12N 15/60 C12R 1:19)

Claims (1)

[Claims] 1. Hind I of a chromosomal DNA containing an N-acylneuraminic acid aldolase gene derived from an N-acylneuraminic acid aldolase-producing bacterium belonging to the genus Escherichia.
II-EcoRI fragment with a size of about 1.2 kb DN
N-acylneuraminic acid aldolase is collected from the culture by culturing a transformant obtained by introducing the recombinant plasmid pMK6 incorporating the A fragment into the above-mentioned chromosomal DNA donor bacterium. A method for producing an acylneuraminic acid aldolase.