JP2627755B2 - Production of acylneulaminate cytidyltransferase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to an acylneuraminate cytidylyltransferase belonging to the genus Escherichia (Acylneuraminatec).
chromosome containing acylneulaminate cytidylyltransferase gene from ytidylyltransferase) -producing bacteria
A new recombinant plasmid incorporating a DNA (hereinafter abbreviated as chromosomal DNA) fragment, a microorganism transformed by introducing the plasmid, and a method for producing acylneulaminate cytidylyltransferase using the microorganism About.

Acylneulaminate cytidylyltransferase [EC 2.7.7.43] is an enzyme that synthesizes cytidine monophosphosialic acid from cytidine triphosphate and sialic acid, and is cytidine monophosphosialic acid, especially cytidine monophospho-N-acetylneuraminic acid. Is useful for the treatment of disorders of nerve stimulation in the central nervous system and peripheral nervous system, and cancer.

(Prior art) Acylneulaminate cytidylyltransferase can be obtained from bovine and porcine submandibular glands and other animal tissues, or Neisseria menigitidis, Escherichia coli (Escher).
It is known to be present in some microorganisms such as ichia coli.

(Problems to be Solved by the Invention) In preparing acylneulaminate cytidylyltransferase from animal tissues, in order to avoid inactivation of the enzyme, the tissue is immediately removed after slaughter and the enzyme is extracted. It is difficult to prepare in large quantities due to the necessity and the need to secure animal tissues as raw materials.

On the other hand, when the microorganism is prepared from the microorganism, large-scale culture of the microorganism is possible, but the content of acylneulaminate cytidylyltransferase in the microorganism is extremely low, and the cytidine triphosphate degrading enzyme is contained in the microorganism. Therefore, when used in the synthesis of cytidine monophosphosialic acid, in order to increase the yield, acylneuraminate cytidylyltransferase is highly purified by ion-exchange chromatography or the like to decompose the enzyme. It has disadvantages such as the need to use it after removal.

Even without such a complicated purification operation, a method for producing acylneulaminate cytidylyltransferase by a microorganism having a high acylneulaminate cytidylyltransferase-producing ability, that is, the enzyme having a high cytidine monophosphosialic acid synthesis yield. There is a demand for the development of a manufacturing method for the above.

(Means for Solving the Problems) The present inventors have conducted various studies to solve the above-mentioned drawbacks, and as a result, have completed the present invention.

That is, the present invention extracts a chromosomal DNA fragment derived from an acylneulaminate cytidylyltransferase-producing bacterium belonging to the genus Escherichia, and integrates the chromosomal DNA fragment into a plasmid to prepare a recombinant plasmid. And obtaining a transformed microorganism by introducing the microorganism into a nutrient medium by introducing the transformed microorganism into a nutrient medium, whereby a significant amount of acylneulaminate cytidylyltransferase is accumulated in the culture.

 Hereinafter, the present invention will be described in detail.

A method for isolating chromosomal DNA from an acylneulaminate cytidylyltransferase-producing bacterium belonging to the genus Escherichia, such as Escherichia coli having acylneulaminate cytidylyltransferase activity, is carried out according to a conventional method, for example, Biochimica et Biophysica Acta Vol. 72, pp619.
-629 (1963).

Incorporation of chromosomal DNA into a plasmid is carried out according to a standard method.
For example, a chromosomal DAN and a plasmid are each cut with a restriction enzyme to prepare a DAN fragment, then both DNA fragments are mixed, and DN
It can be performed by treating with A ligase.

As the plasmid used as the vector DNA, a plasmid having an appropriate selection marker and having multiple copies is suitable, and examples thereof include pBR322, pUC18, and lambda phage.

As restriction enzymes, for example, BamH I, EcoR I, Hind
III, Pst I, Sal I and the like are exemplified, and Hind III is preferably used. Further, as the DNA ligase, for example, T4-DNA ligase derived from T4 phage is suitably used.

Next, the recombinant plasmid obtained by the above method is subjected to a conventional method, for example, Molecular Cloning, A Laboratory Manua.
l, Cold Spring Harbor Laboratory pp250-253 (19
82) The calcium chloride treatment method described in
The cells are introduced into microorganisms belonging to the genus Escherichia as host cells, for example, into Escherichia coli by a rubidium chloride treatment method or the like.

A strain containing the desired recombinant plasmid (ie, a prammid incorporating a chromosomal DNA fragment carrying the genetic information of acylneulaminate cytidylyltransferase) is obtained by treating the cells with toluene and using the suspension as an enzyme sample. Conventional methods, for example, Methods in Enzymology Vol. 8, pp208
The assay can be selected and obtained by assaying according to the method described in -210 (1966).

As a microorganism containing the recombinant plasmid thus obtained, for example, Escherichia coli KJ537 (Microtechnical Laboratory No. 9682) can be mentioned.

The microorganism of the present invention obtained as described above accumulates a significant amount of acylneulaminate cytidylyltransferase in the culture when cultured.

As a medium used for culturing the microorganism of the present invention, any of a natural medium or a synthetic medium containing a carbon source, a nitrogen source, an inorganic salt and the like can be used. As the carbon source, for example, glucose, fructose, sucrose and the like, and as the nitrogen source, for example, ammonium chloride, ammonium nitrate,
In addition to inorganic nitrogen such as ammonium sulfate, organic nitrogen such as yeast extract, peptone, and meat extract can be used. Also,
As the inorganic salt, for example, addition of magnesium chloride, manganese chloride or the like is effective. The concentration of the carbon source used is 0.05
-5WT%, preferably 0.5WT%, nitrogen source concentration 0.05-5WT
%, Preferably 0.5 WT%, and about 0.005 WT% of the inorganic salt.

The cultivation is carried out under aerobic conditions such as shaking culture or aeration and stirring culture, and the culture temperature is 20-42 ° C, preferably 37 ° C, and the culture pH is 6-8, preferably 7; The culture time is usually completed in 12 to 48 hours.

Next, cells cultured by a method such as centrifugation are collected from the above culture solution, and further, when acylneulaminate cytidylyltransferase is extracted from the cells, ultrasonic treatment, French press treatment, freeze-thaw treatment The cells are disrupted by, for example, extracting the enzyme. Furthermore, when purifying the acylneulaminate cytidylyltransferase from the cell lysate extract, a purified enzyme can be obtained by a conventional method such as ammonium sulfate precipitation, ion exchange chromatography, or gel filtration.

In the production of cytidine monophosphosialic acid such as cytidine monophospho N-acetylneuraminic acid, the cultured cells collected by a method such as centrifugation are treated with various surfactants and the treated cells are directly treated. By using,
Also, by using the above-mentioned extract of crushed bacterial cells without any treatment, highly efficient production from cytidine triphosphate and sialic acid can be achieved.

(Examples) Hereinafter, the present invention will be described specifically with reference to examples.

Example 1 (1) Preparation of chromosomal DNA Escherichia coli ATCC13027 having an acylneulaminate cytidylyltransferase activity was prepared using L
After shaking culture in 200 ml of B-medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH 7) at 37 ° C. for 16 hours, the cells were separated by centrifugation. Thus obtained
From 1.2 g of cells (wet weight), use the phenol method [Biochimica
et Biophysica Acta Vol. 72, pp619-629 (196
3)], chromosomal DNA was extracted to obtain about 1.6 mg of chromosomal DNA.

(2) Preparation of Recombinant Plasmid Incorporating Chromosomal DNA Fragment 2 μg of chromosomal DNA obtained in (1) was added to the restriction enzyme Hind I
II was reacted at 37 ° C. for 2 hours, fragmented, treated with phenol, and then treated with ether to obtain a chromosomal DNA fragment solution. Plasmid pUC18 1 to be used separately as a vector
μg was treated with Hind III at 37 ° C. for 2 hours.
And others [Science Vol. 196, pp 1313-1319 (1977)]
, Followed by phenol treatment and then ether treatment to obtain a HindIII-cut pUC18 solution.

The thus obtained plasmid pUC18 fragment solution and the above chromosomal DNA fragment solution were mixed, and treated with T4-DNA ligase at 10 ° C. for 16 hours to prepare a recombinant plasmid.

(3) Transformation of Escherichia coli with the recombinant plasmid.

Escherichia coli JM109, which does not have acylneulaminate cytidylyltransferase activity, is LB
-30 mM cells obtained by shaking culture at 37 ° C for 3 hours in 20 ml of medium.
The suspension was suspended in 10 ml of a calcium chloride solution and kept at 0 ° C. for 1 hour. Then, the cells were separated by centrifugation, and 2 ml of 30 m
Resuspended in M calcium chloride solution. Suspension of this cell
To 0.2 ml of the recombinant plasmid solution prepared in (2) was added, and the mixture was kept at 0 ° C. for 1 hour, and then heat-treated at 42 ° C. for 90 seconds to incorporate the plasmid into the cells of the cell and transform. . Next, 1 ml of L was added to the suspension of the transformed cells.
After adding B-medium and leaving at 37 ° C. for 1 hour, ampicillin 5
0 mg / L, 5-bromo-4-chloro-3-indolyl-β-
It was spread on an agar plate of LB-medium supplemented with 30 mg / L of thiogalactopyranoside and cultured at 37 ° C for 18 hours.

(4) Isolation of a transformant capable of producing acylneulaminate cytidylyltransferase.

The white colonies formed on the agar plate of (3) were
After shaking culture at 37 ° C. for 16 hours in a B-medium, the cells were separated by centrifugation.

The obtained cells were suspended by adding 0.5 ml of distilled water and 5 μl of toluene, and 0.35 ml of the suspension was used for Method.
The presence or absence of acylneulaminate cytidylyltransferase activity was determined by the method described in S. En Enzymology Vol. 8, pp. 208-210 (1966). Thus, a transformant Escherichia coli KJ537 (Microtechnical Laboratories No. 9682) having acylneulaminate cytidylyltransferase-producing ability was obtained.

The recombinant plasmid p carried by this transformant KJ537
A restriction enzyme cleavage map prepared by agarose gel electrophoresis of KJC7 is as shown in FIG. 1, and one chromosomal DNA fragment was incorporated in the plasmid.

Example 2 A recombinant plasmid pK was obtained from the transformant KJ537 obtained in Example 1 by the phenol method in the same manner as in Example 1.
JC7 was extracted and treated with HindIII and then with T4-DNA ligase in the same manner as in Example 1 to obtain a new recombinant plasmid.

Escherichia coli JM109 was transformed with the new recombinant plasmid in the same manner as in Example 1 to obtain a transformant Escherichia coli KJ404 (Microtechnical Laboratory No. 9681).

The recombinant plasmid p carried by this transformant KJ404
The restriction map of KJC77R prepared by the same method as in Example 1 is shown in FIG. 2, and the plasmid contained two chromosomal DNA fragments.

Comparative Examples 1 and 2 and Examples 3 and 4 The strains listed in Table 1, ie, Escherichia coli ATCC1302
7 (strain having acylneulaminate cytidylyltransferase activity as the prior art) in Comparative Example 1, Escherichia coli JM109 (host strain of the recombinant plasmid of the present invention having no acylneulaminate cytidylyltransferase activity, The strain used as a strain) was used in Comparative Example 2, Escherichia coli KJ537 (the strain of the present invention obtained in Example 1) was used in Example 3, and Escherichia coli KJ404 (the strain of the present invention obtained in Examples) was used. Example 4 and 1.
6 L of LB-medium was inoculated and cultured with shaking at 37 ° C. for 16 hours.
After completion of the culture, isolate the cells by centrifugation, and add 40 ml
Was resuspended in 50 mM Tris-HCl buffer (pH 8) and subjected to French press. Then, each was centrifuged to separate the bacterial cell extract, and the same buffer was added to bring the total volume to 50 ml.
Was prepared.

Using 10 μl of the prepared extract, Methods in Enz
ymology Vol. 8, pp. 208-210 (1966), acylneulaminate cytidylyltransferase activity (enzyme activity for producing 1 μmole of cytidine monophospho-N-acetylneuraminic acid per hour is defined as one unit). , Hereinafter abbreviated as enzyme activity unit).
Table 1 summarizes the results obtained by converting 1.6 L into the unit culture solution of LB-medium.

As is evident from these results, the strain of the present invention has an acylneulaminate cytidylyltransferase activity per unit culture that is 6.5-fold (KJ537) to 20-fold (KJ404) that of the conventional strain (ATCC13027). Production was made efficient.

Example 5 1.6 L of Escherichia coli KJ404 obtained in Example 2 was used.
Of LB-medium, and cultured with shaking at 37 ° C. for 16 hours. The cells were separated from this culture by centrifugation, suspended in 40 ml of 50 mM Tris-HCl buffer (pH 8), subjected to French press treatment, and then centrifuged to separate the cell extract. The obtained bacterial cell extract was adjusted to 50 ml by adding the same buffer.

Then, ammonium sulfate was added to the prepared extract, and ammonium sulfate was added to 50%.
The fraction which precipitates at saturation is collected. This fraction was treated with 10 mM Tris-HCl buffer (pH 7.5) containing 10 mM magnesium chloride.
It was dissolved in 0 ml and dialyzed against 5 L of the same buffer for 24 hours. Dialysate 1
1 mM phosphate buffer (pH 7.6) containing 0 mM magnesium chloride
Hydroxylapatite equilibrated in step (40ml, diameter 3cm)
And eluted with a concentration gradient of 1 mM phosphate buffer (pH 7.6) containing 10 mM magnesium chloride and 150 mM phosphate buffer (pH 7.6) containing 10 mM magnesium chloride. The active fraction (40 ml) of this eluate was collected and dialyzed against 5 L of 10 mM Tris-HCl buffer (pH 7.5) containing 10 mM magnesium chloride for 24 hours. Next, this dialysate contains 10 mM magnesium chloride
Pass through an ion exchange resin column (50 ml, 1.5 cm in diameter, trade name DEAE-Toyopearl, manufactured by Tosoh Corporation) equilibrated with 10 mM Tris-hydrochloric acid buffer (pH 7.5), and pass through 10 mM Tris containing 10 mM magnesium chloride. Elution was carried out with the same concentration gradient containing hydrochloric acid buffer (pH 7.5) and 0.5 M potassium chloride. Collect the active fraction of this eluate and contain 10 mM magnesium chloride
It was dialyzed against 5 L of 10 mM Tris-HCl buffer (pH 7.5) for 24 hours.

The dialysate is freeze-dried, and 60 mg of a powdered acylneulaminate cytidylyltransferase having a specific activity of 36 units / mg is prepared.
I got

Reference Example 1 Hereinafter, an example of producing cytidine monophospho N-acetylneuraminic acid using acylneulaminate cytidylyltransferase prepared from the transformed microorganism of the present invention will be described.

1.6 L of Escherichia coli KJ404 obtained in Example 2
Of LB-medium, and cultured with shaking at 37 ° C. for 16 hours. After completion of the culture, the cells were separated by centrifugation, resuspended in 40 ml of 50 mM Tris-HCl buffer (pH 8), and subjected to a French press. This is centrifuged to separate the cell extract,
The same buffer solution as above was added to the obtained bacterial cell extract to adjust the volume to 50 ml. Acylneulaminate cytidylyltransferase activity of the prepared cell extract was measured in the same manner as in Example 3, and the result was 280 units / ml. This 50
18 ml (approximately 5000 units) of the cell extract prepared in
1 mmole of acetylneuraminic acid, 1 mmole of cytidine triphosphate, 400 ml of 1 M Tris-HCl buffer (pH 9) and 40 ml of 1 M magnesium chloride were mixed, and water was added to make a total volume of 2000 ml.
And After reacting at 37 ° C. for 4 hours, the solution is passed through an ion exchange resin (carbonate type, 600 ml, trade name: Dowex 1, manufactured by Dow Chemical Co., Ltd.), washed with water, and continuously subjected to a concentration gradient of 0 to 1 M triethylammonium hydrogen carbonate. Elution was performed. The fraction containing cytidine monophospho N-acetylneuraminic acid was concentrated under reduced pressure, and a 5-fold amount of ethanol was added to the concentrated solution to collect a precipitate. By vacuum drying, 4.672 g of a white powder of cytidine monophospho N-acetylneuraminic acid ammonium salt having an HPLC purity of 93% was obtained. The yield was 72%.

Comparative Reference Example 1 An example of the production of cytidine monophospho N-acetylneuraminic acid using acylneulaminate cytidylyltransferase prepared from Escherichia coli ATCC13027 will be described below.

Escherichia coli ATCC13027 was inoculated into 12.8 L of LB-medium and cultured with shaking at 37 ° C. for 16 hours. After completion of the culture, the cells were collected by centrifugation, resuspended in 320 ml of 50 mM Tris-HCl buffer (pH 8), and subjected to a French press. This was centrifuged to separate the cell extract, and the obtained cell extract was adjusted to 400 ml by adding the same buffer. Acylneulaminate cytidylyltransferase activity of the prepared cell extract was measured in the same manner as in Example 3, and was found to be 14 units / ml. Next, 360 ml (approximately 5000 units) of the prepared cell extract, 1 mm N-acetylneuraminic acid,
ole, 1 mmole of cytidine triphosphate, 400 ml of 1M Tris-HCl buffer (pH 9) and 40 ml of 1M magnesium chloride were mixed, and water was added to make a total volume of 2000 ml. The reaction was carried out at 37 ° C. for 4 hours, and the same treatment as in Reference Example 1 was carried out.
1.431 g of white powder of cytidine monophospho N-acetylneuraminic acid ammonium salt having a purity of 68% was obtained. 22% yield
Met. Since this white powder still contains a large amount of cytidine monophosphate, which is a decomposition product of cytidine triphosphate as a raw material, 10 ml of this powder was used for repurification.
It was dissolved in 10 mM Tris-HCl buffer (pH 7.5), passed through Dowex 1 again, and eluted with a concentration gradient of triethylammonium hydrogen carbonate. The cytidine monophospho N-acetylneuraminic acid fraction was precipitated with ethanol and then dried under vacuum to obtain 0.655 g of cytidine monophospho N-acetylneuraminic acid ammonium salt having an HPLC purity of 88%. Final yield was 10%.

(Effect of the Invention) A chromosome containing an acylneulaminate cytidylyltransferase gene derived from an acylneulaminate cytidylyltransferase-producing bacterium belonging to the genus Escherichia
By preparing a recombinant plasmid incorporating the DNA fragment and transforming a microorganism belonging to the genus Escherichia with the recombinant plasmid, the acylneulaminate cytidylyltransferase activity is higher than that of a microorganism having the conventional activity. Strong new microorganisms are obtained.

By culturing the transformed microorganism in a nutrient medium, a significant amount of acylneulaminate cytidylyltransferase can be accumulated in the culture.

That is, the productivity of acylneulaminate cytidylyltransferase can be improved.

Further, upon production of cytidine monophosphosialic acid such as cytidine monophospho N-acetylneuraminic acid, the content of acylneulaminate cytidylyltransferase in the microorganism is high, that is, the acylneulaminate cytidylyltransferase activity is strong. Even if the enzyme in the microorganism is not purified as in the prior art, the microorganism of the present invention can be used without treatment by merely treating the microorganism with a surfactant or by crushing and extracting the enzyme from the microorganism. Can also produce cytidine monophosphosialic acid in high yield.

[Brief description of the drawings]

FIG. 1 and FIG. 2 are restriction enzyme cleavage maps of the recombinant plasmids pKJC7 and pKJC77R of the present invention, respectively, as analyzed by agarose gel electrophoresis.

Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical indication (C12N 1/20 C12R 1:19) (C12N 9/12 C12R 1:19)

Claims (2)

(57) [Claims] 1. A recombinant plasmid comprising a chromosomal DNA fragment containing an acylneulaminate cytidylyltransferase gene derived from an acylneulaminate cytidylyltransferase-producing bacterium belonging to the genus Escherichia. 2. A recombinant plasmid comprising a chromosomal DNA fragment containing an acylneulaminate cytidylyltransferase gene derived from an acylneulaminate cytidylyltransferase-producing bacterium belonging to the genus Escherichia, is transformed into a microorganism belonging to the genus Escherichia. A transformed microorganism characterized by having been introduced.