JPH0731473A - Production of n-acylneuraminic aldolase - Google Patents

Abstract

PURPOSE:To produce the subject aldolase on an industrial scale by transducing a specific recombinant plasmid into a specified variant strain and culturing the resultant transformant. CONSTITUTION:A recombinant plasmid containing a chromosomic DNA fragment containing an N-acylneuraminic aldolase gene, derived from a microorganism, belonging to the genus Escherichia and capable of producing the N- acylneuraminic aldolase and integrated thereinto is transduced into a variant strain (FERM-P-8519) of Escherichia coli varied so as to express the productivity of the N-acylneuraminic aldolase in the absence of an inducer. The resultant transformant is then cultured to collect the N-acylneuraminic aldolase from the culture. Thereby, the objective aldolase is obtained.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a microorganism belonging to the genus Escherichia and transformed by introducing a recombinant plasmid containing the N-acylneuraminic acid aldolase gene,
The present invention relates to a method for producing N-acylneuraminic acid aldolase.

[0002]

2. Description of the Related Art N-acylneuraminate aldolase (N-acylneuraminate aldolase) is also known as sialic acid aldolase and has the enzyme number E. C. 4.1.3.3. In the systematic name, N-acylneuraminic acid: pyruvate 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.

[0003]

[Chemical 1]

The present inventors have found that when the known bacteria belonging to the genus Escherichia and several other genera are first cultured in the presence of sialic acid, the N-acylneuraminic acid aldolase is easily produced on an industrial scale. It was found that the enzyme can be produced and the technology for producing the enzyme was established (Japanese Patent Publication No. 56-54153).
No. 11111146).

However, in the above-mentioned established method, the addition of sialic acid to the medium is indispensable, and this sialic acid itself has the disadvantage that it requires complicated operations and the like and is expensive. Moreover, unless this sialic acid is added, N
-Acylneuraminic acid aldolase was either not produced or produced only in a very small amount, and could not be industrially implemented at all. That is, the microorganism used in the above method itself cannot substantially express the N-acylneuraminic acid aldolase-producing ability in the absence of sialic acid.

The inventors of the present invention have solved the point that the use of sialic acid, which is the biggest drawback of the above method, is essential, and produce a large amount of N-acylneuraminic acid aldolase on an industrial scale without using the sialic acid. We have conducted intensive research to develop technologies that can be done. As a result, the fact that certain mutant strains belonging to the genus Escherichia among the microorganisms used in the above-mentioned method produce a significant amount of the target enzyme even in a culture medium containing no inducer such as sialic acid and its analogs. And completed the invention based on this finding (Japanese Patent Laid-Open No. 60-18438).
4 publication).

In subsequent studies, the present inventors also extracted a chromosomal DNA fragment containing the N-acylneuraminic acid aldolase gene from the microorganisms belonging to the genus Escherichia used in the above-mentioned established method, and extracted it from Escherichia coli. -Successful in constructing a recombinant plasmid by incorporating it into a Collie vector, and a N-acylneuraminic acid aldolase-producing bacterium belonging to the genus Escherichia transformed with the plasmid or a strain lacking the enzyme is the same as the above mutant strain. In addition, the fact that a desired enzyme can be produced in a large amount in a medium without addition of an inducer such as sialic acid was found, and the invention related to this finding was completed (Japanese Patent Application No. 59-181250).

[0008]

The object of the present invention is to produce a very excellent enzyme which exceeds the productivity of the target enzyme found not only in the above-mentioned conventional techniques but also in the technique newly developed by the present inventors. It is to establish a more improved transformed microorganism having an ability and a technique for producing a target enzyme by the microorganism.

[0009]

According to the present invention, a mutant strain of Escherichia coli mutated to express N-acylneuraminic acid aldolase-producing ability in the absence of an inducer is a N strain belonging to the genus Escherichia. -Introducing a recombinant plasmid into which a chromosomal DNA fragment containing the N-acylneuraminic acid aldolase gene derived from an acylneuraminic acid aldolase-producing bacterium has been introduced, culturing the resulting transformant, and collecting N-acyl neuraminic acid aldolase from the culture. A method for producing an N-acylneuraminic acid aldolase is provided.

According to the present invention, when the above-mentioned mutant strain developed by the present inventors is used as a host and a specific plasmid developed previously is introduced for transformation, the transformant strain is truly surprising. In particular, the fact that it expresses a very excellent ability to produce a target enzyme, which is completely unexpected from any of the mutant strain used as a host and the transformant described above, that is, the transformant according to the present invention produces the target enzyme. It was completed based on the discovery of the fact that Noh will be dramatically improved. Therefore, according to the method of using the above-described transformant of the present invention, sialic acid, which is troublesome to prepare, and a derivative of N-acylneuraminic acid aldolase such as an analog thereof are added to the medium without culturing normal microorganisms. By using the culture medium, a very large amount of the target enzyme, which has been impossible in the past, can be easily produced and collected on a large scale on an industrial scale.

The method for producing the above transformant strain used in the method of the present invention will be described in detail below.

In the present invention, it is important to use a mutant strain belonging to the genus Escherichia as a host. Regarding the mutant strain, as described in detail in JP-A-60-184384, various known Escherichia microorganisms can be used.
It can be obtained by applying various known mutation treatment means. An example of the above-mentioned mutant strain that is particularly preferably used in the present invention is Escherichia coli IF033.
Escherichia coli M8328 obtained by mutating 01 as a parent strain with nitrosoguanidine
(Microtechnical Laboratory No. 832, FERM BP-832) can be exemplified.

The transformed microorganism of the present invention is a mutant strain obtained by similarly mutating various mutant microorganisms having the ability to produce N-acylneuraminic acid aldolase in the medium containing sialic acid, such as the above-mentioned mutant strain. It is obtained by introducing a specific plasmid containing the acid aldolase gene.

The above plasmid is an N-acylneuraminic acid aldolase-producing N-derived bacterium belonging to the genus Escherichia.
-A chromosomal DNA fragment containing an acylneuraminic acid aldolase gene is incorporated into an Escherichia coli vector. The bacterium belonging to the genus Escherichia used as the chromosomal DNA donor is preferably one having a high N-acylneuraminic acid aldolase-producing ability such as Escherichia coli (E. coli), but the N-acylneuraminic acid aldolase-producing ability is preferable. There is no particular limitation as long as it has, and various known bacteria can be used.

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 a usual restriction endonuclease, but is not particularly limited to this method, and unless the N-acylneuraminic acid aldolase gene is cleaved,
For example, a method of physically applying a shearing force and cutting may be used. Chromosome DN using restriction endonuclease
In carrying out the method for cleaving A, if reaction conditions that cause complete cleavage are adopted, various restriction endonucleases having no cleavage site in the target N-acylneuraminic acid aldolase gene can be used, and All types of restriction endonucleases can be used when employing reaction conditions that result in only partial cleavage. 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, any of the commonly used vectors can be used, but especially Escherichia
Collie-based vectors are preferred. Examples of the above vector include, for example, Col E1 strain, pSC101 strain, p
BR322 strain, pACYC177 strain, pCR1
, R6K strains, lambda phage strains and the like.

The above-mentioned chromosomal DNA and vector DNA are ligated by a generally 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.

The thus obtained recombinant plasmid (a conjugate of the donor chromosomal DNA fragment and the vector) is then introduced into a mutant strain belonging to the genus Escherichia which is a host (recombinant plasmid recipient). The method for introducing the above-mentioned plasmid into the host bacterium to perform transformation is typically a transformation method using competent cells [Mol. Gen. Gene.
t., 167 , 251 (1979)] and the like. The present invention is not particularly limited to this method, and any other known various methods can be adopted.

Ability to carry a target chromosomal DNA fragment containing the target N-acylneuraminic acid aldolase gene from the transformant obtained by the above method and to produce the enzyme in a significant amount in the absence of an inducer such as sialic acid. Selective isolation of the target microorganism having a, by any ordinary method, for example, selective genetic clones on the desired chromosome or the trait of the vector or a clone of a bacterium having both of them. It can be carried out by a method using a medium that can grow.

Thus, a large amount of N- was added to the medium containing no sialic acid.
A target transformant having the ability to produce acylneuraminate aldolase can be obtained.

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. Usually, the liquid medium is more 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 the present enzyme is generally an intracellular enzyme, collection and purification of the present enzyme from a culture can be carried out according to a usual method for collecting and purifying the intracellular enzyme. As one particularly preferable representative example, for example, cells are collected from the culture solution by a method such as centrifugation, and the obtained cells are subjected to ultrasonic treatment, grinding treatment using glass beads, or French press treatment. A method of crushing and extracting the enzyme can be exemplified. The extract obtained above can be treated by a conventional method such as ammonium sulfate salting-out method, ion exchange chromatography, gel filtration method, etc., and thus purified N-acylneuraminic acid aldolase can be obtained.

[0025]

EXAMPLES The present invention will be described in more detail below with reference to examples. The activity of N-acylneuraminic acid aldolase is determined by the method of Burnett et al. [JEG Barnett, DLCori
na, and G. Rasool, Biochemical Journal, 125 , 275 (1
971)], and 1 unit of the enzyme activity means the amount of enzyme that produces 1 micromol of N-acetylmannosamine per minute at a reaction temperature of 37 ° C.

[0026]

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. to a logarithmic growth phase, and then chloramphenicol at a final concentration of 100 μg / ml was added. The cells were 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 Amount of demineralized 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 the above (1) was taken, and the restriction endonuclease HindIII was reacted at 37 ° C. for 30 minutes, 60 minutes, or 120 minutes, respectively, to partially transform the DNA. After cutting, the reaction was terminated by heat treatment at 65 ° C. for 10 minutes.

On the other hand, the vector pBR322 was completely cleaved with the restriction endonuclease Hind III, and then treated with alkaline phosphatase to obtain DN of pBR322.
The A fragment was prepared.

The above chromosomal DNA fragment and D of pBR322
5 μg of NA fragment was mixed, and DNA strand ligation reaction was performed at 10 ° C. for 16 hours using DNA ligase derived from T ₄ phage in the presence of ATP and dithiothreitol. After heat treatment at 65 ° C for 10 minutes, 2 volumes of ethanol was added to the reaction solution to precipitate the DNA after the ligation reaction,
A Tris buffer solution containing 10 mM of EDTA (disodium ethylenediaminetetraacetate) (10 mM, pH8.
0) Dissolved in 0.5 ml.

(4) Transformation with Recombinant Plasmid DNA From the Escherichia coli K12C600 strain, a N-acylneuraminic acid aldolase-deficient strain induced by a nitrosoguanidine mutation treatment was grown in L medium (10 ml) to the mid-logarithmic growth phase. 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, then immediately at 42 ° C for 2
A heat pulse of minutes was applied to allow the DNA to be internalized by the 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-mentioned minimum medium plate is sialic acid 2
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, leucine 20 mg, threonine 20 mg and thiamine 1 mg were dissolved in 1 l of pure water, and the pH was adjusted to 7.0. 1 ampicillin
It was prepared by adding so that the concentration became 0 μg / ml.

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

(5) Self-cloning of a plasmid carrying the genetic information of N-acylneuraminic acid aldolase The transformant obtained in (4) above was cultured in 100 ml of L medium, and chloramphenic acid was added 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
Research, 7 , 1513-1523 (1979)], a solution containing pBR322 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 (0.7% agarose, 90 V), the band of pMK2 was cut out under UV 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 ml of 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, ampicillin resistance and N-acylneuraminic acid aldolase activity in the cells are examined, and transformant RC-K12C60
0 / 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 in the same manner as in (5) above, and two kinds of restriction endonucleases were added to this. Eco RI and Hind III were allowed to act simultaneously 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.

On the other hand, the vector pBR322 was completely cleaved with restriction endonucleases Eco RI and Hind III at the same time, and then treated with alkaline phosphatase to obtain pBR.
322 DNA fragments were prepared.

The above-mentioned pMK2 DNA fragment and pBR322
5 μg of the DNA fragment of Example 1 was mixed, and a DNA ligase derived from T ₄ phage was used in the presence of ATP and dithiothreitol to ligate the DNA chains at 10 ° C. for 16 hours to prepare a recombinant plasmid.

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

The bacterial cells thus obtained are treated with ampicillin 10
Cultivate on a minimal medium plate containing μg / ml and separate the resulting colonies to obtain ampicillin resistance and intracellular N
-Acylneuraminate aldolase activity was examined to obtain a transformant RC-K12C600 / pMK6.

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

The restriction enzyme digestion map of pMK6 is shown in FIG. This is derived from pBR322 and is Pst I, Pvu
II, Sal I and Bam HI DNA fragments each having a cleavage site (Hind III-Eco RI fragment, 4321 bp)
And a chromosomal DNA fragment derived from an N-acylneuraminic acid aldolase-producing bacterium (Hind III-Eco RI fragment, about 1.2 k
b) is linked at the cleavage sites of Eco RI and Hind III, and has a size of about 5.5 kb.

The Escherichia coli K12C600 strain carrying the above plasmid pMK6 can be obtained from the Institute of Microbial Science and Technology of the Institute of Industrial Science as "Escherichia coli K12C.
It has been deposited under the name "600 / pMK6", and the deposit number is Micromachine Research Institute No. 833.

(7) Preparation of transformant of the present invention From the transformant obtained in (6) above, pMK6 was taken out in the same manner as in (5) above, and pMK6 was prepared in the same manner as in (4) above. It was incorporated into Collie M8328 strain.

That is, after the Escherichia coli M8328 strain was grown in L medium to the mid-logarithmic growth phase, a Tris buffer (50 mM, pH, containing 50 mM calcium chloride) was used.
Competent (capable of DNA uptake) cells were prepared by washing twice with 7.0). To this competent cell suspension, 1 μg of pMK6 obtained above was added, and the mixture was kept at 0 ° C for 30 minutes, then immediately at 42 ° C for 2
A heat pulse of minutes was applied to allow the DNA to be internalized by the cells.

Next, this cell suspension was inoculated into L medium and 3
After culturing at 7 ° C. for 2 hours to complete the transformation reaction, cells were collected, washed, and the resuspension was treated with ampicillin 40.
Spread on L medium plate containing μg / ml and
Cultured for a day.

The colonies produced as described above were picked up, ampicillin resistance and N-acylneuraminic acid aldolase activity in the cells were examined, and the transformant strain. coliM8328 /
pMK6 was obtained.

The above-mentioned transformant strain was obtained from the Institute of Microbial Science and Technology of the Institute of Industrial Science, "Escherichia coli M8328 / pM.
It has been deposited under the name "K6", and the deposit number is Micromachine Research Institute of Microbiology No. 8519 (FERM p-8519).

(8) Production of N-acylneuraminic acid aldolase by the transformant of the present invention The transformant obtained in (7) above is an Escherichia coli K12C600 strain that is a chromosomal DNA donor containing the N-acylneuraminic acid aldolase gene. And transformant Escherichia coli K12C600 / pMK6 transformed with pMK6 introduced thereinto, Escherichia coli M8328 strain which is a parent strain (mutant strain) of the transformant of the present invention, and Escherichia coli which is a parent strain of the M8328 strain. N in the sialic acid-free medium (yeast extract medium) in comparison with each of the Collie IFO3301 strains
-The acylneuraminic acid aldolase productivity was investigated.

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

<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. 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
Suspend in 0 ml and sonicate to disrupt cells,
The enzyme in the bacterial cells was extracted, and the precipitate and the supernatant were separated by centrifugation to obtain a crude enzyme solution as an extract (supernatant).

The results of measuring the activity of the obtained enzyme solution are shown in Table 1 below.

[0057]

[Table 1]

From Table 1 above, it is clear that by using the transformant of the present invention, a significant amount of N-acylneuraminic acid aldolase can be obtained in a sialic acid-free medium. The target enzyme-producing ability of the transformant of the present invention is the transformant Escherichia coli K12C60 transformed by introducing pMK6 into a chromosomal DNA donor bacterium containing the N-acylneuraminic acid aldolase gene.
0 / pMK6 and the Escherichia coli M8328 strain, which is the parent strain of the transformant of the present invention, show that the enzyme production ability is very remarkable, which is quite unexpected.

The crude enzyme solution can be made into a crude enzyme powder by salting it 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. ⁶ Identification number Office reference number FI technical display location // (C12N 9/88 C12R 1:19) (C12N 1/21 C12R 1:19) (C12N 15 / 09 C12R 1:19) C12R 1:19)

Claims (1)

[Claims] 1. A mutant strain of Escherichia coli mutated to express N-acylneuraminic acid aldolase-producing ability in the absence of an inducer, which is an N-acylneuraminic acid aldolase-producing strain belonging to the genus Escherichia.
-N-characterized by introducing a recombinant plasmid into which a chromosomal DNA fragment containing an acylneuraminate aldolase gene has been introduced, culturing the resulting transformant, and collecting N-acylneuraminate aldolase from the culture.
A method for producing an acylneuraminic acid aldolase.