JPS62122584A - Production of n-acylneuraminic aldolase - Google Patents

Abstract

PURPOSE:To easily obtain the titled enzyme on an industrial scale, by transforming a specific microorganism e.g. with a chromosome DNA fragment containing an N-acylneuraminic aldolase (N-A) gene and culturing the transformed microorganism. CONSTITUTION:A mutant (A) such as Escherichia coli M8328 is produced by a mutagenic treatment of e.g. Escherichia coli belonging to Escherichia genus. An N-A producing strain of Escherichia genus is treated by phenol, etc., to obtain a chromosome DNA, which is cut with a restriction enzyme, etc. The obtained chromosome DNA fragment is inserted and linked to a vector DNA [e.g. strain of ColEI] to obtain a recombinant plasmid (B). The component B is introduced into the strain A to form a transformed strain (C). The strain C is cultured in a liquid medium containing a carbon source, a nitrogen source, etc., and free from sialic acid at 20-45 deg.C and 6-9pH for 10-50hr under aeration and agitation. N-A produced and accumulated in the cell of the strain C is collected from the cell.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel microorganism belonging to the genus Escherichia that has been transformed by introducing a recombinant plasmid containing an N-acylneuraminic acid aldolase gene, and N
- A method for producing acylneuraminic acid aldolase.

Conventional technology N-acylneuraminic acid aldolase (N-acy+n
eurainate aldolase') is
Also known as sialic acid aldolase, enzyme number 1 of the Enzyme Committee of the International Union of Biochemistry: , C, 4, 1, 3, 3,
The systematic name is N-acylneuraminic acid:pyruvic acidase (N-acylneuraminic acid).
ate: an enzyme called pyruvate lyase). This enzyme, as shown in the reaction formula below,
It is an enzyme that catalyzes the decomposition and synthesis reactions of sialic acid (N-acylneuraminic acid).

Sialic acid 1N-acylmannosamine + pyruvate When the present inventors culture known bacteria belonging to the genus Escherichia and several other genera in the presence of sialic acid, the above-mentioned N-acylneuraminic acid aldolase discovered that it could be easily produced on an industrial scale, and established a manufacturing technology for this enzyme (Japanese Patent Publication No. 54153/1983, Patent No. 1)
No. 111346).

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 was added, N-acylneuraminic acid aldolase would not be produced or would only be produced in a very small amount, making it impossible to carry out industrial implementation of the basolateral process.

That is, the microorganisms used in the above method cannot substantially exhibit N-acylneuraminic acid producing ability in the absence of sialic acid.

The present inventors have solved the biggest drawback of the above method, which is that it requires the use of sialic acid, and have developed a technology that can produce large amounts of N-acylneuraminic acid aldolase on an industrial scale without using the sialic acid. As a result of intensive research to develop the microorganisms used in the above method, we have found that mutant strains obtained by mutagenic treatment of those belonging to the genus Escherichia can be used in a medium without the addition of inducers such as sialic acid or its analogs. It was discovered that a significant amount of the target enzyme was produced, and based on this knowledge (Japanese Patent Laid-Open Publication No. 184384/1984 for the purpose of completing the invention).

In subsequent studies, the present inventors also extracted a chromosomal DNA fragment containing the N-acylneuraminic acid aldolase gene from a microorganism belonging to the genus Escherichia used in the established method, and extracted it from Escherichia coli. In addition to successfully creating a recombinant plasmid by integrating it into a system vector, the N-acylneuraminic acid aldolase-producing bacteria belonging to the genus Escherichia or a strain deficient in the enzyme transformed with the plasmid were transformed in the same way as the mutant strain described above. He also discovered that it was possible to produce the desired silicon in a medium without the addition of inducing substances such as sialic acid, and completed an invention based on this knowledge (Japanese Patent Application No. 181-250-1981)
issue).

Problems to be Solved by the Invention The purpose of the present invention is to provide an extremely superior enzyme production ability that surpasses that of the conventional technology described above, as well as the technology newly developed by the present inventors. The object of the present invention is to establish an improved transformed microorganism having the following properties and a technology for producing a target enzyme using the microorganism.

Means for Solving the Problems According to the present invention, there is provided a mutant strain of horses belonging to the genus Escherichia that has been mutated to express the ability to produce N-acylneuraminic acid aldolase in the absence of an inducer, A transformed microorganism characterized in that it has been introduced with a recombinant plasmid into which a chromosomal DNA fragment containing an N-acylneuraminic acid aldolase gene derived from an N-acylneuraminic acid aldolase-producing bacterium is introduced, and the microorganism is cultured. Provided is a method for producing N-acylneuraminic acid aldolase, which comprises collecting N-acylneuraminic acid aldolase from a culture.

According to the present invention, when the above-mentioned mutant strain previously developed by the present inventors is used as a host and transformed by introducing a specific plasmid developed previously, the transformed strain surprisingly shows The fact that the transformed strain according to the present invention expresses an extremely excellent ability to produce the desired enzyme, which is completely unexpected from either the mutant strain used as a host or the transformed strain, This work was completed based on the discovery that the system was improved in terms of performance. Therefore, according to the method using the transformed mutant strain of the present invention, it is possible to easily produce and collect a target enzyme in extremely large amounts on an industrial scale, which was previously impossible, using a normal microbial culture medium. I can do it.

The method for producing a transformed strain according to 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. The mutant strain can be obtained by applying various known mutation treatment methods to various known microorganisms of the genus Escherichia, as detailed in the aforementioned Japanese Patent Application Laid-Open No. 60-184384. As an example of the above-mentioned mutant strain that is particularly preferably used in the present invention, Escherichia coli M8328 (E. No. 832, FERM BP-832).

The transformed microorganism of the present invention includes the above-mentioned mutant strain, as well as
It can be obtained by introducing a specific plasmid containing the N-acylneuraminic acid aldolase gene into a mutant strain obtained by similarly mutating various microorganisms capable of producing N-acylneuraminic acid aldolase in a medium supplemented with sialic acid.

The above plasmid is produced by integrating 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 into an Escherichia coli vector. The bacteria of the genus Escherichia used as the chromosomal DNA donor here include, for example, Escherichia coli (E.

It is preferable to use bacteria that are highly productive of N-acylneuraminic acid aldolase, such as E. coli, but there is no particular limitation, and any known various bacteria can be used as long as they have the ability to produce N-acylneuraminic acid aldolase. .

Preparation of chromosomal DNA from the above-mentioned N-acylneuraminic acid aldolase-producing bacteria can be carried out using a conventional method, such as a method using phenol (3aito-M 1ura method, Bio
chim, Biophys, Acta, ,
72. 619 (1963)) etc.

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, and as long as the N-acylneuraminic acid aldolase gene is not cleaved, for example, the chromosomal DNA can be cleaved by applying a physical shearing force. It is also possible to use other methods.

When carrying out a method of cleaving chromosomal DNA using restriction endonucleases, when adopting reaction conditions that cause complete cleavage, various restriction endonucleases that do not have a cleavage site in the target N-acylneuraminic acid aldolase gene may be used. 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-mentioned vectors include, for example, the Co1E1 strain,
pscroll strain, pBR322 strain, p ACY
Examples include the Cl77(7) strain, the pCRl strain, the R6 strain, and the lambda phage strain.

The above-mentioned chromosomal DNA and vector DNA can be linked by a commonly used method, such as cutting the donor chromosome and vector with the same restriction endonuclease, and then linking them together using DNA ligase. However, the method is not limited to this method, and any other method may be used.

The recombinant plasmid thus obtained (donor chromosomal DNA
The conjugate of the fragment and the vector is then introduced into the mutant strain belonging to the genus Escherichia, which is the host (recombinant plasmid recipient). The method for introducing the above-mentioned plasmid into the host bacterium for transformation is typically, for example, a transformation method using competent cells (Mo1. 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.

The target N-
Selective isolation of a target microorganism that possesses a donor chromosomal DNA fragment containing the acylneuraminic acid aldolase gene and has the ability to produce this enzyme in significant amounts in the absence of inducers such as sialic acid does not require any special method. Both can be carried out by conventional methods, for example, by using a medium that can selectively grow bacterial clones that have the desired genetic trait on the chromosome, the trait possessed by the vector, or a combination of both.

In this way, it is possible to obtain the desired transformed strain having the ability to produce the well-known N-acylneuraminic acid aldolase in a sialic acid-free 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 nitrogen sources include ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium phosphate, ammonium hydroxide, ammonium tartrate,
Examples include 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 amorphous compounds include potassium phosphate, dipotassium phosphate, sodium phosphate, disodium phosphate, magnesium sulfate, magnesium chloride, potassium chloride, sodium chloride, ferrous sulfate, ferrous chloride, and ferrous sulfate. Examples include diiron, ferric chloride, 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 usually 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 culture for usually 10 to 50 hours, the activity of the target N-acylno-ilamate aldolase reaches its maximum.

Since this enzyme is generally an intracellular enzyme, the enzyme can be collected and purified from a culture according to a conventional method for collecting and purifying intracellular enzymes. As a particularly preferable representative example, for example, Kokutai is collected from the above-mentioned culture solution by a method such as centrifugation, and the obtained Kokutai is crushed by ultrasonication, grinding using glass beads, French press processing, etc. , a method for extracting enzymes can be exemplified. Further, the extract obtained above can be treated by conventional methods such as ammonium sulfate salting out method, ion exchange chromatography, gel filtration method, etc., and thus purified N-acylneuraminic acid aldolase can be obtained.

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples. still,
The activity of N-acylneuraminic acid aldolase was determined by the method of Barnett et al. (J, E, G.

3arnett, Q, 1. Corina, a
nd G, Ra5ool.

Biochemical Journal, 1
25, 275 (1971)), and one unit of the enzyme activity refers to the activity of decomposing 1 micromole of N-acetylneuraminic acid per minute at a reaction temperature of 37°C.

Example 1 (1) N-acylneuraminic acid aldolase producing strain E
, chromosomal DN from coli K12C600 strain
Preparation of A Escherichia coli (E,
E.coli) K12C600 strain was cultured with shaking at 37°C for about 3 hours, and the cells in the logarithmic growth phase were collected.

<L medium composition> Peptone 1g/dQ Yeast extract 0.5Q/dQ Glucose 0.1 (1/dQ NacQ 0.5Q/dQ Adjusted to pH 7.2) The above bacterial cells were subjected to NA extraction using the phenol method. The final chromosomal DNA of 3.8 mfJ was extracted and purified.

(2) Preparation of vector DNA and cutting with restriction enzymes Plasmid pBR322 DNA as a vector was prepared as follows. That is, first, one of the 12 strains of Escherichia coli carrying pBR322 as a plasmid was inoculated into a GPM medium with the following composition, and after culturing at 37°C until the logarithmic growth phase, chloramphenicol was added at a final concentration of 100 μG/m12. and further cultured overnight.

<GPM composition> Glucose 10g Peptone 10°N)-1t CQ 1(+Na2
HPOt12H2015, 2g KH2POt 3gNa CQ
3QNa2Sot
0.115! JMgCQ2 ・6H200,
083G Yeast Extract 19 Demineralized water The above procedure was performed to produce a large amount of plasmid DNA in the cells.

Collect bacterial cells 16 hours after adding chloramphenicol,
Treat with lysozyme/SDS to lyse, 30000X (
The superior groove was obtained by ultracentrifugation for 1.1 hour. The plasmid DNA was then concentrated to a final 700 μg by cesium chloride-ethidium bromide equilibrium density gradient centrifugation.
The plasmid DNA of DBR322 was fractionated and collected.

(3) Introduction of chromosomal DNA fragment into vector (1)
) was taken and reacted with restriction endonuclease HindI [I for 30 minutes, Co, or 120 minutes at 37°C to partially cleave the DNA, and then heat-treated at 65°C for 10 minutes. The reaction was stopped.

On the other hand, vector pBR322 was completely cleaved using restriction endonuclease Hindln, and then treated with alkaline phosphatase to prepare an o BR322 DNA fragment.

The above chromosomal DNA fragment and l] BR322 DNA fragment 5
μ9 and in the presence of ATP and dithiothreitol, using DNA ligase derived from T4 phage, 1
The DNA strand ligation reaction took 16 hours at 0°C. After heat treatment at 65° C. for 10 minutes, twice the volume of ethanol was added to the reaction solution to precipitate the DNA after the completion of the ligation reaction, and the DNA was collected.

(4) Transformation with recombinant plasmid DNA An N-acylneuraminic acid aldolase-deficient strain induced from Escherichia coli strain 120,600 by nitrosoguanidine mutation treatment was grown in 10 ml of medium until mid-logarithmic growth stage, and then calcium chloride was added. Competent cells (having the ability to take up DNA) were prepared by washing twice with Tris buffer (50mM, ol-17.0) containing 50mM. Add 0.1 ml of the DNA solution obtained in (3) above to 0.4 ml of this competent cell suspension.
42°C immediately after adding a
A 2-minute heat pulse was applied to allow the DNA to be incorporated into the cells.

Next, this cell suspension was inoculated into a medium, statically cultured at 37°C for 2 hours to complete the transformation reaction, harvested, washed, and the resuspension was transferred to a minimal medium plate. Smear, 3
The cells were cultured at 7°C for 2 days.

In addition, the above minimal medium plate contains 2 g of sialic acid, <NH
-) 1a of 2 SO-, 7g of 2 HPO4, K2
Pot (D2Q, MO804・7H20(7)0
．． 1(], 20ma of roroine, 20mg of threonine
and thiamine 11 (J) were dissolved in 1Q of pure water, the pH was adjusted to 7.0, and 20 g of agar was added to sterilize the solid medium. Ampicillin was added at 10 μg/ml to prepare XIIIJ.

The colonies generated above were harvested, and ampicillin (A
p) Resistance and intracellular N-acylneuraminic acid aldolase activity were investigated, and the transformed strain RC-H1/p MK
2 (approximately 14.2 kb) 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 medium 100%, 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 B1rnboia+ and Doly (Nucleic Ac1ds Re5ear) was performed.
pBR322 plasmid (hereinafter referred to as [, I) MK2J, which carries the genetic information of N-acylneuraminic acid aldolase] was
A solution containing the following was prepared.

This solution was subjected to agarose gel electrophoresis (agarose 0.7
The pMK2 band was cut out under ultraviolet irradiation, placed in a dialysis tube, electrophoresed again, and DNA was extracted from the gel. After removing ethidium bromide, 2 volumes of ethanol were added to cause precipitation, and 90 μg of the obtained I) MK2 was dissolved in 5 mM Tris-HCl buffer (pH 7.5).

Next, in the same manner as in (4) above, Escherichia coli Kl 2C600 strain was made to have DNA uptake ability.
The above pMK2 was incorporated. Ampicillin 10 μ of the bacterial strain obtained in this way! It was cultured on a minimal medium plate containing If/m12, the resulting colonies were isolated, and the ampicillin resistance and intracellular N-acylneuraminic acid aldolase activity were examined to determine the transformed strain RC-Kl 2C600.
/p'MK2 was obtained.

(6) Subcloning of N-acylneuraminic acid aldolase gene From the transformed strain obtained in (5) above, 10 μq of plasmid pMK2 was taken by the same method as in (5), and this was treated with two restriction endonucleases, EcoRI and l. -1i
After a partial cleavage reaction was carried out at 37° C. for 1 hour by simultaneously acting with ndll[, the reaction was stopped by heat treatment at 65° C. for 10 minutes.

On the other hand, vector 0BR322 was completely cleaved using restriction endonucleases EcoRI and HindI [I] and then treated with alkaline phosphatase to obtain I)B.
A DNA fragment of R322 was prepared.

The above pMK2 DNA fragment and 5 μg of pBR322 DNA fragment were mixed, and in the presence of ATP and dithiothreitol, using DNA ligase derived from T4 phage,
A recombinant plasmid was prepared by performing a DNA strand ligation reaction at 10°C for 16 hours.

Then, in the same manner as in (4) above, Escherichia Colli K
After the 12C600 strain was endowed with DNA uptake ability, the recombinant plasmid prepared above was allowed to take up the strain.

The bacterial cells thus obtained were cultured on a minimal medium plate containing 10μ9/- of ampicillin, the resulting colonies were isolated, and ampicillin resistance and intracellular N-acylneuraminic acid aldolase activity were examined to determine the transformed strain RC-
12C600/I) MK6 was obtained.

This transformed strain possesses a recombinant plasmid into which a chromosomal DNA fragment containing the N-acylneuraminic acid aldolase gene has been integrated, and the plasmid is
It is called MK6J.

The restriction enzyme cleavage map of pMK6 is shown in FIG. This is derived from pBR322, PSt engineering, pvu ■, 3
A DNA fragment having a cleavage site that is cleaved with al-E and BamHI, respectively, and a chromosomal DNA fragment derived from an N-acylneuraminic acid aldolase-producing bacterium, were cleaved with EC0RI and H
It is ligated at the cleavage site of indlI[, approximately 5.5k
It has a size of b.

In addition, Escherichia spp. carrying the above plasmid pMK6
The 12C600 strain for Escherichia collie was sent to the National Institute of Microbiology, Agency of Industrial Science and Technology.
It has been deposited under the name K6, and its deposit number is MicroKenjo Deposit No. 833.

(7) Preparation of the transformed strain of the present invention pMK6 was extracted from the transformed strain obtained in (6) above in the same manner as in (5) above, and pMK6 was extracted in the same manner as in (4) above. It was incorporated into

That is, after growing Escherichia coli M8328 strain in L medium to the mid-logarithmic growth phase, 50 m
Tris-M buffer containing M (50mM).

Competent cells (having the ability to take up DNA) were prepared by washing twice with pH 7.0). 1 μg of pMK6 obtained above was added to this competent cell suspension.
was added and held at 0°C for 30 minutes, then immediately heated to 42°C.
A 2 minute heat pulse was applied to allow DNA to be incorporated into the 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 with ampicillin 40 μg/mfJ.
The cells were spread on a medium plate containing the following, and cultured at 37°C for 1 day.

The colonies generated above were harvested, and ampicillin (A
p) Resistance and intracellular N-acylneuraminic acid aldolase activity were investigated, and transformed strains E and coli M832
8/I) MK6 was obtained.

The above transformed strain has been deposited with the National Institute of Microbiology, Agency of Industrial Science and Technology under the name Escherichia coli M8328/p MK6, and its deposit number is Escherichia coli M8328/p MK6.
No. 519 (FERM P-8519).

(8) Production of N-acylneuraminic acid aldolase by the transformed strain of the present invention The transformed strain of the present invention obtained in (7) above was transferred to 120,600 strains of Escherichia coli, which is a chromosomal DNA donor bacterium containing the N-acylneuraminic acid aldolase gene. and Escherichia coli strain M8328 which is the parent strain (mutant strain) of the transformed strain of the present invention and the parent strain of the M8328 strain. In contrast to each of the Escherichia coli IFO3301 strains, their sialic acid-free medium (yeast extract medium)
The productivity of N-acylneuraminic acid aldolase was investigated.

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

<Yeast extract medium composition> Yeast extract 20g Succinic acid 10g Pure water Amount to make the total 19 (pH 6,0) Each of the above-mentioned media was inoculated with each microorganism, and shaken culture was performed at 30°C for 24 hours. Thereafter, the bacterial cells were collected from the culture medium by centrifugation, and 25mM phosphate buffer (1) 87.5) 100mQ
The cells were suspended in water and subjected to ultrasonic treatment to disrupt the cells, the enzyme inside the bacterial cells was extracted, and the precipitate and 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.

Table 1 From Table 1 above, according to the use of the transformed strain of the present invention,
It is clear that a significant amount of N-acylneuraminic acid aldolase can be obtained in a sialic acid-free medium. In addition, the ability to produce the target enzyme observed in the transformed strain of the present invention is as follows:
12C600/pM was transformed into a transformed Escherichia coli strain by introducing pMK6 into a chromosomal DNA donor bacterium containing the N-acylneuraminic acid aldolase gene.
Escherichia K6 and the parent strain of the transformed strain of the present invention.
It can be seen that the production ability of the enzyme in each of the coli M8328 strains is quite unexpected and very remarkable.

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 oxygen powder can be purified by means such as ion exchange chromatography and gel filtration to obtain a further purified enzyme preparation.

[Brief explanation of drawings]

Figure 1 shows the recombinant plasmid pMK used in the present invention.
6 shows a restriction enzyme cleavage map of No. 6. (that's all)

Claims (2)

[Claims] (1) Belongs to the genus Escherichia, and N
- A mutant strain that has been mutated to express the ability to produce acylneuraminic acid aldolase, and is derived from an N-acylneuraminic acid aldolase-producing bacterium belonging to the genus Escherichia.
- A transformed microorganism characterized in that it has been introduced with a recombinant plasmid into which a chromosomal DNA fragment containing an acylneuraminic acid aldolase gene has been integrated. (2) Belongs to the genus Escherichia, and N
- A mutant strain that has been mutated to express the ability to produce acylneuraminic acid aldolase, and is derived from an N-acylneuraminic acid aldolase-producing bacterium belonging to the genus Escherichia.
-N- characterized in that a transformed microorganism into which a recombinant plasmid into which a chromosomal DNA fragment containing an acylneuraminic acid aldolase gene has been introduced is introduced, and N-acylneuraminic acid aldolase is collected from the culture.
A method for producing acylneuraminic acid aldolase.