JP2632373B2 - DNA fragment containing β-galactosidase producing gene and plasmid incorporating the DNA fragment - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION INDUSTRIAL APPLICATION The present invention relates to a β-derived from Lactobacillus bulgaricus.
DNA fragment containing galactosidase producing gene, and the DNA
It relates to a plasmid in which the fragment has been incorporated into a vector.

2. Description of the Related Art In recent years, genetic engineering techniques, mainly recombinant DNA experimental techniques, have rapidly developed.

In particular, as basic research on recombinant DNA experimental technology,
Expression vectors have been actively developed, and various expression vectors have been constructed particularly in Escherichia coli systems. By incorporating a DNA fragment containing the gene of interest into these expression vectors, substance production and expression in the host are performed.

Using this recombinant DNA experimental technique, a thermostable β-galactosidase gene derived from Bacillus stearothermophilus was inserted into an expression vector to form a recombinant DNA, and Bacillus stearothermotherm was transformed with the Bacillus subtilis transformed with the recombinant DNA. A method for producing a heat-resistant β-galactosidase derived from Filus (Japanese Patent Application Laid-Open No. 61-81788), and a recombinant DNA obtained by incorporating a thermostable β-galactosidase gene derived from the thermophile Thermos aquaticus into an expression vector, A method for producing thermostable β-galactosidase derived from Thermus aquaticus in Escherichia coli transformed with recombinant DNA (Japanese Patent Application Laid-Open No. 62-208285) is disclosed.

β-galactosidase is an enzyme also called lactase, and β-galactosidase is a disaccharide contained in milk of mammals.
It is also known that they act on the 1,4 bond to produce glucose and galactose, and that their synthesis produces oligosaccharides in which galactose is 1,6-linked to lactose. Accordingly, β-galactosidase has been actively used for the purpose of improving the quality of milk and dairy products and developing new uses. Further, β-galactosidase has been used as a labeling enzyme in enzyme immunoassays.

β-galactosidase is used in plants, animal small intestine, and
Widely distributed in microorganisms such as yeast, mold and bacteria,
Its nature varies from source to source. Among them, Lactobacillus bulgaricus, a lactobacillus, has been used as a starter for lactic acid bacteria in fermented milk, and the properties of β-galactosidase contributing to lactic acid fermentation have been studied. It is very difficult to produce β-galactosidase using Lactobacillus bulgaricus because it is complicated and the operation of separating and purifying the enzyme is complicated. Moreover, at present, no attempt has been made to produce a large amount of Lactobacillus bulgaricus-derived β-galactosidase using recombinant DNA experimental techniques.

Problems to be Solved by the Invention The present inventors, the chromosome of lactobacilli Lactobacillus bulgaricus capable of producing β-galactosidase, by using a restriction enzyme to remove a DNA fragment containing a β-galactosidase producing gene, By producing a plasmid in which a DNA fragment was incorporated into a vector DNA, it was possible to produce a large amount of β-galactosidase derived from Lactobacillus bulgaricus, and thus completed the present invention.

Therefore, it is an object of the present invention to provide a plasmid in which a DNA fragment containing a gene for producing β-galactosidase of Lactobacillus bulgaricus is incorporated into a vector DNA.

 Hereinafter, the present invention will be described in detail.

Means for Solving the Problems The chromosomal DNA of the lactobacillus Lactobacillus bulgaricus producing β-galactosidase in the present invention can be isolated as follows.

After culturing and collecting the lactobacillus Lactobacillus bulgaricus SBT0034 strain, a mutanolysin solution is added and the mixture is incubated for a certain period of time. Next, after adding an SDS solution to lyse, DNA is extracted by phenol and chloroform treatment. DNA
Was recovered by ethanol precipitation, treated with RNase,
Obtain chromosomal DNA.

Chromosomal DNA is completely cut with restriction enzymes. The restriction enzyme is desirably: restriction enzyme Sal I, which is subjected to agarose electrophoresis to collect and purify a DNA fragment having a size of about 3 kb to 10 kb. On the other hand, any vector may be used as long as it is a vector suitable for use in the present invention, but Escherichia coli plasmid pBR329 is preferable. This vector is cleaved with the same restriction enzyme as that used for cleaving the chromosomal DNA, for example, restriction enzyme SalI, and the DNA fragment is ligated thereto using T4 ligase. In this way, a plasmid having the β-galactosidase-producing DNA gene incorporated into the vector is obtained. An example of a restriction map of this plasmid is shown in FIG. 1 (see Example 1).

This plasmid pBG1 has a plasmid pLZ15 (28.3 kb) containing a β-galactosidase gene derived from Lactobacillus casei which is conventionally known as about 8.3 kb (FEMS Microbiolo
gy Letters, Vol. 44, No. 2 (1987), pp. 173-177), is easy to integrate into host cells, and is stable after integration.

The recombinant plasmid obtained as described above was
Transform Escherichia coli deficient in the galactosidase gene. Selection of Escherichia coli harboring a vector DNA into which a DNA fragment containing a β-galactosidase producing gene of Lactobacillus bulgaricus SBT0034 strain was incorporated
After culturing on an agar plate medium containing bromo-4-chloro-3-indolyl-β-D-galactopyranoside (hereinafter referred to as X-Gal) and ampicillin, X-Gal is degraded by β-galactosidase. Thus, by selecting colonies that exhibited a blue color.

Extraction of a plasmid from Escherichia coli can be performed by the following conventional method. The Escherichia coli carrying the plasmid is collected by culture. After adding a lysozyme solution to the cell suspension and keeping it warm for a certain period of time, an SDS solution is added to lyse the cells. After adding sodium chloride solution and leaving it on ice for a certain time,
The supernatant was obtained by centrifugation, and polyethylene glycol 60
Precipitate the DNA by adding 00 and collect. The DNA is dissolved in a buffer and subjected to ethidium bromide-cesium chloride equilibrium density gradient ultracentrifugation to obtain plasmid DNA. The plasmid DNA thus obtained can be transformed again into E. coli.

The DNA fragment containing the β-galactosidase-producing gene obtained as described above was used for the Lactobacillus lactobacillus.
The origin of the chromosomal DNA of Bulgaricus is confirmed by Southern blot hybridization, and the β-galactosidase activity is confirmed by activity staining.

Example 1) Preparation and Cleavage of Chromosomal DNA from Lactobacillus Lactobacillus bulgaricus SBT0034 Strain: Lactobacillus lactobacillus bulgaricus SBT0034 strain (Microtechnical Laboratory No. 10081) was added to 100 ml of MRS BROTH (DIFC
O) and cultured overnight at 37 ° C. to collect the cells. STM cells
Buffer (10 mM Tris-malate, 1 M sucrose, pH 6.
After washing twice in 5), the cells were suspended in 2 ml of the same buffer. 200 μl of mutanolysin (500 μg / ml) is added to this, and
After incubating for 3 minutes, Tris-SDS (0.1 M Tris-HCl, 0.
3M of 1M NaCl, 1% SDS, pH 9.0) was added, and the mixture was kept at 65 ° C for 15 minutes. Phenol was added thereto, the mixture was stirred, centrifuged to collect the upper layer, and chloroform: isoamyl alcohol (24: 1) was added for extraction. To the obtained chromosomal DNA solution, 2 volumes of cold ethanol was added, and the mixture was collected by centrifugation. TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
Dissolved in 5 ml.

Next, 100 μl of RNase (2 mg / ml) was added, and the mixture was kept at 37 ° C. for 1 hour. The obtained chromosomal DNA was dissolved in 2 ml of TE buffer.

Take an appropriate amount of the chromosomal DNA solution, add the restriction enzyme Sal I thereto, react at 37 ° C., cut completely, electrophorese on 1% agarose, and extract the DNA fraction having a size of about 3 kb to 10 kb. The product was recovered by electrophoretic elution, phenol treatment, chloroform extraction, and ethanol precipitation. The recovered DNA was dissolved in a TE buffer.

2) Cleavage and dephosphorylation of vector DNA: 0.2 μg of vector DNA pBR329 was added to Sal I1 restriction enzyme.
10 mM Tris-HCl (pH 8.0), 7 mM M
After reacting at 37 ° C. for 1 hour in a reaction solution of gCl ₂ , 150 mM NaCl, and 7 mM 2-mercaptoethanol, the mixture was recovered by phenol treatment, chloroform extraction, and ethanol precipitation. The recovered DNA was dissolved in a TE buffer.

20 μl of a 10-fold concentration of BAP buffer (0.5 M Tris-HCl, pH 8.0) was added to the Sal I-digested DNA solution (containing 1 μg of DNA), and sterile distilled water was further added to make the whole 200 μl. To this, 0.5 unit of Escherichia coli alkaline phosphatase was added and reacted at 60 ° C. for 1 hour, followed by phenol treatment and ether extraction. To the extracted DNA solution, 25 μM of 3M sodium acetate was added, and recovered by ethanol precipitation. The recovered DNA was dissolved in a TE buffer.

3) Insertion of chromosomal DNA fragment into vector DNA: chromosomal DNA fragment and vector DNA described in 1) and 2) above
Were mixed at a ratio of about 3: 1 and ligated using a DNA ligation kit (Takara Shuzo) to obtain a recombinant plasmid. The ligation method followed the instructions of the ligation kit.

4) Transformation of recombinant plasmid into Escherichia coli: Add the recombinant plasmid described in the above 3) to competent cells of Escherichia coli JM105 strain, leave on ice for 30 minutes, and treat at 42 ° C for 2 minutes. did. This was added to 1.5 ml of LB medium (0.5% sodium chloride, 1% tryptone, yeast extract).
0.5%) and cultured at 37 ° C. for 1 hour. This culture was mixed with ampicillin (50 mg /) and X-Gal (40 mg /
) Was spread on an LB agar medium containing) and cultured overnight at 37 ° C to obtain a transformant of E. coli resistant to ampicillin.

5) Selection of E. coli producing β-galactosidase: Among the Escherichia coli transformants obtained as described in 4) above, the produced β-galactosidase decomposed X-Gal to give a blue color. Colonies were selected.

6) Extraction of plasmid from Escherichia coli: Extraction of the plasmid from the Escherichia coli transformant producing β-galactosidase obtained in the above section 5) was carried out by the following conventional method.

Escherichia coli carrying the plasmid is collected by culture and wet weight 2 g
Were suspended in 5 ml of a buffer solution (25% sucrose, 50 mM Tris-hydrochloric acid, pH 8.0), and a lysozyme solution (5 mg /
1 ml, 0.25 M EDTA (pH 8.0) 2 ml and RNase (5 mg / m
l) 0.2 ml was added and left on ice for 5 minutes. 3 at 37 ° C
After heating for 1 minute, 1 ml of 10% SDS was added, and the mixture was kept at 37 ° C. for 1 minute. Further, 2.5 ml of 5M NaCl was added, and the mixture was left on ice for 3 hours, and then centrifuged to extract an upper layer (cleared lysate). An equal volume of a 20% polyethylene glycol 6000 solution was added and left overnight at 4 ° C. to precipitate the plasmid.

The plasmid was recovered by centrifugation, dissolved in a small amount of TE buffer, and subjected to ethidium bromide-cesium chloride equilibrium density gradient ultracentrifugation to obtain a plasmid. The plasmid obtained by incorporating the DNA fragment containing the β-galactosidase producing gene from Lactobacillus bulgaricus SBT0034 obtained in this way was named pBG1,
The restriction map is shown in FIG.

7) Southern blot hybridization: In order to confirm that the DNA fragment containing the β-galactosidase producing gene incorporated into pBG1 was derived from the chromosomal DNA of Lactobacillus bulgaricus SBT0034, Southern blot hybridization was performed. I went.

Transfer of the DNA to the membrane was in a conventional manner. The probe used was biotin-dUTP (BRL), the hybridization used a blue gene kit (BRL), and the experimental method followed the kit instructions.

As a result of the above Southern blot hybridization, the probe was a Sal1 I digest of pBG1 and a SalI-Pst of pBG1.
DNA cut portion integrated into pBG1 of the I-cut and the chromosomal DN of Lactobacillus lactobacillus bulgaricus SBT0034 strain
The DNA fragment integrated with pBG1 in the digestion product of the restriction enzyme A with SalI hybridized to a DNA portion of the same size, but the host bacteria E. coli JM105 strain (2) and β-
It did not hybridize with the chromosomal DNA of Escherichia coli HB101 strain (1) having galactosidase activity.

From the above, it was clarified that the DNA fragment containing the β-galactosidase producing gene integrated into pBG1 was derived from the chromosomal DNA of Lactobacillus lactobacillus vulgaricus SBT0034 strain.

8) β-galactosidase activity staining: β-galactosidase produced by Escherichia coli JM105 strain carrying pBG1 is different from Lactobacillus lactobacillus bulgaricus.
The activity was confirmed by activity staining to be consistent with β-galactosidase produced by the SBT0034 strain.

Escherichia coli JM105 strain (holding pBG1), Escherichia coli JM105 strain (holding only vector DNA pBR329), Escherichia coli HB101 strain and Lactobacillus lactobacillus bulgaricus SBT0034 strain were collected and cultured, and 10 mM Tris-HCl buffer (pH 8.0) was used. ). This was subjected to an ultrasonic crusher to collect and concentrate the intracellular extract, followed by SDS-polyacrylamide electrophoresis. The gel after electrophoresis was immersed in an X-Gal solution to perform activity staining.

As a result, β-galactosidase activity was detected in 1, 3 and 4, but no activity was detected in Escherichia coli JM105 strain carrying only vector DNA pBR329.

The β-galactosidase activity of the Escherichia coli JM105 strain carrying pBG1 is detected with the same electrophoretic value as the activity of the Lactobacillus lactobacillus bulgaricus SBT0034 strain, and is detected with a different electrophoretic value from the activity of the Escherichia coli HB101 strain. Was.

From the above, it was confirmed that β-galactosidase produced by Escherichia coli JM105 strain carrying pBG1 was identical to β-galactosidase produced by Lactobacillus lactobacillus bulgaricus SBT0034 strain.

Therefore, when this result is combined with the result of the above item 7),
The DNA fragment containing the β-galactosidase producing gene incorporated into pBG1 is derived from the chromosomal DNA of Lactobacillus lactobacillus bulgaricus SBT0034,
It is concluded that the β-galactosidase produced by this gene is identical to the β-galactosidase produced by Lactobacillus lactobacillus bulgaricus SBT0034 strain.

[Brief description of the drawings]

FIG. 1 shows a restriction map of a plasmid incorporating a DNA fragment containing a β-galactosidase producing gene according to the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical indication (C12N 9/38 C12R 1:19) (56) Reference Kor. J. Appl. Microbiol. Bioeng. , Vol. 14, No. 1 (1986), p. 75-84 FEMS Microbiology Letters, Vol. 44, No. 2 (1987), p. 173-177

Claims (6)

(57) [Claims] (1) Lactobacillus bulgaricus (Lactobac)
(Illus bulgaricus) -derived DNA fragment containing a β-galactosidase producing gene. 2. A DNA fragment obtained by cutting the chromosome of Lactobacillus bulgaricus with a restriction enzyme SalI and containing a β-galactosidase producing gene. 3. Lactobacillus bulgaricus is Lactobacillus bulgaricus SBT0034 (Microtechnical Laboratory No. 10081).
The DNA fragment according to claim 1 or 2, wherein 4. A plasmid having the ability to produce β-galactosidase, wherein the DNA fragment according to claim 1 is incorporated into a vector. 5. The plasmid according to claim 4, wherein the vector is Escherichia coli plasmid pBR329. 6. An approximately 8.3 kb plasmid pBG1 which is represented by the following restriction map and has β-galactosidase expression ability.