JPH02222675A - Mutant bacillus subtilis - Google Patents

Abstract

NEW MATERIAL:Bacillus subtilis producing an enzyme forming maltotetraose. USE:It is used to produce maltotetraose which is used as a clinical reagent or a functional food material. PREPARATION:For example, a donor bacterium which is capable of producing an enzyme forming maltotetraose such as Pseudomonas saccharophila is cultured, the culture mixture is centrifuged to collect cell bodies of the microorganism. The cell bodies are subjected to bacteriolysis to collect DNA in a usual manner and the DNA is cut with a restriction enzyme and the segments are bonded to the vector to give a recombinant DNA. The recombinant DNA is introduced into Bacillus subtilis to effect transformation. Then, the microorganism is cultured to measure the enzyme activity of maltotetraose formation and select the strain of high capability of producing maltotetraose whereby a bacillus subtilis including the gene of the enzyme forming maltotetraose is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel Bacillus subtilis into which a novel plasmid incorporating a maltotetraose-generating enzyme gene has been introduced.

[Conventional technology]

Maltotetraose producing enzyme (Ec3.2.1.60.
Ex.

maltotetraohydrolase) is 19
Pseudomonas szutzzeri (Pseudomonas
It was first discovered in the culture fluid of A. tutzert). In recent years, it has been revealed that the maltotetraose-producing enzyme is also produced by Pseudomonas saturophila.

[Problem to be solved by the invention]

Maltotetraose is in increasing demand as a clinical reagent and functional food material. However, the amount of maltotetraose-producing enzyme produced by conventional microorganisms is low and unstable, and the culture medium contains many impurities, making it difficult to separate and purify the maltotetraose-producing enzyme.

The present invention provides a novel recombinant Bacillus subtilis in which the maltotetraose-producing enzyme gene is incorporated into various vectors and introduced into a host microorganism, thereby facilitating the separation and purification of the maltotetraose-producing enzyme.

[Means to solve the problem]

In order to achieve the above object, the present inventors cloned a maltotetraose-producing enzyme gene from a maltotetraose-producing enzyme-producing bacterium, and then incorporated the gene into various vectors and introduced it into a host microorganism. The present invention was completed by successfully causing the obtained recombinant microorganism to produce maltotetraose-producing enzyme.

Specifically, the present invention relates to Bacillus subtilis that produces a maltotetraose-producing enzyme.

The present invention will be specifically explained below.

The DNA of a donor microorganism capable of producing a maltotetraose-producing enzyme can be prepared by culturing the donor microorganism, collecting the resulting culture by centrifugation, and then lysing it.

As a bacteriolysis method, treatment with a cell wall lytic enzyme such as lysozyme, ultrasonication, etc. are used. Further, if necessary, other enzyme agents such as protease and ribonuclease, and surfactants such as sodium lauryl sulfate are used in combination. Furthermore, freezing and thawing treatment may be performed. In order to separate and purify DNA from the lysate thus obtained, conventional methods include phenol extraction, protein removal treatment, protease treatment, ribonuclease treatment, and alcohol precipitation.

This can be carried out by appropriately combining methods such as centrifugation.

DNA can be cut by ultrasonication, restriction enzyme treatment, or the like. After the cleavage, a modifying enzyme such as phosphatase or DNA polymerase is used as necessary. Furthermore, the base sequence at the end of the DNA fragment can be changed by using various linkers and adapters.

From a mixture of cut DNA fragments of various lengths, only fragments of optimal length can be obtained by sucrose density gradient centrifugation, extraction from electrophoresed gels, etc.

As a vector, a phage or a plasmid that can autonomously propagate in a host microorganism is suitable.

The method for joining DNA fragments and vector fragments is
Recombinant DNA is created by allowing DNA ligase to act on the A fragment and the vector fragment.

Any host microorganism may be used as long as the recombinant DNA is stable, can grow autonomously, and can express its characteristics.

The method for introducing the recombinant DNA into the host microorganism is a known method, for example, when the host microorganism is Bacillus subtilis, the competent cell method (Gryczan, T.) is used.

J., Contente, S. and Dubn.
au+ o, l J, Bacteriol,.

134, 318. (1978)) or the protoplast method (Chang.

S,, Cohen, S, N, + Mo1. ge
n, Genet, +168+111 (1979)
) etc. can be adopted.

In the case of λ phage DNA, in vitro testing method (Horn, B., Methods in
Enzymology.

68, 299. (1979)) to form λ phage particles, and add these λ phage particles to a culture suspension of Escherichia coli to obtain a special transducing phage that has the ability to produce maltotetraose-producing enzyme. .

A method for selecting a transformed microorganism into which recombinant DNA has been introduced is to culture it in a liquid selection medium and measure the maltotetraose-producing enzyme activity in the culture solution. As the liquid selection medium, a minimal medium or a medium supplemented with an anti-inflammatory substance is used as appropriate depending on the marker on the vector.

To measure enzyme activity, add starch solution to the culture solution and heat at 40°C.
After incubation, the produced maltotetraose is identified and quantified using thin layer chromatography or high performance liquid chromatography.

The resulting maltotetraose-producing enzyme-producing bacteria were cultured at 37°C in a liquid selective medium and subjected to a known method such as the alkaline extraction method (Birnboim, H, C, and Do
ly+J,, Nucleic As1ds Res,
, ? , 1513. (1979)).

〔Example〕

Next, the present invention will be explained in detail with reference to examples.

Example 1 Cloning of maltotetraose producing enzyme gene Bushutomonas saccharophila (Pseudomonas saccharophila
IAM 1504 was placed in a liquid medium containing soluble starch (1 g of soluble starch, polypeptone I
g, 0.1 g of monopotassium phosphate, 0.28 g of dipotassium phosphate, 100 d of water, pH 7.0)
The bacteria were cultured at 0°C for 48 hours, centrifuged to collect and washed, and the obtained bacteria were collected using the method of 5aito and Miura (
Saito, H. and Miura, Bio
chi+s, Bkophys, Acta+ 72+
619 (1964)) to isolate chromosomal DNA. The obtained DNA was dissolved in Tris-HCl/EDTA buffer, restriction enzyme Sau 3A1 (manufactured by Takara Shuzo Co., Ltd.) was added, and the DNA was partially digested at 37°C.
A chromosomal fragment of approximately 2 Kb or more was separated and obtained using sugar density gradient ultracentrifugation.

Phage λL47 (manufactured by Amarjam) was used as a vector, and λL4 cut with Baa Hl (manufactured by Zenshuzo Co., Ltd.) was used.
7 DNA and the chromosomal fragment of approximately 2 Kb or more obtained from the aforementioned Bushtomonas sanrophila strain IAM 1504 were mixed and ligated by adding T4 ONA ligase (manufactured by Zenshuzo Co., Ltd.) (Weiss, B. , 5ab
lon, A.

J., Live, T., R., Fareed, G.
, C., and Richardson.

C,C,,J,Biol,Chew,, 243.
4543 (1968)), and the treated solution was added with an in vitro batching kit (manufactured by Zenshuzo Co., Ltd.) to incubate the treated solution.
Vitro Packaging Fugu method (l(orn, B,, M
methods in Enzymology.

residence, 299. (1979)), the DNA was introduced into phage particles. The phage particles were added to a bacterial suspension of Escherichia coli L95, and 1% soluble starch was added.

Lambda medium containing 1.2% agar (10g Bactodributon)
, 12.5 g of NaC dissolved in 12 ml of water) at 37°
After culturing in C. and spraying an iodine solution onto the plaques that appeared, it was possible to isolate phages that did not cause an iodine reaction as maltotetraose-producing enzyme-producing phages.

The resulting maltotetraose-producing enzyme-producing phage was isolated and cultured with Escherichia coli tag 66 in lambda medium at 37°C. The culture solution was centrifuged to remove host cells, polyethylene glycol was added to aggregate the phage particles, and the phage particles were collected by centrifugation to obtain a new phage. This phage was λGF102
It was named.

The phage particles were dialyzed against a phage suspension buffer containing 50% formamide, and λGF102 phage D
Got NA. Phage λ, GF102 ONA is 46,
The physical map (restriction enzyme cleavage map) is shown in FIG. 1 with a size of 4 Kb. Here, the white part is derived from vector λL47, and the black part is a chromosome fragment. All abbreviations are restriction enzyme cleavage recognition sites.

The λGF102 phage DNA obtained here was dissolved in Tris-HCl/EDTA buffer, and the restriction enzyme Sau 3AI
was added and partially decomposed at 37°C. Chromosome fragments of approximately 2 Kb or more are separated from the degraded product using silica density gradient ultracentrifugation.
The clone was obtained and recloned using plasmid pHY300PLK. Plasmid pHY300PLK (manufactured by Yakult) is 4.9 Kb and is ampicillin resistant (Ap'
) and tetracycline resistance (Tc'), and is cleaved at one site by the restriction enzyme Ba11H1.

After pHY300PIJ, which had been cut at one site with Ram Hl, was treated with alkaline phosphatase (manufactured by Zenshuzo Co., Ltd.), the chromosome fragments of approximately 2 Kb or more obtained from the λGF102 phage were mixed, and T4 DNA ligase was added to perform ligation. Using this treatment solution, the competent cell method (
Ishiwa, H., and 5hibahara
H,,Jpn,J.

Genet, 60.235. (1985)).

The resulting transformed cells were treated with a selective medium of 1% soluble starch and 20 μg/d of tetracycline.

Agar medium containing 1.6% agar (10 g of Bactolibuton,
5g of yeast extract, 5g of NaCit dissolved in water)
The cells were cultured at 37°C to obtain a tetracycline-resistant strain. This tetracycline-resistant strain was treated with tetracycline 20
μg/IR1, containing 1% soluble starch at 37°
The cells were cultured at C for 24 hours, and the presence or absence of maltotetraose-generating enzyme activity in the culture solution was examined by thin layer chromatography.

In thin layer chromatography, the culture solution is diluted with n-butanol/n
- 60″C in a solvent system of propatool/water (315/4)
After performing upward development 12 times, sulfuric acid was sprayed to search for the presence of maltotetraose, and a maltotetraose-producing enzyme-producing strain was successfully isolated.

The resulting maltotetraose-producing enzyme-producing strain was isolated and cultured at 37°C in a medium containing 20 μg/d of tetracycline. Centrifuge the culture solution to collect bacteria.
After washing, the isolated bacterial cells were subjected to alkaline extraction method (Birnboi
m, H, C, and Doly, Jet Nucl
eic^5ids Res,, 7.1513. (19
79)) to obtain a new plasmid. This plasmid was named plasmid pGF11.

Plasmid pGF11 is 8. The physical map of the size of OKb is shown in Figure 2. Here, the white part is derived from the vector pHY300PL, and the black part is the chromosome fragment part. A11l r indicates ampicillin resistance, Tc r indicates tetracycline resistance, and all other abbreviations are restriction enzyme cleavage recognition sites. be.

Bacillus subtilis ISW transformed with plasmid pGF11
1214-1) GPII has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERM P-10412.

::(7) Cut pGFll DNA with Xhol (manufactured by Takara Shuzo Co., Ltd.) to obtain a 2.3 Kb fragment. This fragment was labeled with 32p, and the chromosomal DNA of Bushtomonas saturophila strain IAM1504, the chromosomal DNA of Bacillus subtilis strain 15W1214, and λGF102 were used.
Southern hybridization with respective Xho1 digests to ON (Southern, E, M,.

J, Mo1. Bio! ,+Box, 503. (197
5)) was performed. Bacillus subtilis strain chromosomal DNA
However, a 2 to 3 Kb band was found that specifically hybridized to the M1504 strain chromosomal DNA and λGF102ON^ to Bushtomonas saturophila ■. From this, it was confirmed that the cloned DNA was derived from Bushtomonas saccharofilae strain IAM1504.

Example 2 Recloning of maltotetraose-generating enzyme gene into Bacillus subtilis strain NA64 using plasmid pUB110EX pGFll obtained in Example 1 was treated with restriction enzyme Xhol. This reaction solution was subjected to 0.7% agarose electrophoresis, and 5,5 Xb DNA and 2.3 Kb DNA containing the maltotetraose-generating enzyme gene were observed
Of A, only 2 and 3 b DNA was recovered and purified using a commercially available DNA purification kit "GENECLEAN" (manufactured by Funakoshi Co., Ltd.).The gene was introduced using Bacillus subtilis N.
^64 was used as the host, and plasmid pUB110EX (4, 5 and b) was used as the vector. pUBlloEX has an Xhol linker (
This is a plasmid derivative inserted with Takara Shuzo Co., Ltd.). 2. This pUBlloEX was treated with Xhol and then treated with alkaline phosphatase, containing the previously recovered and purified maltotetraose purification enzyme gene. :lb DN
The A fragments were mixed and ligated by adding T40N^ ligase.

Using this treatment solution, Bacillus subtilis strain NA64 was subjected to the competent cell method (Gryczan+ T, J, .
Content.

S., and Dubnau, D., J. Bact.
eriol, 134+318° (197 g)).

The obtained transformed cells were transferred to a selective medium, LG medium containing 1% soluble starch and kanamycin lOag/ml (10 g of batatotributone, 5 g of yeast extract, NaCj
25 g, glucose 2 g dissolved in 11 water) at 37°
The cells were cultured at C for 24 hours, and the presence or absence of maltotetraose-generating enzyme activity in the culture solution was examined by thin layer chromatography in the same manner as in Example 1. As a result, we were able to isolate seven maltotetraose-producing enzyme-secreting strains.

The obtained maltotetraose-producing enzyme producing strain was incubated at 37°C in LG medium containing 10 tt g/d of kanamycin.
The culture solution was centrifuged to collect the bacteria, and after washing, the plasmid was isolated from the isolated bacteria by an alkaline extraction method. This plasmid was named plasmid pH3DO1.

Plasmid pH5DQI has a size of 6.9 Kb, and its physical map is shown in FIG. Here, the white part is derived from vector pUB110EX, the black part is a chromosome fragment part, and Km'(neo') indicates kanamycin resistance and neomycin resistance. All other abbreviations are restriction enzymes.

Bacillus subtilis N transformed with plasmid pH5DO1
A64-pH5DO1 has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERM P-10413.

(Effects of the Invention) According to the present invention, a new recombinant microorganism is obtained in which a maltotetraose-generating enzyme gene is inserted into various vectors and introduced into a host microorganism, and this recombinant microorganism stably produces a polytetraose-generating enzyme having maltotetraose-generating enzyme activity. It was discovered that peptides can be produced. According to the present invention, it is possible to increase the supply amount of a polypeptide having maltotetraose-generating enzyme activity, which is of great significance.

[Brief explanation of the drawing]

Figure 1 is a physical map of phage λGF102, Figure 2 is a physical map of plasmid pGF11, and Figure 3 is a physical map of plasmid pGF11.
The figures each show a physical map of plasmid p)15001. No.

Claims (1)

[Claims] Bacillus subtilis produces maltotetraose-producing enzyme.