JPH0533028B2 - - Google Patents

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-generating enzyme (EC3.2.1.60,
exo-maltotetraohydrolase) was developed by Robyt in 1971.
It was first discovered in the culture fluid of Pseudomonas stutzeri by Ackerman and Ackerman. In recent years, it has been revealed that the maltotetraose-producing enzyme is also produced by Pseudomonas saccharophila .

[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
The gene was integrated into various vectors and introduced into host microorganisms, and the resulting recombinant microorganisms were successfully made to produce maltotetraose-producing enzymes,
The invention has been completed.

That is, the present invention relates to Bacillus subtilis transformed by introducing a plasmid incorporating DNA encoding a maltotetraose-generating enzyme derived from Pseudomonas satucarophylla.

The present invention will be specifically explained below.

The DNA of a donor microorganism capable of producing maltotetraose-producing enzyme is obtained by culturing the donor microorganism,
It can be prepared by centrifuging the obtained culture to collect the bacteria, and then lysing the bacteria. 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. To separate and purify DNA from the lysate obtained in this way, methods such as phenol extraction, protein removal treatment, protease treatment, ribonuclease treatment, alcohol precipitation, and centrifugation may be combined as appropriate according to conventional methods. This can be done by

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 to create recombinant DNA by allowing DNA ligase to act on the DNA fragments and vector fragments.

The host microorganism may be any host microorganism that has stable recombinant DNA, is capable of autonomous growth, and can express its characteristics, and Bacillus subtilis is used in the present invention.

The method for introducing recombinant DNA into host microorganisms is
Known methods, for example, when the host microorganism is Bacillus subtilis, the competent cell method (Gryczan, TJ, Contente, S. and Dubnau,
D., J. Bacteriol., 134 , 318, (1978) or the protoplast method (Chang, S., Cohen, SNM,
Mol.Gen, Genet., 168 , 111 (1979)) etc. can be adopted.

For λ phage DNA, in vitro packaging method (Hohn, B., Methods in
Enzymology, 68, 299, (1979)) to form λ phage particles, and add these λ phage particles to a suspension of cultured bacteria of Escherichia coli for special transduction that possesses maltotetraose-producing enzyme production ability. You can get Fage.

The method for selecting transformed microorganisms into which recombinant DNA has been introduced involves culturing them in a liquid selection medium and measuring the maltotetraose-producing enzyme activity in the culture solution. Depending on the marker on the vector, a minimal medium or an antibiotic-supplemented medium is appropriately used as the liquid selection medium.

To measure enzyme activity, add starch solution to the culture solution,
After incubation at 40°C, 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 known methods such as alkaline extraction (Birnboim, HCand
Doly, J., Nucleic Asids Res., 7, 1523,
(1979)).

〔Example〕

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

Example 1 Cloning of maltotetraose-generating enzyme gene Pseudomonas saccharophila TAM 1504 was cultured in a liquid medium containing soluble starch (1 g of soluble starch, 1 g of polypeptone, 0.1 g of monopotassium phosphate, 0.28 g of dipotassium phosphate, water) 100ml, PH7.0) at 30℃ for 48 hours, centrifuged to collect bacteria, washed, and the obtained bacteria were used according to the method of Saito, Miura (Saito, H. and
Miura, K., Biochim. Biophya. Acta, 72 , 619
(1963)) isolated chromosomal DNA. The obtained DNA was dissolved in Tris-HCl/EDTA buffer, and the restriction enzyme Sau 3Al (manufactured by Takara Shuzo Co., Ltd.) was added.
After partial digestion at 37°C, chromosome fragments of approximately 2 kb or more were isolated and obtained from the digested product using sucrose density gradient ultracentrifugation.

Phage λL47 (manufactured by Amersyam) was used as a vector, and λL47 DNA cut with Bam HI (manufactured by Takara Shuzo) was mixed with a chromosome fragment of approximately 2 kb or more obtained from the Pseudomonas saccharofila strain IAM 1504, and T4 DNA Ligase (manufactured by Takara Shuzo Co., Ltd.) was added to perform the ligation process (Weiss, B.,
Sablon, AJ, Live, TR, Fareed, GCand
Richardson, CC, Biol.Chem., 243 , 4543
(1968)). An in vitro packaging kit (manufactured by Takara Shuzo Co., Ltd.) was added to the treatment solution to perform the in vitro packaging method (Horn, B., Methods in
Enzymology, 68 , 299, (1979)).
DNA was introduced into phage particles. The phage particles were added to a bacterial cell suspension of Escherichia coli WL95, which contained 1% soluble starch and 1.2% agar.
Medium (10 g of bactotryptone, 2.5 g of NaCl, 1 part of water)
The phages that did not undergo an iodine reaction were isolated as maltotetraose-producing enzyme-producing phages by spraying an iodine solution onto the plaques that appeared.

The resulting maltotetraose-producing enzyme-producing phage was isolated and transformed into Escherichia coli WL66.
and cultured at 37°C in lambda medium. 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 new phages. This phage was named λGF102.

The phage particles were dialyzed against a phage suspension buffer containing 50% formamide to obtain λGF102 phage DNA. Phage λGF102DNA
The physical map (restriction enzyme cleavage map) is 46.4 kb and is shown in Figure 1. 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 obtained λGF102 phage DNA was dissolved in Tris-HCl/EDTA buffer and treated with restriction enzymes.
Sau3AI was added and partially decomposed at 37°C. A chromosomal fragment of approximately 2 kb or more was isolated and obtained from the digested product using sucrose density gradient ultracentrifugation, and the plasmid PHY300PLK was obtained.
Re-cloning was attempted using Plasmid PH
Y300PLK (manufactured by Yakult) is 4.9 kb, has ampicillin resistance ( ^Apr ) and tetracycline resistance (Tc ^r ), and can be cleaved at one site with the restriction enzyme Bam HI.

After treating PHY300PLK cut at one site with Bam HI with alkaline phosphatase (manufactured by Takara Shuzo Co., Ltd.), the approximately
Chromosome fragments of 2 kb or more were mixed and ligated by adding T4 DNA ligase. Using this treatment liquid,
Using Bacillus subtilis strain ISW1214 as a host,
Competent cell method (Ishiwa, H., and
Shibahara H., Jpn.J.Genet., 60 , 235, (1985)
The plasmid was introduced by.

The obtained transformed bacterial cells were transferred to L medium (10 g of bactotryptone, 5 g of yeast extract, and 5 g of NaCl dissolved in 1 part water) containing 1% soluble starch, 20 μg/ml tetracycline, and 1.6% agar. 37
A tetracycline-resistant strain was obtained by culturing at ℃. This tetracycline-resistant strain was treated with tetracycline.
37 in L medium containing 20μg/ml, 1% soluble starch
The cells were cultured at ℃ for 24 hours, and the presence or absence of maltotetraose-generating enzyme activity in the culture solution was examined by thin layer chromatography.

For thin layer chromatography, the culture solution is incubated twice at 60°C in a solvent system of n-butanol/n-propanol/water (3/5/4), and then sulfuric acid is sprayed to remove maltotetraose. We searched for the presence of maltotetraose and were able to isolate a maltotetraose-producing enzyme-producing strain.

The resulting maltotetraose-producing enzyme-producing strain was isolated and cultured at 37°C in L medium containing 20 μg/ml of tetracycline. The culture solution was centrifuged to collect bacteria, and after washing, the isolated bacteria were extracted using an alkaline extraction method (Birnboim, HCand Doly, J., Nucleic
Acids Res., 7, 1513, (1979)), the plasmid was isolated and a new plasmid was obtained. This plasmid was named plasmid pGF11.

Plasmid pGF11 has a size of 8.0 kb, and its physical map is shown in FIG. Here, the white part is derived from vector PHY300PLK, and the black part is a chromosome fragment. Ap ^r indicates ampicillin resistance, Tc ^r indicates tetracycline resistance, and all other abbreviations are restriction enzyme cleavage recognition sites.

Bacillus transformed with plasmid pGF11
Bacillus subtilis ISW1214−
pGF11 was sent to the Institute of Microbial Technology, Agency of Industrial Science and Technology.
It has been deposited as FERM P-10412.

This pGF11 DNA is cut with XhoI (manufactured by Takara Shuzo Co., Ltd.) to obtain a 2.3 kb fragment. This fragment was labeled with ³² P and
IAM1504 strain chromosomal DNA, Bacillus subtilis
ISW1214 strain chromosomal DNA and λ GF102
Southern hybridization with each XhoI digest of DNA (Southern, EM, J. Mo1.
Bio1., 98 , 503, (1975)). Bacillus・
Pseudomonas subtilis strain IAM1504 strain chromosomal DNA and λ
Haubridizes specifically to GF102DNA
A 2.3 kb band was found. From this, it was confirmed that the cloned DNA was derived from Pseudomonas satsukalophylla strain IAM1504.

Example 2 Recloning of maltotetraose-generating enzyme gene into Bacillus subtilis strain NA64 using plasmid pUB11OEX pGF11 obtained in Example 1 was treated with restriction enzyme XhoI. This reaction solution was subjected to 0.7% agarose electrophoresis, and 5.5kb DNA and 2.3kb DNA containing the maltotetraose-generating enzyme gene were observed.
Of the DNA, only 2.3 kb DNA was recovered and purified using a commercially available DNA purification kit GENECLEAN ^™ (manufactured by Funakoshi). For gene introduction, Bacillus subtilis NA64 was used as host, and plasmid pUB11OEX was used.
(4.5kb) was used as a vector. pUB11OEX is a plasmid derivative in which an XhoI linker (manufactured by Takara Shuzo Co., Ltd.) is inserted into the EcoRI site of plasmid pUB11O. This pUB11OEX was treated with XhoI and then treated with alkaline phosphatase, containing the previously recovered and purified maltotetraose purification enzyme gene.
The 2.3 kb DNA fragments were mixed and ligated by adding T4 DNA ligase. Using this treatment solution, the competent cell method was applied to Bacillus subtilis strain NA64 (Gryczan, TJ, Contente, S. and Dubnau,
D., J. Bacterio 1., 134 , 318, (1978)).

The obtained transformed bacterial cells were used as selective medium 1
LG medium containing % soluble starch and 10 μg/ml kanamycin (10 g bactotryptone, yeast extract
5g, NaCl5g, glucose 2g dissolved in 1 part water)
The cells were cultured at 37° 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 a maltotetraose-producing enzyme-secreting strain.

The obtained maltotetraose-producing enzyme producing strain was grown in LG medium containing 10 μg/ml of kanamycin for 37 days.
The cells were cultured at 0.degree. C., the culture solution was centrifuged to collect the bacteria, and after washing, plasmids were isolated from the isolated cells by an alkaline extraction method. This plasmid is plasmid PH
It was named SDO1.

Plasmid PHSDO1 has a size of 6.9 kb, and its physical map is shown in Figure 3. Here, the white part is derived from the vector pUB11OEX, the black part is the chromosome fragment part, and Km ^r (neo ^r ) indicates kanamycin resistance and neomycin resistance.
All other abbreviations are restriction enzymes.

Bacillus subtilis NA64-PH transformed with plasmid PHSDO1
SDO1 is located at the Institute of Microbial Technology, Agency of Industrial Science and Technology.
It has been deposited as FERM P-10413.

〔Effect of the invention〕

According to the present invention, a novel recombinant microorganism is obtained by incorporating a maltotetraose-producing enzyme gene into various vectors and introduced into a host microorganism, and the recombinant microorganism stably produces a polypeptide having maltotetraose-producing enzyme activity. I found it. 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]

FIG. 1 shows a physical map of phage λ GF102, FIG. 2 shows a physical map of plasmid pGF11, and FIG. 3 shows a physical map of plasmid PHSD01.

Claims (1)

[Claims] 1. Bacillus subtilis was transformed by introducing a plasmid into which DNA encoding maltotetraose-producing enzyme derived from Pseudomonas satucarophylla was introduced.