JPH0640827B2 - Novel plasmid, method for constructing the same, and novel microorganism containing the plasmid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel plasmid, more specifically, a plasmid incorporating a deoxyribonucleic acid (DNA) fragment carrying genetic information involved in secretory production of alkaline CGTase, The present invention relates to a method for constructing a plasmid and a novel microorganism containing the plasmid.

[Conventional technology]

Cyclodextrin is a non-reducing substance in which 6 to 8 or more glucose molecules are cyclically linked by α-1,4 bonds. CGTa, an enzyme that produces cyclodextrin
se is classified as a transferase as EC 2.4.1.16. Conventionally, this CGTase is a bacterial species belonging to the genus Bacillus, for example, Bacillus macerans, Bacillus circulans, Bacillus megaterium, Bacillus megaterium.
Bacillus stearothermophilu
s) or the like, or a bacterial species belonging to the genus Klebsiella, for example, Klebsiella pneumoniae or the like, which is obtained by aerobically culturing in a suitable medium having a neutral or weakly acidic pH [“Enzyme Handbook”: Akabori Shiro Supervision, published by Asakura Shoten (Showa 41) and “Amylase”: Reference by Michinori Nakamura, published by Academic Publishing Center (1986), etc.] and aerobic bacteria belonging to the genus Bacillus aerobically in a suitable pH alkaline medium. Method obtained by culturing the cells (Horikoshi, K., Akiba, T.
"Alkalophilic Microorganisms", Academic Center (1
982) and Japanese Patent Publication No. 52-93).

However, in the fermentation production of enzymes by these microorganisms, the culture time is long, scale-up is difficult, and the amount of enzyme production is very small, which makes separation and purification difficult. On the other hand, research on the production of CGTase using genetic engineering was carried out by using the Bacillus macerans-derived CGTase in Bacillus subtilis.
subtilis) and Escherichia col
i) cloned research (Toshino Takano, Kunio Yamane et al .; 7th Annual Meeting of the Molecular Biology of Japan, 1984) and from Bacillus macerans and Bacillus stearothermophilus
A study of cloning CGTase into Bacillus subtilis and Escherichia coli (Tomoo Kubota, Yoshio Tsujisaka et al .; Japan Fermentation Engineering Conference 1985) has already been announced.
No studies have been found on the cloning of an alkaline CGTase from an alkalophilic bacterium belonging to the genus Bacillus into Bacillus subtilis.

[Problems to be solved by the invention]

The present invention is an alkaline solution that solves the above-mentioned problems of the prior art.
It is intended to provide a novel plasmid incorporating a deoxyribonucleic acid (DNA) carrying specific genetic information involved in secretory production of CGTase, a method for constructing the same, and a novel microorganism transformed with the plasmid. .

[Means for solving problems]

A plasmid is an extrachromosomal gene found in the cells of microorganisms, which has recently been used as a means for gene recombination in microorganisms, and its importance in research in the field of the fermentation industry has been increasing.

In recent years, as shown in the examples of amino acids and peptides, studies have been conducted on plasmids incorporating DNA that carries the genetic information involved in the specific requirement for the growth of microorganisms and the ability to produce metabolites, and some The plasmid has been introduced into the host microorganism, and a transformant thereof has been obtained.

The present inventor succeeded in constructing a novel plasmid into which a DNA carrying the genetic information involved in secretory production of alkaline CGTase, which is a metabolite of a microorganism belonging to the genus Bacillus, was incorporated, and the plasmid was further transformed into Bacillus subtilis. (Bacillus subtilis) Bacillus subtilis, a new and useful transformant introduced into the strain 1A-289.
We succeeded in obtaining lus subtilis) 1A-289 (pTUB703) and completed the present invention.

Hereinafter, the present invention will be described in detail.

The host microorganism used in the present invention is a plasmid (vector DN) having a form capable of growing in cultured cells, such as pUB110 known as a Bacillus plasmid.
In A), a microorganism belonging to the genus Bacillus, for example, Bacillus sp. It is a microorganism belonging to the genus Bacillus represented by Bacillus subtilis, which can contain a plasmid into which a DNA fragment prepared from # 1011 bacterium (Microtechnology Research Institute No. 8685) has been incorporated.

Examples of the microorganism containing a DNA that carries the genetic information involved in the secretory production of alkaline CGTase used in the present invention include Bacillus sp. # 1011 bacterium
No.) can be given. Bacillus sp. The # 1011 bacterium is a microorganism isolated from soil collected in Fukuroi City, Shizuoka Prefecture, and has the following mycological properties. In addition, the test and classification method of the mycological properties are described in “Aerobic Sporeforming Bacteria”.
”(United State Department of Agriculrture, Nov.1
952 by NRSmith, REGordon & FEClark)] and "Bergese's Manual of Detergent Native Bacteriology"['Bergry's Manual of Datermi
native Bacteriology, 1957)].

(Mycological Properties of Bacillus sp. # 1011 B) Form 1 On a plate agar medium having a composition (pH 10.0) containing 15 g of starch, 10 g of polypeptone, 5 g of yeast extract, 1 g of K ₂ HPO ₄ , 0.2 g of MgSO ₄ .7H ₂ O, 10 g of sodium carbonate and 15 g of agar per form 1 ( However, sodium carbonate is autoclaved separately from other components and then added to the medium.), And the observed morphology is shown in Table 1.

B. Growth condition Table 2 shows the growth condition of the # 1011 bacteria in various media. In addition, since there was almost no growth in the media pH 7.0 and no change was observed, 1% sodium carbonate was added to the media.
% To make pH 10.0.

C. Physiological Properties Tables 3-1 and 3-2 show the results of the test on the # 1011 bacteria by adding 1% of sodium carbonate to each of the physiological test media.

Bacillus sp. The # 1011 bacterium is an aerobic spore bacterium when the above-mentioned various bacteriological properties are summarized. Therefore, it is clear that the # 1011 bacterium is a microorganism belonging to the genus Bacillus. It was clearly distinguished from known bacterial species in that the pH was 8 to 12, and it was concluded that it is appropriate to identify it as a new bacterial species of alkalophilic cells.

It is incorporated into the vector DNA. Bacillus sp. # 1
The DNA fragment prepared from 011 bacterium is a DNA carrying the genetic information involved in the secretory production of alkaline CGTase, which is a metabolite of the Bacillus bacterium, and contains the vector DN
As A, in addition to those extracted from naturally occurring,
A part of DNA other than the part necessary for growth may be partially deleted, for example, pE194 strain, pC194 strain,
Examples include the pUB110 system. Also, the above vector D
As a method for incorporating the DNA fragment into NA, any known method can be applied. For example, a suitable restriction enzyme (Endonu
Clease) is selected and treated to cleave at a specific site of DNA, then mixed with vector DNA treated with a restriction enzyme that forms a complementary base sequence at the cleavage site, and re-ligated with ligase. The thus obtained ligated product of the DNA fragment and the vector DNA was subjected to the protoplast transformation method [Chang, S., Cohen, SN ,: Mol. Gen. Gene.
t. 168, 11 (1979)] and introduced into the cells of a bacterium of the genus Bacillus, which is a recipient bacterium, and propagated until it is stable as a genetic trait, a transformant having a desired genetic trait and a vector DNA trait is obtained. can get.

The transformants obtained in the examples described below are created by the following two-step operation. However, the operation of is optional. It is also possible to directly incorporate a DNA fragment prepared from # 1011 bacterium into vector DNA such as pUB110.

DNA of λD69 phage as a bacteriophage vector DNA [Silhavy TJ, Berman ML, Enquist
LW: Experiments with Gene Fusions "(Cold Spring
See Harbar Laboratory (1984) p. 247, which is a restriction end
38.8k with one cleavage site by onuclease BamHI
It is the DNA of b. A restriction enzyme digestion map is shown in FIG. ], And the Bacillus sp. In vitro packaging method [(Horn, B., Murray, K .: Proc. Natl. Acad. Sc
iUS, 74 .3259 (1977)] to prepare a new transducing phage particles λD69-TU110 obtained by.

A novel pTUB703 plasmid obtained by using pUB110 as the plasmid vector DNA and incorporating the DNA fragment prepared from the above-mentioned transduced phage particles was used as a Bacillus subtilis strain 1A-289 strain [Catalog of Strains 2nd
edition (1982): The Bacillus Genetic Stock Cente
r, (The Ohio STate Uni.Dep of Microbiology), genetic traits: aroI906, met B5, sacA321, amy E] to create a new microorganism by the protoplast transformation method,
This is Bacillus subtilis 1A-289 (pTUB703) [Bac
illus subtilis 1A-289 (pTUB703)]. A restriction enzyme digestion map of the pTUB703 plasmid of Bacillus subtilis 1A-289 (pTUB703) is shown in FIG.

As is clear from FIG. 3, this plasmid was used as pTUB11.
0 At the BamHI site of the restriction site of plasmid DNA, the Bacillus sp. # 1011 is a circular DNA molecule incorporating an alkaline CGTase DNA fragment that carries the genetic information involved in the secretory production of alkaline CGTase, namely pUB1
It is a novel circular DNA molecule of 7.2 Kb consisting of 10 plasmid DNA and DNA of about 2,600 base pairs.

Next, the mycological properties of the Bacillus subtilis 1A-289 (pTUB703) were the same as those of the Bacillus subtilis 1A-289 strain, which is a DNA recipient, and kanamycin resistance and alkalinity.
Identical except for CGTase productivity (Catalog of Stra
ins 2nd edition (1982): The Bacillus Genetic Stock
Center, (The Ohio State Uni.Dep of Microbiology,
Genetic traits: aroI906, met B5, sacA321, amy E), and another characteristic is the characteristic of pTUB703 plasmid, that is, the characteristic of CGTase to be extracellularly accumulated by the pTUB703 plasmid which carries the genetic information of alkaline CGTase-producing ability. The feature is that it is added.

Secretory production of alkaline CGTase by Bacillus subtilis 1A-289 (pTUB703) was observed about 12 hours after the start of culture and its production was sustained for a long time as shown in Examples below (first In contrast, the Bacillus sp. As shown in FIG. 2, the secretory production of alkaline CGTase by the # 1011 bacterium reaches a maximum 3 days after the start of culture. Therefore, the present invention provides the Bacillus subtilis 1A.
-289 (pTUB703), that is, a microorganism capable of secreting and producing alkaline CGTase, which has never existed before, can be produced extremely advantageously,
It is novel and useful in terms of providing.

This means that the DNA of about 2,600 base pairs contained in the pTUB703 plasmid obtained by the present invention confers the secretory productivity of metabolites to the host.

The method for constructing the plasmid of the present invention and the transformant containing the plasmid, Bacillus subtilis 1A-2, are described below.
The method for producing 89 (pTUB703) and the effect of the transformant will be described with reference to Examples.

Example [Example 1] (1) Chromosome D having genetic information of alkaline CGTase-producing ability
Preparation of NA An alkalophilic Bacillus sp. Which has the ability to produce and accumulate alkaline CGTase outside the cells. # 1011 bacterium (Microtech Lab. No. 8685) was used as a medium [15 g starch per 1 part, peptone
10 g, yeast extract 5 g, K ₂ HPO ₄ 1 g, MgSO ₄ .7H ₂ O 0
Composition containing 0.2 g and adjusted to pH 10.0 with 10 g of sodium carbonate] at 40 ° C. for 12 hours with shaking culture to collect cells in the late logarithmic growth phase, and then use the Saito, Miura method (Saito, H., Miura,
K .: Biochem. Biophys. Acta, 72 , 619, (1964)].
About 5 mg NA was obtained.

(2) Insertion of chromosomal DNA fragment into phage DNA 300 μg of chromosomal DNA obtained in (1) above was taken, restriction endonuclease Sau3AI (Takara Shuzo Co., Ltd.) was added, and the mixture was incubated at 0 ° C. for 30 minutes.
It was allowed to react for minutes and partially cut. Furthermore, a chromosomal DNA fragment of about 7 to 12 Kb was separated and purified by a sucrose density gradient ultracentrifugation method to prepare a DNA fragment. Bacteriophage λD
20 μg of 69 DNA (sold from the Institute of Medical Science, University of Tokyo: restriction enzyme digestion map is shown in FIG. 4) was completely digested with restriction endonuclease BamHI (manufactured by Takara Shuzo Co., Ltd.) The fragments were mixed and DN derived from T4 phage
DN by A ligase (manufactured by Takara Shuzo Co., Ltd.) at 10 ° C for 24 hours
The A chain is ligated and the in vitro packaging kit (Amersham International) is used to
This was used as a transducing phage particle. Λ medium containing 1% starch [10 g of bactotryptone (DIfco), NaCl 2.
Composition containing 5 g] Escherichia coli SM on agar plate
32 shares sold by the Institute of Medical Science, University of Tokyo, Genetic trait: SA
500, his, pyrD, lons100, gal, strA) were grown at 37 ° C. and infected with the above-mentioned transducing phage particles.
Active transducing phage λD69-TU110 particles were obtained. PEG6000 after culturing the transducing phage particles with Escherichia coli 211SM32 strain in λ medium 1
Law (KRYamamoto, BM alberto, R. Benzinger, L, Lowhorn
e & G. Treiber: Virolgy, 40,734 (1970)], the transduced phage DNA was extracted and purified to obtain about 50 Kb of DNA.
150 μg was obtained.

(3) Insertion of the transduced phage DNA fragment into a plasmid vector Take 50 μg of the DNA obtained in (2) above, add Sau3AI,
The mixture was reacted at 30 ° C. for 30 minutes and partially cut. Furthermore, a DNA fragment was prepared by separating and purifying a transducing phage DNA fragment of about 2 to 9 Kb by a sucrose density gradient ultracentrifugation method. On the other hand, kanamycin resistance (Km
^r ) and ampicillin resistance (Amp ^r ), pUB110 plasmid DNA [Bethesda Research Laboratories (USA), restriction enzyme digestion map is shown in FIG. 5. "DNA C
loning Vol.II "IRL PRESS (Oxford washin gton DC) b
y DMGlover 1985 ”] completely decomposed with BamHI
BAD (; Bacterial Alkela
nd Phosphatase) treatment and mixing with the DNA fragment
Plasmid DNA in which a DNA fragment was incorporated by ligation reaction of DNA chains for 24 hours at 4 ° C with 4DNA ligase
Was built.

(4) Transformation with a plasmid carrying the secretory production gene of alkaline CGTase Using the solution of the plasmid DNA obtained in (3) above, a protoplast transformation method using Bacillus subtilis 1A-289 strain as a host [(Chang, S ., Cohen, SN: Mol. Gen. Gene
t. 168, 111, (1979)].
Was introduced into the cells, starch 1%, kanamycin 150 μ
DM3 medium containing g / ml and agar 0.8% (5% casamino acid
100 ml, 1M sodium citrate (pH 7.3) 500 ml, 3) 5% K ₂
HPO ₄ 50 ml, 1.5% KH ₂ PO ₄ 50 ml, 10% yeast extract 50
ml, 20% glucose 25 ml, 1M MgCl ₂ 20 ml, 2% bovine serum albumin 5 ml) and cultivated at 37 ℃, kanamycin resistant Bacillus subtilis 1A-289 (pTUB703) having alkaline CGTase activity Got

[Example 2] The transformant Bacillus subtilis 1A-289 (pTUB703) obtained in (4) of Example 1 was treated with LGKm medium (tryptone (Difco) 10 g, yeast extract (Difco) 5 g, NaCl 5 per 1).
g Composition containing 2 g of glucose and 10 mg of kanamycin] 100
The culture was performed with shaking in a 500 ml Sakaguchi flask containing 37 ml at 37 ° C. The cell growth (cell mass) was measured by the absorbance (OD) at 660 nm. The enzyme activity of alkaline CGTase is 0.05 m
1 dissolved in M / 10 glycine-NaCl buffer (pH 10.5)
After adding 0.5 ml of a% soluble starch solution and reacting at 40 ° C. for 2 hours, the pH was neutralized with acetic acid, and the mixture was heated at 100 ° C. for 10 minutes. 50 μg of glucoamylase was added thereto to decompose unreacted starch at 40 ° C. for 1 hour, and the amount of glucose produced was measured by the dinitrosalicylic acid method. In addition, as a control, a solution prepared by adding water instead of the enzyme solution was treated in exactly the same manner as above, and the difference between the two was used as the amount of cyclodextrin produced. Here, 1 unit refers to the amount of enzyme that produces 1 mg of cyclodextrin by the above method (Japanese Patent Publication No. Sho 52).
-93 gazette).

The cell amount of the transformed strain reached its maximum 12 hours after the start of the culture (shown in FIG. 1), and the extracellular alkaline CGTase activity reached the maximum at approximately 24 hours. Produced alkaline
CGTase is stable.

As a comparison, the alkalophilic Bacillus sp. The # 1011 bacterium (Microtechnology Research Institute, No. 8685) was cultured and the alkaline CGTase activity was measured. 1 per starch 15 g, polypeptone 10 g, yeast extract 5 g, H ₂ H
A medium (pH 10.0) containing 1 g of Po ₄ , 0.2 g of MgSO ₄ .7H ₂ O, and 10 g of sodium carbonate was inoculated and cultured at 40 ° C. with shaking.

The amount of bacterial cells reached the maximum 24 hours after the start of the culture, and the activity of extracellular alkaline CGTase (shown in Fig. 2) gradually increased 24 hours after the inoculation, and reached the maximum at 72 hours. Reached

[Example 3] The transformant Bacillus subtilis 1A-289 (pTUB703) was subjected to the same procedure as in Example 2 by centrifuging the culture medium for 24 hours after the start of culture to remove the bacteria and prepare the culture supernatant. After ammonium sulfate was added to 100 ml of the solution for salting out, the precipitate was dissolved in water and dialyzed against running water overnight to obtain an alkaline CGTase enzyme solution. Put 5% starch gelatinization solution (containing 10 mM CaCl ₂ ) 1 adjusted to pH 10.5 with N / 10 glycine-NaCl buffer solution, add 25 ml of the above enzyme solution, and perform an enzyme reaction at 65 ° C for 6 hours. did. After completion of the reaction, the precipitate was removed by centrifugation to remove the precipitate, and the product was precipitated by adding trichlorethylene. After washing the precipitate with water, the trichlorethylene was heated to remove, and recrystallized from a 60% propyl alcohol aqueous solution. About 2 g of white crystals were obtained. When the crystals were analyzed by Avicel thin layer chromatography, they were separated with a developing solution of n-butanol: propanol: water (3: 5: 4) by the ascending method twice and then 1
% Iodine methanol solution was atomized, and spots showing a color peculiar to α- and β-cyclodextrin which coincided with the Rf value of the reagent were observed.

〔The invention's effect〕

A plasmid incorporating a DNA fragment that carries the genetic information involved in the secretory production of alkaline CGTase of the present invention, by incorporating this into Bacillus subtilis by a genetic engineering method and transforming it, alkaline CGTase can be obtained in a short culture time. A new microorganism, Bacillus subtilis, to be produced can be created, and by using this microorganism, it has become possible to industrially and efficiently produce a large amount of CGTase.

[Brief description of drawings]

FIG. 1 shows Bacillus subtilis 1 used in the method of the present invention.
The extracellular alkaline CGTase production activity by A-289 (pTUB703) was shown in FIG. 2 is a graph showing extracellular alkaline CGTase production activity by # 1011 bacterium. Figure 3 shows Bacillus subtilis 1A-289 (pTUB703).
Is a restriction enzyme digestion map of the plasmid (pTUB703) of
The white parts are derived from pUB110 plasmid DNA, the black parts are Bacillus sp. # 1011 chromosomal DNA fragment (alkaline CGTase DNA fragment) is shown. FIG. 4 is a restriction enzyme digestion map of bacteriophage λD69. Further, FIG. 5 is a restriction enzyme digestion map of the vector DNA pUB110.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12R 1:07) (C12N 1/21 C12R 1:19) (C12N 9/10 C12R 1:19)

Claims (16)

[Claims] 1. A Bacillus subtill.
s) as a host, the Bacillus (Bacill)
us) microbial alkaline cyclomaltodextrin glucanotransferase (Cyrclomaltodext
rin glucanotransferase, hereinafter referred to as alkaline CGTase. ), Which is responsible for the genetic information involved in secretory production of Bacillus sp. Deoxyribonucleic acid (DN) derived from the # 1011 bacterium (Microtechnology Research Institute, No. 8685)
A) A plasmid incorporating the fragment at its restriction site. 2. The plasmid according to claim 1, wherein the plasmid is pTUE703. 3. The plasmid according to claim 1, wherein the plasmid vector is DNA of pUB110 plasmid. 4. The plasmid according to claim 1, wherein the restriction site is a BamHI site. 5. The plasmid according to claim 1, wherein the microorganism belonging to the genus Bacillus is a microorganism belonging to an alkalophilic bacterium. 6. A microorganism belonging to an alkalophilic bacterium is Bacillus sp. The plasmid according to claim 5, wherein the plasmid is # 1011 bacterium (Ministry of Industrial Science, No. 8685). 7. Alkaline CG of a microorganism belonging to the genus Bacillus
Bacillus sp. Which gives the secretory productivity of Tase and has the following restriction map. # 1011 bacterium
No.)-Derived DNA fragment using a restriction enzyme, limiting the vector DNA of the plasmid with a restriction enzyme that does not interfere with the genetic information involved in the secretory production of alkaline CGTase of said DNA fragment. The Bacillus sp. Carrying the genetic information involved in the secretory production of alkaline CGTase, which consists of recombining the DNA fragment into the restriction site of the vector DNA of the plasmid and extracting the recombined plasmid, and has the following restriction enzyme map: ． # 1011 A method for constructing a plasmid incorporating a DNA fragment derived from the bacterium. 8. The method for constructing a plasmid according to claim 7, wherein the plasmid is pTUB703 plasmid. 9. A Bacillus sp. Which has the ability to secrete alkaline CGTase and has the following restriction map. A DNA fragment derived from # 1011 bacterium (Microtechnology Research Institute No. 8685) is directly prepared by decomposing the bacterium's DNA with a restriction enzyme. How to build. 10. An alkaline CGTase secreting and producing ability is provided,
Bacillus sp. Having the following restriction map. # 1011
A DNA fragment derived from a bacterium (Microtechnology Research Institute, No. 8685) was digested with a restriction enzyme to obtain a DNA fragment, which was once recombined into λD69 bacteriophage DNA to produce CGTa.
After preparing the transducing phage particles capable of producing se,
The method for constructing a plasmid according to claim 7, which is prepared by degrading the DNA with the restriction enzyme. 11. pUB1 as a plasmid vector DNA
10. The method for constructing a plasmid according to claim 7, wherein the DNA of 10 plasmids is used. 12. The method for constructing a plasmid according to claim 7, wherein a BamHI site is used as the restriction site. 13. A restriction enzyme (Endonuc) for preparing a DNA fragment.
8. The method for constructing a plasmid according to claim 7, characterized in that Sau3AI is used as a lease). 14. The method for constructing a plasmid according to claim 7, wherein the microorganism belonging to the genus Bacillus belongs to an alkalophilic bacterium. 15. A microorganism belonging to an alkalophilic bacterium is Bacillus sp. The method for constructing a plasmid according to claim 14, characterized in that it is # 1011 bacterium (Ministry of Industrial Science, No. 8685). 16. Bacillus sp. That carries the genetic information involved in the secretory production of alkaline CGTase. A plasmid pTUB characterized by the following restriction enzyme map, in which a DNA fragment derived from the # 1011 bacterium (Microtechnology Research Institute, No. 8685) was incorporated.
Bacillus subtilis 1 A-289 (pTUB703) [Bacillus subt containing 703 and capable of producing alkaline CGTase
illis 1A-289 (pTUB703)].