JPH0640826B2 - 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, CGTase.
The present invention relates to a plasmid incorporating a deoxyribonucleic acid (DNA) fragment that carries the genetic information involved in the extracellular production of the above, a method for constructing the 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 stearothermophilus (Bacillus stearothermophilus), etc., or a bacterial species belonging to the Klebsiella genus, for example, Klebsiella
A method of aerobically culturing Newmore (Klebsiella pneumoniae) or the like in an appropriate medium having a neutral or weakly acidic pH ["Enzyme Handbook": supervised by Shiro Akahori, published by Asakura Shoten (Showa 41) and "Amylase": See Nakamura Michinori, Academic Society Publishing Center (1986) etc.) and a method of aerobically culturing an alkalophilic bacterium belonging to the genus Bacillus in a suitable pH alkaline medium [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 using Bacillus macerans-derived CGTase as Bacillus subtilis (Bacillus subtilis).
s subtilis) and Escherichia coli clones (Toshiya Takano, Kunio Yamane et al .; 7th Annual Meeting of the Molecular Biology of Japan, 1984) and Bacillus macerans and CGTase from Bacillus stearothermophilus
Studies on cloning into subtilis and Escherichia coli (Tomoo Kubota, Yoshio Tsujisaka et al .; Japan Fermentation Engineering Conference in 1985) have already been published. No studies of cloning into are found. Furthermore, the above-mentioned academic conference report in which CGTase was cloned into the Escherichia coli strain did not show the fact that the transformed Escherichia coli strain produced a large amount of the enzyme in the culture supernatant, and the transformed strain was industrially used. In order to obtain the enzyme, it is necessary to collect the bacterial cells after culturing, and once destroy the bacterial cells by means such as ultrasonic waves to separate and purify the intracellular enzyme. It is not suitable as an enzyme for foods and medical products because the presence of toxic substances and insoluble particles of the above becomes a problem.

[Problems to be solved by the invention]

The present invention solves the above-mentioned problems of the prior art by incorporating a novel plasmid incorporating deoxyribonucleic acid (DNA) carrying specific genetic information involved in extracellular productivity, a method for constructing the same, and transformation with the plasmid. The purpose is to provide a novel microorganism.

[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 of 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 preparing a novel plasmid into which a DNA carrying genetic information involved in extracellular 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 Escherichia coli.・ Escherichia coli, a new and useful transformant introduced into HB101 strain, Escherichia coli (Escherichia coli)
We succeeded in obtaining ia coli) HB101 (pTUE202) and completed the present invention.

Hereinafter, the present invention will be described in detail.

The host microorganism used in the present invention is a colicin E ₁ factor known as a plasmid of the genus Escherichia,
A plasmid (vector DNA) having a trait capable of growing in the cultured cells is added to a microorganism belonging to the genus Bacillus as an exogenous gene DNA, for example, Bacillus sp. # 1011
Escherichia, which can contain a plasmid into which a DNA fragment prepared from a bacterium (Microtech Lab. No. 8685) has been incorporated.
It is a microorganism belonging to the genus Escherichia represented by Escherichia coli.

Examples of the microorganisms used in the present invention, which contain DNA carrying the genetic information involved in the extracellular production of alkaline CGTase, 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 Bacteri”.
a ”(United State Department of Agriculture, Nov.195
2 by NRSmith, REGordon & F.E. Clark)] and "Bergese's Manual of Determinative Bacteriology"["Bergry's Manual of Daterminat
ive 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 that carries the genetic information involved in the extracellular production of CGTase, which is a metabolite of the Bacillus bacterium, and the naturally-occurring vector DNA was extracted. In addition to the above, a part of DNA other than the part necessary for proliferation may be partially deleted, and examples thereof include ColE ₁ strain, pMB9 strain, pBR322 strain, pSC101 strain and the like.

Further, as a method for incorporating the DNA fragment into the vector DNA, any known method can be applied. For example,
Select and treat appropriate restriction enzyme (Endonuclease)
A method is used in which the vector DNA cleaved at the A specific site, then treated with a restriction enzyme that forms a complementary nucleotide sequence at the cleavage site, is mixed and religated with ligase. The DNA fragment and the vector DNA thus obtained
The ligation product of E. coli is introduced into the cells of the recipient microorganism Escherichia by the transformation method and propagated until it becomes stable as a genetic trait, whereby a transformant having both the desired genetic trait and the vector DNA trait is obtained. To be

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 the DNA fragment prepared from the # 1011 bacterium into vector DNA such as pBR322.

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.Sci.
US, 74 .3259 (1977)] to prepare a new transducing phage particles λD69-TU110 obtained by.

Using pBR322 as the plasmid vector DNA, the novel pTUE202 plasmid obtained by incorporating the DNA fragment prepared from the above-mentioned transducing phage particles into Escherichia coli HB101 strain (Escherichia coli 12 strain and Escherichia coli B strain (Hybrid strain) and a transformation reaction to create a new microorganism, which is Escherichia coli HB 101 (pHUE 202) [Escherichia coli H
B101 (pTUE 202)]. Said Escherichia
The restriction enzyme digestion map of the pTUE202 plasmid of E. coli HB101 (pTUE202) is shown in FIG.

As is clear from FIG. 3, this plasmid is pBR322.
At the BamHI site of the restriction site of the plasmid DNA, the Bacillus sp. # 1011 is a circular DNA molecule incorporating an alkaline CGTase DNA fragment, which carries the genetic information involved in extracellular production of alkaline CGTase, namely pBR3
It is a novel circular DNA molecule of 9.1 Kb consisting of 22 plasmid DNA and DNA of about 4,700 base pairs.

Next, the Escherichia coli HB 101 (pTUE 202)
Mycologically, Escherichia coli, which is a DNA recipient,
Properties of HB 101 strain, ampicin resistance, alkaline CGTa
Same except for se productivity, but [Molecular Cloning
See A Laboratory Manual, p. 504 (1982), Genetic traits:
F^-hsdS20 (r , M ), Rec A13, ara-14, proA2, lacY
1, galK2, rpsL20 (Sm^r), xyl-5, mtl-1, supE44, λ^-〕,other
As a characteristic of pTUE 202 plasmid,
PTUE202 plasmid that carries the genetic information of the ability to produce soluble CGTase
Has the property of producing CGTase extracellularly and accumulating it.
The feature is that it is added.

The extracellular production of alkaline CGTase by Escherichia coli HB 101 (pHUE 202) was observed about 12 hours after the start of the culture, and the production amount was maintained for a time (see Example 1). On the other hand, the Bacillus sp. As shown in FIG. 2, the extracellular production of alkaline CGTase by the # 1011 bacterium reaches a maximum after 3 days from the start of culture. Therefore, the present invention relates to the above Escherichia coli H.
B 101 (pTUE 202), that is, novel and useful in terms of creating and providing a microorganism which has the ability to produce an enzyme protein extracellularly, which has been heretofore none, and which can produce CGTase extremely advantageously It is equipped with.

This means that the DNA of about 4,700 base pairs contained in the pTUE 202 plasmid obtained by the present invention imparts the extracellular productivity of metabolites to the host.

The method for constructing the plasmid of the present invention and the transformant containing the plasmid, Escherichia coli HB 1 are described below.
The method for producing 01 (pTUE 202) 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
10g, yeast extract 5g, K ₂ HPO ₄ 1g, MgSO ₄ · 7H ₂ O
In a composition containing 0.2 g of sodium carbonate and adjusted to pH 10.0 with 10 g of sodium carbonate), shake culture was carried out at 40 ° C. for 12 hours, and cells in the late logarithmic growth phase were collected, and then the Saito, Miura method (Saito, H., Miura,
K .: Biochem. Biophys. Acta, 72 , 619, (1964)) D
DNA extraction and purification by NA extraction method
A about 5 mg 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, The University of Tokyo) was completely digested with the restriction endonuclease BamHI (Takara Shuzo Co., Ltd.) and a part of the digested DNA fragment was mixed with the DNA fragment of T4 phage ( Takara Shuzo Co., Ltd.)
The DNA strands were ligated at 10 ° C. for 24 hours, and used as transducing phage particles using an in vitro packaging kit (manufactured by Amersham International). Lambda medium containing 1% starch [per bactotryptone (Difc
o) Composition containing 10 g and NaCl 2.5 g] Escherichia coli SM32 strain (sold by the Institute of Medical Science, University of Tokyo, genetic trait: SA500, his, pyrD, lons 100, gal, str) on an agar plate
A) was grown at 37 ° C., and transduced phage λD having alkaline CGTase activity was selected from a large number of plaques obtained by infecting the same with the above-mentioned transduced phage particles.
69-TU110 particles were obtained. After culturing this transducing phage particle with the Escherichia coli SM32 strain in the λ medium of 1, the PEG6000 method [KRYamamoto, BM alberto, R. Benzing
er, L, Low horne & G. Treiber: Virology, 4
0, 734 transducing phage D by (1970)]
NA was extracted and purified to obtain about 150 μg of about 50 Kb DNA.

(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 transducing phage DNA of about 2 to 9 Kb by sucrose density gradient ultracentrifugation
The fragment was separated and purified to prepare a DNA fragment. On the other hand, pBR322 with tetracycline resistance (Tet ^r ) and ampicillin resistance (Amp ^r ) used as a plasmid vector
Plasmid DNA (manufactured by Bethesda Research Laboratories (USA)) was completely digested with Bam HI and heat-treated at 65 ° C for 10 minutes to obtain BAP (; Bacterial Alkaline Phosphatase).
After the treatment, it is mixed with the above DNA fragment, and the DNA chain is ligated with T4 DNA ligase at 10 ° C. for 24 hours to give DN.
A plasmid DNA incorporating the A fragment was constructed.

(4) Transformation with a plasmid carrying the extracellular production gene of alkaline CGTase Escherichia coli HB 101 strain, which is a hybrid strain of Escherichia coli K-12 strain and Escherichia coli B strain, was mixed with L medium (tryptone (Difco ) 10 g, yeast extract (Difco) 5 g, and NaCl 5 g and adjusted to pH 7.0) 50 ml), inoculated with shaking culture at 37 ° C., grown to the late logarithmic growth phase, and then collected. This was sequentially suspended in a solution of 0.1 M CaCl ₂ at a final concentration under ice cooling to obtain competent cells. The plasmid DNA solution obtained in (3) was added to this cell suspension, and the mixture was reacted under ice cooling for 30 minutes,
The plasmid DNA was incorporated into the cells by heat shock at 42 ° C. for 2 minutes. Then, this cell suspension was separately inoculated into the L medium, shake-cultured at 37 ° C. for 60 minutes, and the transformation reaction [Lederberg, EM, Cohen, SN; J.
Bacteriol. 119, 1072 (1974)], and the resulting transformants were subjected to LSAmp agar plates (tryptone (Difco) 10 per plate).
g, yeast extract (Difco) 5 g, starch 10 g, NaCl 5 g, ampicillin 50 mg, agar 15 g) at 37 ° C.,
Escherichia coli HB 101 (pTUE 202) having ampicillin resistance and alkaline CGTase activity was obtained.

[Example 2] The transformant obtained in Example 1 (4), Escherichia
E. coli HB 101 (pTUE 202) was added to LAmp medium [10 g of trypsin (Difco), 5 g of yeast extract (Difco), NaCl per 1 ml].
Composition containing 5 g and 50 mg ampicillin] 500 containing 100 ml
The culture was carried out at 37 ° C. with shaking in a ml volume Sakaguchi flask. Cell growth
(Amount of cells) was measured by the absorbance (OD) at 660 nm. The enzymatic activity of alkaline CGTase was determined by adding 0.5 ml of 1% soluble starch solution dissolved in M / 10 glycine-NaCl buffer (pH 10.5) to 0.05 ml of enzyme solution and reacting at 40 ° C for 2 hours. The pH was brought to K and heated at 100 ° C for 10 minutes. 50 to this
After the unreacted starch was decomposed at 40 ° C. for 1 hour by adding μg of glucoamylase, the amount of glucose produced was measured by the dinitrosalicylic acid method. Also, as a control, water was added instead of the enzyme solution and treated in exactly the same manner as above,
The difference between the two was used as the amount of cyclodextrin produced. Here, 1 unit means the amount of enzyme that produces 1 mg of cyclodextrin by the above method (see Japanese Patent Publication No. 52-93).

The amount of bacterial cells in the transformant reached a maximum 12 hours after the start of culture (shown in Fig. 1), and the extracellular alkaline CGTase activity reached a maximum in approximately 24 hours. Produced alkaline
CGTase is very 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. 15g starch, 10g polypeptone, 5g yeast extract, H ₂ HP
o ₄ 1 g, was inoculated into the medium (pH 10.0) containing MgSO ₄ · 7H ₂ O 0.2g, sodium carbonate 10 g, and cultured with shaking at 40 ° C..

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 Escherichia coli HB101 (pTUE 202) was subjected to the same operation as in Example 2 by centrifuging a culture solution for 24 hours after the start of culture to remove the bacteria to prepare a culture supernatant 100. After ammonium sulfate was added to ml for salting out, the precipitate was dissolved in water and dialyzed against running water overnight to obtain an alkaline CGTase enzyme solution. Add 5% starch gelatinization solution (containing 10 mM CaCl ₂ ) 1 adjusted to pH 10.5 with N / 10 glycine-NaCl buffer solution to the flask, 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 18 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
A% iodine methanol solution was atomized, and spots showing a color peculiar to α- and β-cyclodextrin corresponding to the Rf value of the reagent were observed.

Silica-based ODS-NH by high performance liquid chromatography
_{2 Using} a column packed with resin (8φ x 250 mm), separating with an eluent of acetonitrile: water (65:35) and detecting with a differential refractometer, the reagents α-, β- and γ-cyclodextrin The product was identified according to the retention time of.

〔The invention's effect〕

The plasmid in which the DNA fragment carrying the genetic information involved in the extracellular production of CGTase of the present invention is incorporated into a microorganism belonging to the genus Escherichia by a genetic engineering technique and transformed, so that the culture time can be shortened. CGTase
It is possible to create a new microorganism belonging to the genus Escherichia that produces, and since the microorganism produces a large amount of CGTase outside the cells, 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 Escherichia coli HB used in the method of the present invention.
Fig. 2 shows the extracellular alkaline CGTase production activity of 101 (pTUE 202) as shown in Bacillus sp. 2 is a graph showing extracellular alkaline CGTase production activity by # 1011 bacterium. Figure 3 shows Escherichia coli HB 101 (pTUE 20
It is a restriction enzyme digestion map of the plasmid (pTUE 202) of 2), the open part is derived from pBR322 plasmid DNA, and the black part is Bacillus sp. # 1011 chromosomal DNA fragment (alkaline CGTase DNA fragment) is shown. FIG. 4 is a restriction enzyme digestion map of bacteriophage λD69.

─────────────────────────────────────────────────── ─── 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 (17)

[Claims] 1. A plasmid which gives extracellular ability to a host belonging to the genus Escherihia, which is Bacillus
Cyclomaltodextrin glucanotransferase from microorganisms belonging to the genus (Bacillus)
lucanotransferase, hereinafter referred to as CGTase. ) Of Bacillus sp. Which carries the genetic information involved in the extracellular production of Bacillus sp. # 1011 bacterium
No. 5) -derived deoxyribonucleic acid (DNA) fragment incorporated into its restriction site. 2. The plasmid according to claim 1, wherein the plasmid is pTUE202. 3. The plasmid according to claim 1, wherein the plasmid vector is DNA of pBR322 plasmid. 4. The plasmid according to claim 1, wherein the host belonging to the genus Escherichia is Escherichia coli. 5. The plasmid according to claim 1, wherein the restriction site is a BamHI site. 6. The plasmid according to claim 1, wherein the CGTase is an alkaline CGTase. 7. The plasmid according to claim 1, wherein the microorganism belonging to the genus Bacillus is a microorganism belonging to an alkalophilic bacterium. 8. A microorganism belonging to an alkalophilic bacterium is Bacillus sp. The plasmid according to claim 7, which is a # 1011 bacterium (Ministry of Microbiology, Ltd., No. 8685). 9. A Bacillus sp. Which gives the extracellular productivity of CGTase and has the following restriction map. A DNA fragment derived from the # 1011 bacterium (Microtechnology Research Institute No. 8685) is prepared using a restriction enzyme, and a restriction enzyme that does not interfere with genetic information involved in the extracellular production of CGTase of the prepared DNA fragment. For the extracellular production of CGTase, which comprises restricting the vector DNA of a plasmid by using Escherichia coli, recombining the DNA fragment with a restriction site of the vector DNA of the restricted plasmid, and extracting the recombined plasmid. Bacillus sp. Which carries the genetic information involved and has the following restriction map. # 1011 A method for constructing a plasmid incorporating a DNA fragment derived from the bacterium. 10. The method for constructing a plasmid according to claim 9, wherein the plasmid is pTUE202 plasmid. 11. pBR3 as a plasmid vector DNA
22. The method for constructing a plasmid according to claim 9, wherein the DNA of the plasmid is used. 12. The method for constructing a plasmid according to claim 9, wherein a BamHI site is used as the restriction site. 13. The method for constructing a plasmid according to claim 9, wherein the CGTase is an alkaline CGTase. 14. A restriction enzyme (Endonuc) for preparing a DNA fragment.
10. The method for constructing a plasmid according to claim 9, wherein Sau3AI is used as a lease). 15. A Bacillus sp. Which gives the extracellular productivity of CGTase and has the following restriction map. The DNA fragment derived from the # 1011 bacterium (Microtechnology Research Institute No. 8685) is directly prepared by decomposing the bacterium's DNA with a restriction enzyme. How to build. 16. A Bacillus sp. Which gives the extracellular productivity of CGTase and has the following restriction map. The DNA fragment derived from the # 1011 bacterium (Microtechnology Research Institute No. 8685) was digested with a restriction enzyme to obtain a DNA fragment with λD.
69 After preparing transducing phage particles capable of producing CGTase by recombination with bacteriophage DNA,
The method for constructing a plasmid according to claim 9, which is prepared by decomposing A with the restriction enzyme. 17. A Bacillus sp. That carries the genetic information involved in the extracellular production of CGTase. # 1011 bacterium
No. 85) containing a DNA fragment derived from Escherichia coli HB101 (pTUE 202) [Escherichia coli HB101 (pTUE 202) containing a plasmid pTUE202 characterized by the following restriction enzyme map and capable of producing CGTase in vitro.
UE 202)].