JPH0142675B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel plasmid, a method for its preparation, and a novel microorganism containing the plasmid.
A new plasmid incorporating deoxyribonucleic acid (DNA) that carries specific genetic information involved in the extracellular production (secretion) of the metabolite xylanase,
The purpose of this invention is to provide a novel microorganism transformed with the plasmid. Plasmids are extrachromosomal genes found within the cells of microorganisms, which are circular molecules of DNA.
In recent years, it has been used as a means of genetically modifying microorganisms, and its importance in research in the field of fermentation industry has been increasing. In recent years, as seen in the examples of amino acids and peptides, DNA carries genetic information related to the specific requirements necessary for the growth of microorganisms and the ability to produce metabolites.
Research has been carried out on plasmids that have integrated the plasmids, and some plasmids have been introduced into host microorganisms and transformed strains have been obtained. The present inventors have succeeded in preparing a new plasmid incorporating DNA carrying genetic information involved in the extracellular production (secretion) of xylanase, a metabolite of a microorganism belonging to the genus Bacillus, and furthermore, The present invention was completed by successfully obtaining a new and useful transformed strain, Escherichia coli HB101 (pCX311), by introducing a plasmid into Escherichia coli HB101 strain. The present invention will be explained in detail below. The host microorganism used in the present invention is colicin, which is known as a plasmid of the genus Escherichia.
A microorganism belonging to the genus Bacillus as a foreign gene DNA is added to a plasmid (vector DNA) in a format that can proliferate in cultured cells, such as factor _E1 .
It is a microorganism belonging to the genus Escherichia represented by Escherichia coli that can contain a plasmid incorporating a DNA fragment prepared from Bacillus C125 (Feikoken Bacteria No. 7344). An example of a microorganism containing DNA carrying genetic information involved in the extracellular production of xylanase used in the present invention is Bacillus C125 (Feikoken Bacteria No. 7344). Bacillus C125 is a microorganism isolated from soil collected in Tsurugashima Town, Iruma District, Saitama Prefecture, and has the following mycological properties. The testing and classification method for mycological properties is based on the “Aerobic Spore Homing” method.
Bacteria” [“Aerobic Spore-forming”
Bacteria” (United State Department of
Agriculture, Nov. 1952 by N.R. Smith, RE
Gordon & F.E.Clark)] and Bergey's Manual of Determinative Bacteriology (1975)
Determinative Bacteriology (1957)]. Γ Mycological Properties a) Morphology 1) It is a bacillus with a size of 0.5-0.7μ x 3.0-4.0μ. 2) Forms spores, size 0.6-0.8μ×
The sporangium is 1.0-1.2μ in oval shape and swollen. 3) Gram staining is positive. b) Growth status in each medium

【table】 c) Physiological properties

【table】

[Table] To summarize the above properties, the Bacillus
Since C125 bacteria is an aerobic spore-forming bacterium,
It is clear that it is a microorganism belonging to the genus Bacillus, but the range in which it can grow is
It was clearly distinguished from known species in that it had a pH of 11.0 and a temperature of up to 55°C, and it was concluded that it was appropriate to recognize it as a new species of alkalophilic bacteria. Bacillus integrated into the vector DNA
The DNA fragment prepared from the C125 bacterium is DNA that carries genetic information involved in the extracellular production (secretion) of xylanase, which is a metabolic product of Bacillus bacteria, and the vector DNA is a naturally occurring DNA fragment. In addition to those extracted from DNA, it is also possible to use DNA that lacks parts of the DNA other than those necessary for proliferation, such as ColE ₁ strain, pMB9 strain,
pBR322 strain, pSC101 strain, R6K strain,
Examples include the Lambda Phage strain. Furthermore, any known method can be applied to incorporate the DNA fragment into the vector DNA. For example, a suitable restriction enzyme (Endonuclease)
Select and treat the DNA to cut the DNA at a specific site, then similarly treated DNA to be used as a vector.
A method is used in which the molecules are mixed with each other and recombined using ligase. The thus obtained conjugate of the DNA fragment and the vector DNA is introduced into the cells of a recipient microorganism of the genus Escherichia by a transformation method and multiplied until it becomes stable as a genetic trait. A transformed strain having both the genetic traits and the traits of the vector DNA can be obtained. The transformed strain obtained in the Examples described below is
pBR322 plasmid DNA as vector DNA
A new pCX311 was obtained by incorporating a DNA fragment prepared from Bacillus C125 into this.
The plasmid was transferred to E. coli HB101 strain (E. coli K-12 strain and E. coli strain K-12).
Escherichia coli HB101 (pCX311) is a new microorganism that can be obtained by a transformation reaction by introducing it into Escherichia coli
HB101 (pCX311)] (referred to as FERMP-7345). The restriction enzyme cleavage map of the pCX311 plasmid of Escherichia coli HB101 (pCX311) is shown in Figure 4. As is clear from Figure 4, this plasmid contains the restriction site of pBR322 plasmid DNA.
A DNA circular molecule in which a xylanase DNA fragment carrying the genetic information involved in the extracellular production (secretion) of xylanase of the Bacillus C125 bacterium is integrated into the Hind site, that is, pBR322 plasmid DNA
It is a novel DNA circular molecule of 9.01Kb consisting of about 4000 base pairs of DNA. Next, the said Escherichia coli HB101
The mycological properties of (pCX311) are the same as those of the DNA recipient strain Escherichia coli HB101, except for penicillin resistance and xylanase productivity [Molecular Cloning A Laboratory
See Manual.p.504 (1982), genetic trait: F ^- , hsd
S20 (r, m), rec A13, ara-14, proA2,
lacY1, galK2, rpsL20 (Sm′), xyl-5, mtl-
1, supE44λ ^- ], As other characteristics, the above pCX311
Its distinctive feature is that it has the additional property of secreting and accumulating xylanase outside of the bacterial cell by means of the pCX311 plasmid, which carries the genetic information for xylanase production ability. As shown in the examples below, the extracellular production (secretion) of xylanase by Escherichia coli HB101 (pCX311) accounts for more than 80% of the total enzyme production, including intracellular production, and its production continues for a long time. amount is sustained. On the other hand, the Bacillus
Extracellular production (secretion) of xylanase by C125 bacteria
In contrast, as shown in FIG. 3, the activity gradually reaches its peak over a long period of time after culture, and thereafter its activity rapidly decreases and lacks long-term sustainability. Therefore, the present invention provides the above-mentioned Escherichia coli
HB101 (pCX311), that is, the novelty and usefulness of creating and providing a microorganism that has the ability to extracellularly produce (secrete) enzyme proteins, which was previously absent, and that can produce xylanase extremely advantageously. It is equipped with. In addition, the produced substances are not only the target single polymer, but also other polymer substances and multiple enzyme proteins, which are each produced (secreted) in large amounts outside the bacterial body and cannot be collected together. can. That is, the Escherichia coli HB101 (pCX311)
As described in the Examples below, penicillinase, alkaline phosphatase, etc., which were conventionally detected only in the bacterial cells, were detected together with xylanase.
It was revealed that each of these was secreted in significant amounts outside the bacterial cells. This means that the approximately 4000 base pair DNA contained in the pCX311 plasmid obtained by the present invention confers on the host the ability to extracellularly produce (secrete) metabolites. . The following describes the method for preparing the plasmid of the present invention, the method for preparing the transformed strain containing the plasmid, Escherichia coli HB101 (pCX311), and the effects of the transformed strain on various enzyme proteins, mainly xylanase. Extracellular production (secretion) will be explained using Examples. Example 1 (1) DNA with genetic information for xylanase production ability
Preparation of Bacillus C125, an alkalophilic bacterium that has the ability to produce and accumulate xylanase outside the bacterial body (Feikoken Bacteria No. 7344), was grown in a medium [(g/): Wrinkle
10.0, yeast extract 5.0, polypeptone 5.0,
K ₂ HPO ₄ 1.0, MgSO ₄ 7H ₂ O 0.2 with Na ₂ CO ₃
After culturing with shaking at 30°C for 19 hours in [adjusted to pH 6.0], the bacteria in the late logarithmic growth stage were collected.
DNA by phenol method DNA extraction method
was extracted and purified to obtain 5 mg of DNA. (2) Insertion of DNA fragment into vector Take 10 μg of chromosomal DNA obtained in (1), add restriction endonuclease Hind, and incubate at 37°C for 5 minutes.
Partial cleavage was performed after reacting for 10, 20, 30, and 60 minutes. On the other hand, pBR322 plasmid DNA with tetracycline resistance (Tet ^r ) and ampicillin resistance (Amp ^r ) used as a vector
(Bethesda Research Laboratories (USA)
After completely cutting the _T4 phage-derived
DNA strand ligation reaction was carried out using DNA ligase at 10°C for 24 hours, and after heat treatment at 65°C for 5 minutes, 3 times the volume of ethanol was added to the reaction solution to precipitate and collect the plasmid DNA into which the DNA had been incorporated. (3) Transformation with a plasmid carrying the extracellular production (secretion) gene of xylanase Escherichia coli HB101 strain, a hybrid strain of Escherichia coli K-12 and Escherichia coli B strains [Molecular Cloning A
See Laboratory Manual p.504 (1982)]
(Genetic trait: F ^- , hsd S20 (r, m), rec
A13, ara-14, proA2, lacY1, galK2, rps
L20(Sm′), xyl-5, mtl-1, supE44λ ^- )
LB medium (10 tryptone (Difco) per 1 pure water
g, yeast extract 5g, glucose 1g, Nacl
(containing 10 g, adjusted to pH 7.0) was inoculated into 10 ml, cultured with shaking at 37°C, grown to late logarithmic growth, and then harvested. Cool this on ice,
Competent cells were made sequentially suspended in solutions with a final concentration of 0.03 M Cacl ₂ . Add the plasmid DNA solution obtained in (2) to this cell suspension, react for 60 minutes on ice, and apply a heat shock at 42°C for 1 to 2 minutes to allow the plasmid DNA to be incorporated into the cells. Ta. Next, this cell suspension was separately inoculated into the LB medium and incubated at 37°C for 3 to 5 days.
After shaking culture for an hour and performing transformation reaction,
Among the transformed strains obtained by collection and washing,
Escherichia coli HB101 (pCX311) (Feikoken Bacterial Serial No. 7345) was obtained. Example 2 The transformed strain obtained in Example 1 (3), Escherichia coli HB101 (pCX311)
(FERM P-7345) was mixed with LB medium (10 g of tryptone, 5 g of yeast extract, 1 g of glucose per plate).
g, glycerol 2g, NaCl 10g, penicillin
Containing 10mg) 500 containing 0.5% xylan in 100ml
Culture was carried out with shaking at 37°C in a ml flask. Cell growth (measurement of bacterial mass) is determined by absorbance at 660 nm (OD)
The enzyme activity was determined by adding 0.1 ml of xylan solution (manufactured by Seikagaku Corporation) and 0.1 ml of 0.2M trismalate buffer at pH 8.0 to 0.05 ml of the enzyme solution and reacting at 40°C for 10 minutes. ,5-dinitrosalic-ylic acid)1
ml of water and react at 100℃ for 5 minutes, then add 4 ml of water.
was added and the absorbance at 510 mμ was measured, and the amount of enzyme that produced a reducing power of 1 mg of xylose per minute was defined as 1 unit (U) of xylanase. FIG. 1 is a graph showing the xylanase activity, ie, the extracellular production (secretion) ability of xylanase, when using a medium prepared by adding 0.5% xylan to the LB medium. The cell mass of the transformed strain reached its maximum 9 to 12 hours after inoculation, and as shown in Figure 1, the extracellular xylanase activity began to increase approximately 6 hours later and reached its maximum 13 hours later. (≒0.35U/ml). The xylanase produced is very stable;
As shown in Figure 1, further culture
The production amount did not decrease even after continuing for hours, reaching more than 80% of the total enzyme activity. On the other hand, intracellular xylanase was slightly observed in the early stage of culture (9 hours after inoculation), but its production accounted for 10% of the total enzyme activity.
%, and the maximum was only 0.13 U/ml, and its activity gradually decreased. Also, Figure 2 shows
This is a graph showing xylanase activity when a medium containing 0.5% wrinkles instead of xylan was used in the LB medium, and almost the same tendency as in the case of FIG. 1 was observed. For comparison, the DNA-donating bacterium Bacillus C125 (Feikoken Bacterial Serial No. 7344)
were cultured and xylanase activity was measured. In the case of Bacillus C125, the medium used [(g/): xylan 10.0, yeast extract 5.0, polypeptone 5.0, K ₂ HPO ₄ 1.0, MgSO ₄ 7H ₂ O 0.2
[adjusted to pH 6.0 with Na ₂ CO ₃ 10] was inoculated into a 500 ml flask containing 100 ml of the solution, and cultured with shaking at 37°C. The extracellular xylanase activity of the culture solution was measured every 8 hours. The activity gradually increased 8 hours after inoculation, and reached its highest level at 48 hours (≒0.5
U/ml), but the activity rapidly decreased thereafter (see Figure 3). In addition, after salting out by adding ammonium sulfate to the culture solution of the transformed strain of Example 1, the precipitate was dissolved in water and dialyzed against running water overnight, and then equilibrated with 20mM phosphoric acid-monosodium phosphate-buffer of pH 4.5. After adsorbing the xylanase to the converted CM cellulose, the xylanase was eluted by changing the salt concentration from 0.1M to 0.7M.
It is eluted at around 0.4M. Collect the active fractions and combine them with Seph-adex G-100 (Seph-adex G-100).
Purified xylanase was obtained by gel filtration using 100). In addition, the culture solution of Bacillus C125 (Feikoken Bacteria No. 7344) was treated and purified in the same manner to obtain the same xylanase. In order to confirm the identity of both xylanases, pH activity, ultracentrifugal analysis, electrophoresis, molecular weight, etc. were measured, and the identity of both was confirmed as follows. a) pH 4 to 5 was adjusted using acetate, pH 5 to 8 was adjusted using trismalate, pH 7 to 9 was adjusted using tris-hydrochloric acid, and pH 9 to 11 was adjusted using glycine caustic soda, and the influence of pH was investigated using the above measurement method. As shown in Figure 5, the pH activities of both enzymes were found to be identical. b) In ultracentrifugation analysis, both enzymes have a sedimentation constant of approx.
It showed a single peak of 3.5S. c) In disk electrophoresis at pH 8.3, both enzymes formed a single band, and electrofocusing with ampholine showed a single peak, with an isoelectric point of 6.3. d) The molecular weight of both enzymes was determined to be approximately 40,000 by the SDS-polyacrylamide method. Example 3 Regarding the extracellularly produced substances of Escherichia coli HB101 (pCX311) (FERM P-7345), Escherichia coli HB101 strain to which the plasmid (pCX311) was not introduced and
Table 1 shows the results of examining enzyme proteins other than xylanase in comparison with the substances produced by Escherichia coli HB101 strain into which the pBR322 plasmid was introduced. The culture conditions were LB medium supplemented with xylan as in Example 2, and 37°C for penicillinase.
The results are obtained by culturing with shaking for 20 hours, and culturing for alkaline phosphatase and β-galactosidase for 15 hours. Furthermore, the enzymatic activities of alkaline phosphatase and β-galactosidase are expressed by measuring the absorbance (OD) at a wavelength of 420 nm.

【table】 [Brief explanation of drawings]

Figures 1 and 2 show the xylanase production activity of Escherichia coli HB101 (pCX311) used in the method of the present invention, and Figure 3 shows the xylanase production activity of Bacillus C125.
3 is a graph showing the xylanase production activity of bacteria. Figure 4 shows Escherichia coli.
This is a restriction enzyme cleavage map of the plasmid (pCX311) of HB101 (pCX311), and FIG. 5 is a graph showing the identity of the pH activities of xylanase produced by Escherichia coli HB101 (pCX311) and Bacillus C125.

Claims (1)

[Scope of Claims] 1. A plasmid that gives a host the ability to produce xylanase in vitro, the restriction site of which is Hind.
A plasmid that incorporates a deoxyribonucleic acid (DNA) fragment that carries the genetic information involved in the extracellular production of xylanase from Bacillus C125. 2. The plasmid according to claim 1, wherein the plasmid is the pCX311 plasmid. 3. The plasmid according to claim 1, wherein the plasmid vector is pBR322 plasmid DNA. 4. The plasmid according to claim 1, wherein the host is a microorganism belonging to the genus Escherichia. 5. The plasmid according to claim 4, wherein the microorganism belonging to the genus Escherichia is Escherichia coli. 6. Preparing a DNA fragment prepared from Bacillus C125 that gives the ability to produce xylanase extracellularly using a restriction enzyme, and interfering with the genetic information involved in the extracellular production of xylanase in the DNA fragment. Restricting the vector DNA of the plasmid using restriction enzymes that do not restrict the restriction sites of the vector DNA of the plasmid
A method for preparing a plasmid incorporating a DNA fragment carrying genetic information involved in extracellular production of xylanase, which comprises recombining the DNA fragment into a site and extracting the recombined plasmid. 7. The preparation method according to claim 6, wherein the plasmid is a pCX311 plasmid. 8. The method for preparing a plasmid according to claim 6, using the DNA of the pBR322 plasmid as the plasmid vector. 9. Escherichia coli HB101 (pCX311) capable of producing xylanase in vitro [Escherichia coli HB101 (pCX311)].