JPH0375153B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention involves culturing a host microbial cell containing a plasmid containing deoxyribonucleic acid (DNA) carrying specific genetic information involved in the extracellular production of bioactive polymeric substances, and culturing the plasmid. The purpose of this method is to produce (secrete) and accumulate the above-mentioned polymeric substance outside the bacterial body using the genetic information of the microorganism, and to collect a significant amount of the produced substance. A method has been proposed to produce relatively low-molecular substances such as amino acids and peptides by introducing a plasmid containing DNA carrying specific genetic information into a host microorganism and culturing this microorganism. Plasmids capable of producing molecular substances have different propagation levels depending on the host microorganism, and it is not possible to achieve a sufficient expression function of the plasmid, and no effective culture method has been established. Conventionally, when a microorganism belonging to another genus is transformed as a host with a plasmid that incorporates DNA carrying the genetic information for producing macromolecular substances, which are metabolites of microorganisms, outside the microbial cell, only specific products can be produced. However, it has not been possible to produce a significant amount of the microorganisms outside the bacterial cells, and this has not been possible even through plasmid-based transformation using microorganisms of the genus Escherichia that can normally be used as hosts. In contrast, the present inventors previously developed a plasmid containing DNA carrying genetic information involved in the extracellular production (secretion) of penicillinase as a polymeric substance that is a metabolite of microorganisms belonging to the genus Bacillus. We discovered a method for culturing this microorganism and succeeded in producing (secretion) a large amount of enzyme proteins such as penicillinase outside of the microorganism, making it possible for the first time to achieve the above objective. did. Furthermore, as a result of intensive research, the present inventor introduced and transformed a microorganism belonging to the genus Escherichia with a plasmid incorporating DNA carrying genetic information involved in the extracellular production (secretion) of xylanase as a polymeric substance. The present invention was achieved by obtaining a new microorganism, finding a method for culturing the microorganism, and successfully producing (secreting) a large amount of polymeric substances represented by enzyme proteins such as xylanase outside the microorganism. It was completed. In the present invention, "polymeric substances" refers to a series of useful bioactive macromolecular substances, including enzyme proteins, antibiotics, etc., which are metabolites produced by microorganisms, as well as bioactive proteins of animal origin. It refers to something. The method of the present invention will be explained in detail below. The microorganism used in the method of the present invention contains a plasmid (extrachromosomal DNA, or vector DNA) in a format that can proliferate in cultured cells, such as the colicin E ₁ factor known as an extrachromosomal gene (plasmid) of the genus Escherichia. , a microorganism belonging to the genus Escherichia represented by Escherichia coli, containing a plasmid incorporating foreign genetic DNA. The foreign gene (DNA fragment) incorporated into the vector DNA is DNA that carries information related to extracellular production (secretion) of a specific polymeric substance, such as xylanase, penicillinase,
Examples include DNA that carries information for producing enzyme proteins such as phosphatase, β-galactosidase, amylase, protease, β-glucanase, etc. and secreting them outside the bacterial cell. The foreign gene DNA is DNA that carries information related to the extracellular production (secretion) of macromolecular substances originating from microorganisms, such as Bacillus.
A DNA fragment prepared from the microorganism belonging to the genus Bacillus C125 (Feikoken Bacterium No. 7344) is advantageously used. 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 test and classification method for mycological properties is "Aerobic Spore Homing Bacteria"
[“Aerobic Spore-forming Bacteria” (Unitd
State Department of Agriculture, Nov.1952
by NRSmith, REGordon & FEClark)
and “Burge Ace Manual of Determinative Bacteriology” (1957)
[Bergey's Manual of 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 x 1.0-
The sporangium is 1.2 μ in oval shape and swollen. (3) Gram staining is positive.

【table】

【table】

[Table] To summarize the above properties, the above-mentioned 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
1.0 and up to 55°C, it was clearly distinguished from known bacterial species, and it was concluded that it was appropriate to recognize it as a new alkalophilic bacterial species. The vector DNA may be extracted from naturally occurring DNA, or may be one in which part of the DNA other than the part essential for proliferation is missing.
Examples include ColE ₁ strain, pMB9 strain, pBR322 strain, pSC101 strain, R6K strain, lambda phage strain, and the like. Further, the foreign gene may be added to the vector DNA.
Any known method can be applied to incorporate DNA. For example, a method is used in which DNA is selected and treated with an appropriate restriction enzyme (Endonuclease) to cleave the DNA, then mixed with similarly treated DNA to be used as a vector, and religated with ligase. The conjugate of the foreign DNA fragment and vector DNA thus obtained is introduced into the cells of a recipient microorganism of the genus Escherichia by a transformation method,
When the vector is grown until the genetic trait is stable, a transformed strain having both the desired genetic trait and the vector DNA trait can be obtained. To culture the microorganism thus obtained, a medium suitable for producing a substance produced by specific genetic information and suitable for the growth of the host microorganism of the genus Escherichia can be used. However, in the method of the present invention, the composition of the medium used includes inorganic salts required for growth,
Grow in a medium that essentially contains the selected carbon source,
It is necessary to continue culturing as it is from the time when the number of bacterial cells reaches the maximum in the same medium until the production and accumulation of macromolecular substances, which are metabolites, substantially cease in the medium. It is. As the inorganic salt, sodium chloride is particularly effective, and potassium chloride can also be used. The medium containing inorganic salts includes carbon sources such as glucose, sucrose, lactose, maltose, glycerol, xylan, etc., nitrogen sources such as aqueous ammonia and ammonium salts, and inorganic ions, as well as amino acids and vitamins as necessary. The influence of the carbon source is especially important, and it is necessary to select and add nutrients as appropriate depending on the substance to be produced. Usually Escherichia. LB medium (tryptone, yeast extract,
Salt), BPB medium (Difco; polypeptone, yeast extract, potassium phosphate), nutrition. Agar medium (Difco0001), tryptone. A basic medium prepared from a saline medium or the like may be used. In the above-mentioned medium that does not contain inorganic salts, more than 80% of the substances are produced within the bacterial cells, so the presence of inorganic salts is essential in order to produce most of the produced substances outside the bacterial cells. The appropriate amount of inorganic salt to be used is approximately 0.5 to 3.0% based on the medium composition. Regarding the influence of the carbon source, particularly excellent results are obtained when 〓 and/or xylan are added to the LB medium, and it is desirable to add each in an amount of about 0.5% based on the medium composition. The culture method can adopt conditions suitable for the growth of microorganisms of the genus Escherichia, such as pH, temperature, oxygen supply, etc., but after inoculating the microorganisms into the medium,
During the period from when the microorganism grows and reaches its maximum cell mass, that is, from the late stage of logarithmic growth until production and accumulation of polymeric substances in the medium substantially cease,
It is necessary to continue culturing in the same medium. The time from when the amount of microorganisms of the genus Escherichia reaches the maximum until production and accumulation of polymeric substances in the medium substantially ceases is approximately 12 to 48 hours. Note that the pH condition is not particularly affected, but a pH range of 5 to 8, particularly a pH of 7, is suitable.
In this way, a significant amount of polymeric substances can be produced (secreted) outside the cells of the microorganism without further adding inorganic salts, carbon sources, and other components required for growth to the medium during culture, which is advantageous. It can be collected. By the method of the present invention, not only a single target polymeric substance but also multiple enzyme proteins are produced (secreted) in large amounts outside the bacterial cells, and can be collected together. . That is, by culturing the Escherichia coli HB101 (pC It was revealed that it was secreted in significant amounts. This is approximately 4000 base pairs of DNA contained in the plasmid (pC×311) described below obtained by the present invention.
This means that the host is endowed with the ability to produce (secrete) metabolites outside the cell. By the culture method of the present invention, in addition to enzyme proteins, antibiotics, polysaccharides, and other polymeric fermentation products can be produced in large quantities, and genetic information for the production of foreign physiologically active polymeric substances can also be obtained. By using a plasmid that has DNA that carries genetic information involved in the extracellular selective production (secretion) of physiologically active polymeric substances, it is possible to suppress physiological effects such as hormone peptides such as insulin and interferon antibodies. Since it can be applied to mass production of active proteins, it will greatly contribute to the industrial fermentation production of useful polymeric substances. The case of xylanase, an enzyme protein, will be explained below as an example of the method of the present invention. First, we will exemplify the preparation of a transformed strain of Escherichia coli using a plasmid incorporating DNA carrying the genetic information for extracellular production (secretion). (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), in a medium [(g/):〓
10.0, yeast extract 5.0, polypeptone 5.0,
K ₂ HPO ₄ 1.0, MgSO ₄ 7H ₂ O 0.2 in Na ₂ CO ₃ 10
After culturing with shaking at 30°C for 19 hours in a medium (adjusted to pH 6.0), the cells were harvested at the late stage of logarithmic growth.
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 10μg of DNA obtained in (1), add restriction endonuclease Hind, and incubate at 37°C for 5 minutes.
It was allowed to react for 20 minutes, 30 minutes, and 60 minutes for partial cleavage. 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)
(manufactured by Hind) was completely cleaved with Hind, heat treated at 65℃ for 5 minutes, mixed with the former, and the DNA strands were ligated using _T4 phage-derived DNA ligase at 10℃ for 24 hours. After 5 minutes of heat treatment,
Two volumes 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, which is a hybrid strain of Escherichia coli K-12 strain and Escherichia coli B strain [Molecular Cloning A
See Laboratory Manual p.504 (1982)]
(genetic traits: ^F- , hsdS20(r ^- _B , m ^- _B ), rec
A13, ara ^- 14, pro A2, lac Y1, gal K2,
rps L20 (Sm ¹ ), xyl-5, mtl-1, sup
E44λ ^- ) was inoculated into 10 ml of LB medium (containing 10 g of tryptone (Difco), 5 g of yeast extract, 1 g of glucose, and 10 g of NaCl per pure water, adjusted to pH 7.0), and cultured with shaking at 37°C.
Bacteria were harvested after growing to the late logarithmic growth stage. This was sequentially suspended in a solution of 0.03M CaCl ₂ at a final concentration under ice cooling to obtain competent cells. Add the plasmid DNA lysate obtained in (2) to this cell suspension, react for 60 minutes on ice, and heat at 42℃ for 1 hour.
A heat shock was applied for ~2 minutes to incorporate the plasmid DNA into the cells. Next, this cell suspension was separately inoculated into the LB medium,
After carrying out a transformation reaction by culturing with shaking at 37°C for 3 to 5 hours, the cells were harvested and washed to obtain a transformed strain.
Among them Esierhia coli HB101
(pCX311) (FERMP-
7345) was obtained. The plasmid of this transformed strain (pC 9.01Kb of novel DNA consisting of paired DNA
It is a circular molecule, and its restriction enzyme cleavage map is
As shown in the figure. Example 1 The transformed strain obtained in (3) above, Escherichia
Cori HB101 (pC×311) (Feikoken Bacillus No. 7345)
(FERMP-7345) was shaken at 37°C in a 500 ml flask containing 0.5% xylan in 100 ml of LB medium (each containing 10 g of tryptone, 5 g of yeast extract, 1 g of glucose, 2 g of glycerol, 10 g of NaCl, and 10 mg of penicillin). Cultured. Cell growth (measurement of bacterial mass is by absorbance (OD) at 660 nm,
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 with pH 8.0 to 0.005 ml of the enzyme solution and reacting at 40°C for 10 minutes.
Add 1 ml of DNS (3,5-dinitrosalicylic acid) and react at 100℃ for 5 minutes, then add 4 ml of water and measure the absorbance at 510 mμ.
The unit is (U). 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 produced xylanase was very stable, and as shown in Figure 1, even if the culture was continued for up to 48 hours, the production amount did not decrease and reached more than 80% of the total enzyme activity. In contrast, 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.
The maximum activity was only 0.13 U/ml, and its activity gradually decreased. In addition, Figure 2 is a graph showing the xylanase activity when using the LB medium to which 0.5% 〓 was added instead of xylan, and almost the same trend as in Figure 1 was observed. It was done. For comparison, the DNA-donor Bacillus C125 (Keiken Bacteria No. 7344) was cultured and the 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 9.0 with Na ₂ CO ₃ 10]] was inoculated into a 500 ml flask containing 100 ml of the phage, and cultured with shaking at 37°C. The extracellular xylanase activity of the culture solution was measured every 8 hours. After inoculation, the activity gradually increased 8 hours after inoculation, and by 48 hours, the activity was the highest (0.5U/
ml), but the activity rapidly decreased thereafter (see Figure 3). Example 2 The influence of inorganic salts in the medium was compared on extracellular xylanase production of Escherichia coli HB101 (pCX311) (FERMP-7345). The culture conditions are the same as in Example 1.
It is similar to That is, the medium of Example 1 was used as a basic medium, and inorganic salts were added thereto and cultured for 14 hours and 20 hours, respectively, to examine the influence of the inorganic salts. The results are shown in Table 1.

【table】

[Table] Example 3 Regarding the extracellularly produced substances of Escherichia coli HB101 (pCX311) (FERM P-7345) (FERM P-7345), Escherichia coli HB101 strain into which the plasmid (pCX311) was not introduced as well as
Table 2 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 the xylan-added LB medium of Example 1, and the culture conditions for penicillinase were 37°C.
The cells were cultured with shaking for 20 hours, and alkaline phosphatase and β-galactosidase were cultured for 15 hours each. In addition, the enzymatic activities of alkaline phosphatase and β-galactosidase are
It is expressed by measuring the absorbance (OD) at 420 nm.

[Table] Reference Example After adding ammonium sulfate to the culture solution of the transformed strain of Example 1 and salting out, the precipitate was dissolved in water and dialyzed against running water overnight, and then diluted with 20mM phosphoric acid-phosphoric acid 1 sodium buffer at pH 4.5. When xylanase is adsorbed onto equilibrated CM cellulose and eluted by changing the salt concentration from 0.1M to 0.7M, it is eluted at around 0.4M. The active fractions were collected and subjected to gel filtration using Sephadex G-100 to obtain purified xylanase. In addition, the culture solution of Bacillus C125 (Feikoken Bacteria No. 7344) was treated and purified in the same manner to obtain a similar xylanase. In order to see the identity of both xylanases, PH
As a result of measurements of activity, ultracentrifugation, electrophoresis, molecular weight, etc., the identity of the two was confirmed as follows. (a) PH4 to 5 was adjusted using acetate, PH5 to 8 was adjusted using trismalate, PH7 to 9 was adjusted using tris-hydrochloric acid, and PH9 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 identity of the PH activities of both enzymes was revealed. (b) In ultracentrifugation analysis, both enzymes have a sedimentation constant of approx.
It showed a single peak of 3.5S. (c) Disk electrophoresis at pH 8.3 revealed that 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.

[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 101 (pCX311) and Bacillus C125.

Claims (1)

[Claims] 1 Incorporating deoxyribonucleic acid (DNA), which carries genetic information involved in the extracellular selective production of xylanase, a metabolite of Bacillus C125 bacteria, or this DNA and DNA encoding a protein other than xylanase. A microorganism belonging to the genus Escherichia containing a plasmid is inoculated into a medium containing an inorganic salt required for growth. By continuing the culture until the production and accumulation of xylanase or xylanase and the protein in the medium stops, the xylanase or xylanase and the protein are released outside the microorganism. A method for culturing microorganisms, characterized by producing.