JPS60126077A - Culture of microorganism - Google Patents

Abstract

PURPOSE:To produce a physiologically active polymeric substance out of the microbial cell, and collect the polymeric substance in high efficiency, by preparing a plasmid integrated with a DNA participating in the extracellular production of a physiologically active polymeric substance, inserting the plasmid in the cell of the host microorganism, and culturing the microorganism. CONSTITUTION:The present invention is exemplified in the case that the physiologically active polymeric substance is xylanase. The DNA fragment capable of producing and accumultaing xylanase out of the microbial cell and collected from Bacillus C125 strain (FERM-P No.7344) is integrated in a vector (DNA of pBR322 plasmid). The plasmid is inserted in the cell of Escherichia coli HB101 to obtain a transformed strain [Escherichia coli HB101 (pCX311) (FERM-P No. 7345, cf. the figure)]. The transformed strain is cultured in a medium containing sodium chloride, etc. as an inorganic salt, and xylan, etc. as a carbon source for about 12-48hr, and xylanase accumulated out of the microbial cell is collected.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a plasmid in which deoxyri, which carries the genetic information of the microorganism involved in the extracellular production of physiological host-molecular substances, has integrated a nucleic acid (DNA) into the host microorganism cell. The purpose is to collect a significant amount of physiological substances by cultivating the cells and producing (secreting) and accumulating them outside the bacterial cells based on this specific genetic information. By introducing the genetic information of the pancreatic foot into the microorganisms of the host soil, and culturing the microorganisms with NA, relatively low molecular weight compounds such as amino acids and -27" However, Grasmid, which has the ability to produce high-molecular substances, has different levels of growth depending on the host microorganism, and is unable to fully express the plasmid. No effective cultivation method has been established.

Conventionally, when another microorganism is transformed as a host with a plasmid that carries the genetic information for producing 1M, a monomer product of a microorganism, outside the bacterial cell, specific production is possible. Only 'fr' can be produced in large quantities outside the bacterial body, but this could not be achieved with microorganisms of the genus Escherichia, which can normally be used as hosts, even by transformation using grass-mids.

This VC: i'J 1. The present inventor has previously developed a plasmid incorporating DNA of a microorganism belonging to Bacillus Kaede (IIi) containing genetic information involved in the extracellular production (secretion) of nicillinase as a product polymeric substance. was introduced into a microorganism called Escherichia 1mPIA, and by finding a method for striping this microorganism, they succeeded in producing (secretion) a large amount of enzyme proteins such as penicillinase outside the microorganism, and achieved the above-mentioned purpose. This is the first time that he has achieved this goal.

Furthermore, as a result of eight researches, the present inventors have discovered that a plasmid 1 containing all the DNA containing a genetic button '(r+1i) related to the extracellular production (secretion) of xylanase as a molecular compound τj.
A new microorganism was obtained by introducing this into a microorganism similar to Escherichia, and a method for culturing the microorganism was discovered, and enzymes such as xylanase were added to the outside of the microorganism. In the present invention, "polymer substances" refer to enzyme proteins that are metabolic products produced by microorganisms, In addition to antibiotics, the term refers to a group of useful bioactive polymer substances, including bioactive proteins of animal origin.

The method of the present invention will be explained in detail below.

The microorganism used in the method of the present invention contains all types of grasmids (extrachromosomal DNA or vector D
It is an Escherichia-like microorganism represented by Escherichia coli, which contains a plasmid that incorporates the foreign rice gene DNA 1 in NA).

A foreign gene (DNA) to be incorporated into the vector DNA
Fragments) are DNAs that carry information involved in the extra-box production (secretion) of specific polymeric substances, such as xylanase, benicillinase, phosphatase, β-galactosidase, amylase, protease, β-glucanase, etc. Examples include DNA that carries information for producing enzyme proteins and secreting them outside the bacterial body.

The foreign genetic DNA is a DNA that carries information involved in the extracellular production (secretion) of microbial-originated macromolecular substances. Kenbokuki No. 73
Advantageously, DNA fragments prepared from Dada are used.

Examples of microorganisms containing DNA carrying genetic information involved in the extracellular production of xylanase used in the present invention include Bacillus Cl,
Google). Bacillus C/25 is a microorganism isolated from soil collected in Tsurugashima Town, Iruma District, Saitama Prefecture, and has the following mycological properties.

In addition, the test and classification method for national properties is ``Aerobic Spore Homing Bacteria''['Aero
blc Spare-formlng Bacterl
a'(United State Department, vn@
nt of Agrlcul, ture, Nov,
/932by N, R, Sm1th, R, E, Gor
Don & F, E, C1ark)) and Hi "Parts Ace Manual Op Determining Bacteriolosso" CI? , t7) (Bergey's Ma
nual of Determinative Bac
It was carried out based on teri-ology (/937)).

a) Morphology/) The size is a rod of 0.5-0.7 μ x 3.0 μ and θμ.

2) Forms spores, size θ, 4-00gμ×1. Q
~7.2μ ovoid, spore-like swollen.

3) Durham staining G: is positive.

b) Growth status in each medium C) Physiological properties and above, the above Bacillus Cl, 25
Since the bacterium is an aerobic monosporous bacterium, Bacillus (
Although it is clear that it is a microorganism with a growth rate of mKm, it is clearly distinguished from known species in that its growth range is up to pl/θ and s°C, and it is a new alkalophilic bacterium. It was concluded that it is appropriate to recognize it as a 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, such as Co I E.
, pMB 9 strain, pBR, l ud strain,
Examples include the pSC10/ line, the R2 line, and the lambda phage line.

Moreover, any known method can be applied to the method of incorporating the foreign rice gene DNA into the vector DNA. For example, a method may be used in which the DNA is digested with an appropriate restriction enzyme (Endonuclease), followed by mixing with ON, A, which has been treated in the same manner and used as a vector, and religating with ligase.

DNA fragments of foreign rice and vector DNA thus obtained
The conjugate is introduced into the cells of a recipient microorganism of the genus Escherichia by the transformation method, and when it grows until it is established as a genetic trait, the desired genetic trait and vector DNA are combined.
A transgenic strain having both of these traits can be obtained. To fully culture the microorganisms obtained in this way, a medium that is incapable of producing substances produced by specific genetic information and that is suitable for the growth of host microorganisms of the genus Escherichia can be used. In the method of the present invention, growth and proliferation are performed in a medium that essentially contains a selected carbon source as well as an inorganic salt required for growth, and the body operculum reaches its maximum size in the same medium. It is necessary to continue the culture from the time until the production and accumulation of madder molecular substances, which are metabolic products, substantially cease in the medium. 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, drumstick, xylan, etc., nitrogen sources such as aqueous ammonia and ammonium salts, and inorganic ions, as well as amino acids, if necessary. Nutrients such as vimemin can be contained, and the influence of the carbon source qlI is particularly important, and it is necessary to appropriately select and add nutrients depending on the substance to be produced. LB medium (tryptone, yeast extract, salt) usually used as a growth medium for Eshie nohia and coli.
, BPB medium (Dlfco; polypeptone, yeast extract, potassium phosphate), nutrients, agar medium (Difco0
00/), Trigton 0 salt medium, etc. may be used as the basic medium.

In the above-mentioned medium that does not contain inorganic salts, substances of gO- or higher 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 ceramic body. Furthermore, the use of inorganic salts is approximately 0.0% with respect to the medium composition. S~3
．． 0% is appropriate.

When carbon source shadow 9ilid, xylan and/or xylan were added to the LB medium, excellent results were obtained in %, and it is desirable to add θ and 5, respectively, to the medium composition.

In the culture method, conditions such as p111 temperature and acid system supply amount that fail to grow Escherichia microorganisms were adopted, and 0 was obtained. However, after inoculating the microorganisms into the medium, the microorganisms grew and It is necessary to continue culturing in the same medium for a period of time from when the growth rate reaches its maximum, that is, from the late stage of logarithmic growth until the production and accumulation of madder molecular substances in the medium substantially ceases.

The time from when the bacterial population of Escherichia microorganisms reaches the maximum VC until the production and accumulation of polymeric substances in the medium substantially stops is within the time range of Habo/Koguza. , p11 condition is not particularly affected, but 9115
A range of ~g, especially p117, is reasonable. Thus, no aa is dehydrated for growth during cultivation. Using salts, carbon sources, and other components as a medium, a significant amount of polymeric substances are produced (secreted) outside the cells of the microorganisms, and the presence of
It can be collected.

According to the method of the present invention, the raw grass material is not only a single target polymer substance, but also multiple enzyme proteins are produced (shunted) outside the bacterial body @grave, and can be collected together. . That is, the above Esierich 7-co1) HB10/(pC
X3//) strain, it was found that xylanase, benicillinase, and alkaline phosphatase, which had previously been detected only inside the bacterial cells, were secreted in significant amounts outside the bacterial cells, as described in the Examples below. revealed.

This is based on the plasmid r (
p c x 3 / / ) +c included approximately 9,0θ
00 base pairs of DNA is used for extracellular reproduction (secretion) of metabolites.
It means that it is endowed with a fully competent host.

Does the culture method of the present invention require antibiotics in addition to enzymes and proteins? ! In fact, polysaccharides and other cylindrical fermentation products can be produced in large quantities, and in addition, NA and bioactive polymeric substances, which carry genetic information for the production of biologically active molecular substances in foreign rice, can be produced in large quantities. By using norasmid, which has DNA that carries genetic information involved in extracellular selective production (secretion), it can also be applied to the Okura bioactive protein production method for hormone peptides such as insulin and physiologically active proteins such as interferon antibodies. It greatly contributes to the industrial fermentation production of useful polymeric substances.

Below, as an example of the method of the present invention, the case of one enzyme protein, xylanase, will be explained. First, a complete example of the preparation of a transformed strain of Escherichia coli using a plasmid incorporating DNA carrying genetic information for extracellular production (secretion) will be described.

(Preparation of DNA with genetic information for xylanase production ability No.) Total medium 1r/l): 10.0%, yeast extract S, 0, poly 4gton S, O, HPO4
/ , θ, Mg5 (J4・riH2Cθ, 62 is Na
z COA / θ to pH 4, θ for 5 tuts, shaken culture for 79 hours at 3θC in Tamonoko, log JJI
After bringing the bacterial cells at the late stage of I field, DN was determined by the phenol method.
DNA was extracted using the A extraction method, extracted, and DNA 5 cape was extracted into 4 samples.

(Insertion of the 21D DNA fragment into the vector. Take 10 μm of the 0NA obtained in (1) and inject it with restriction endonuclease H1n.
Add dllllk and at 37℃ for 3 minutes, 70 minutes, 20 minutes, 30 minutes.
The mixture was allowed to react for 1 and θ minutes and was partially cleaved. On the other hand, tetracycline resistance (Tetr) used as a vector
and ampicillin resistance (Amp')'
R322f Lasmi de DNA (Bethesd
a Re5earch Laboratories (
(USA)) was completely cut off with Hindll and 45C
After heat treatment for 5 minutes, the former was mixed with the former, and the DNA strands were ligated at 70℃ using T4 phage-derived DNA ligase for 3 minutes.
Grasmid DNA'e was incorporated by adding twice the volume of ethanol. 1) Total NA precipitate was collected.

(3) Transformation with a plasmid carrying the extracellular production (secretion) gene of xylanase Escherichia coli K-/2 strain and Escherichia coli strain 3
Escherichia, which is 98 shares of No. 1 Egrid strain,
coli strain H8107 (Molecular Clonln
gAL Laboratory Manual p, 301I
(/9 g2)] (Genetic form g: F-1h
sds,! θ(r-8, m-8), rec A/,
J', ara/II, ρroA2, IacY/,
gal Niko, rps L 2θ(Sm'), xyl
-3゜mtl-/, sup E 1111λ-)iL
B medium (pure water/) was added (D
Contains lfco) / θ, Intoxicating extract sy, glucose/f, Na1l/θr', pH'7
．． 0 to l! 14IR Shimono) / θ punishment, 37C
After culturing with shaking, the cells were harvested after growing to late logarithmic expansion. The final concentration was θ, 03 M under cooling with water.
The cells were sequentially suspended in a CaC22 bath solution to obtain competent cells. This cell m = plasmid D obtained in (2) in the solution
Add a bath of NA and react with AO under water cooling, &2C
,/~ by applying a heat shock to the Grasmid D.
NA was incorporated into all cells. Next, this cell suspension was separately inoculated into the LB medium and incubated at 37°C for 3~! After culturing with shaking for r hours to perform a transformation reaction, the Mice bacteria were washed to obtain transformed microorganisms, from which Escherichia coli Ha/θ/ (pcx, 1//) (Feikoken-Kyodai 73'
1S) (FERMP-RI3DAS) was obtained. This transformed strain, Grasmid (pCXJ//), contains a DNA circular molecule incorporating a xylanase DNA fragment carrying the genetic information involved in the extracellular production (secretion) of L xylanase, that is, 98R312f lasmid DNA and approximately 1It, θθO. It is a novel 0/Kb NA circular molecule consisting of DNA with 1 base pair, and its restriction enzyme cleavage map is as shown in the figure.

Example/Transformed strain obtained in (3) above, Escherichia coli He/θ/ (pC,
No.) (FERMP-73gS) was mixed with LB medium (Trigton/θv1 yeast extract 51, glucose/1,
Glycerol ur, NaCt/θ), Penicillin 10K) SθO containing 0 and S xylan in 10Cme
Shaking culture was carried out at 37°C in an Mg flask. Cell growth (bacterial violet measurement is performed using A A o nm luminous intensity (00
), and the enzyme activity is 90 by adding 0.005 enzyme solution to xylanase (biochemical M? ℃
DNS (3,5-dinitrosalicyl acid) /
After adding rrd;f and reacting at 700℃ for S minutes, add ml of water and measure the absorption kk at 510 mμ, and calculate the amount of enzyme that produces the reducing power of xylose/Tny in 7 minutes as xylanase/unit (U). did.

FIG. 1 shows that the LB medium has 0. It is a graph showing xylanase activity, that is, xylanase extracellular production (C secretion) ability when using a medium containing S% Gysilane.

Cell 1 of the pre-Hpi transformed strain reached its maximum level at 9 hours after inoculation, and as shown in Figure 1, the extracellular xylanase activity continued to increase for approximately 6 hours. At first, it tentatively reached the maximum for 73 hours (ki θ, 33 U/me).

If the xylanase produced is extremely
As shown in Figure VC, an additional seven-year-old culture (IL-11
Even if the production was continued for up to g hours, the production amount did not decrease and reached 0% or more of the total enzyme activity. On the other hand, although some intracellular xylanase was observed in the early stage of culture (7 hours after inoculation), its production was about 10% of the total enzyme activity, and the maximum was only 0°/3 U/trte. , and its activity gradually decreased.

Furthermore, Fig. 2 is a graph showing the xylanase activity when using a medium in which 0° left side rice was added instead of xylan to the LB medium, which is different from the case in Fig. 1. The direction was recognized. As a comparison, the DNA donor bacillus Bacillus C/Ida (Feikoken Bacterial Serial No. 73 tag) was cultured and the xylanase activity was measured.

In the case of Bacillus C/, 25 bacteria, use J Alley ((
r/l): xylan 1060, yeast extract S, 0, polypone S, θ, 2HPO4/, (/%MgSO
4. 7H20, 0,2k Na2Co3/p at θ
H-ro. [adjusted to 0]/θQ ml was inoculated into a SOO-sized flask and cultured with shaking at 37°C.

The extracellular xylanase activity of the culture solution was measured at the beginning of the day. After inoculation, the activity gradually increased at 9 g hours, and reached the maximum activity (C0.3 U/d) at 9 g hours, but the activity rapidly decreased thereafter (see Figure 3).

Example A+J 8 Eschierhia coli HB/θ/ (PCX
3//) (FERMP
The extracellular xylanase production of ``-73iis'' was compared with that of ``1Ik-'' without salt in the medium. The culture conditions are the same as in Examples.

That is, the medium of Example/ was added to the basal medium and the salt-free fc.
The effect of the inorganic salt was investigated by culturing for 1 hour and 10 hours after the addition of the inorganic salt.

The results are shown in Table 7.
//) (FERM P-
Regarding the extracellular #substance of 73gS), Grasmid (p
Escherichia coli HB without introducing CX3/ / )
/θ strain and Escherichia coli H8107 strain into which ρF3R32,2 Grasmid was introduced,
Table 2 shows the results of examining enzyme proteins other than xylanase.

In addition, in LB medium supplemented with xylan according to the culture conditions h1 Example, 4-nisirinase was cultured with shaking at 37° C. for θ hours, and alkaline phosphatase and β-galactosidase were cultured for 75 hours. In addition, the enzymatic activities of alkaline phosphatase and β-galactosidase are determined by the optical density (OD) at q2θnm.
It is measured and expressed.

(Example No.) After salting out by adding ammonium sulfate to the culture solution of the transformed strain in Example /, it was dissolved in precipitated gold water, dialyzed against night running water, pII filtered, and the left YuQ'rn M phosphate-CM cellulose Vr equalized with phosphate/soda buffer.

After increasing the absorption, xylanase is eluted after xylanase by changing the salt concentration from θ,/M to 0.7M. Active classification money m-[-t77:? Dull filtration with Tsukusu G-10θ (5ephadcx G-/θO) was performed to obtain sheath-mounted xylanase. Also,
The Bacillus Cl2S play (Feikoken Bacterial Serial No. 734tlI)
The same xylanase was obtained by treating and purifying the entire homogenate from the culture of No. 1).

In order to confirm the identity of both xylanases, p11 activity, ultracentrifugation analysis, electrophoresis, molecular weight, etc. were measured, and the identity of both was confirmed as follows.

a) pH4t-31” as acetate, pH5-g n
) IJ Su? Ray), pH 7-9 was adjusted using Tris-HCl, p119-// was adjusted using glycine caustic soda, and as a result of calculating the influence of pH using the above-mentioned distance measurement method, as shown in Figure S, the pH activity of both enzymes was adjusted. Sameness was made clear and powerful.

b) In ultracentrifugation analysis, the sedimentation constant of both doxions was approximately 3.5.
3 single peaks were shown.

c) Both enzymes form a single band in disk electrophoresis at pHg 3, and electrofocusing with ampholine shows a single peak, with a peak at the isoelectric point. It was on 3.

d) The molecular weight of both enzymes was determined by the 5DS-polyacrylamide method to be about 202θθθ.

[Brief explanation of drawings]

Figures 1 and 2 show the xylanase production activity of Escherichia coli He/θ/ (pCX3//) used in the method of the present invention, and Figure 3 shows the xylanase production activity of Bacillus CI2! is a graph showing the xylanase production activity of bacteria. Figure 9 shows Escherichia coli He/θ/ (pCX3
This is a limit # cumulative cutting map of Grasmid CpCX3//) of //), and Fig. 5 C, E. coli lθ/ Cp
A small graph shows the identity of the pH activity of xylanase produced by CX3//) and Nogutilus Clu5. % Applicant RIKEN Figure 3 Culture period (hγ) Figure 4 □: ρBR322 Plasnid DNA Muroha until Kusho 8a Bakares C425 DNA fragment (kiss length-ze' DNA fragment) Figure 5 E, coQ and HB 101 (pcX311)-1→
Indication of Mr. Kazuo Wakasugi, Director-General of the Microbial System Torres CI2!5j I2 Title of Patent Application No. 232507 of 1988 Relationship with the person who performs the method of culturing microorganisms Applicant name (679) RIKEN Implantation Order Figures 1 and 2 will be corrected as shown in the attached sheet during the Japanese Imma 1 spontaneous release. Procedural amendment 59.10. -5 Showa year, month, day 1, case description 1982 Patent Application No. 23250'7 2, title of the invention Method for culturing microorganisms 3, relationship with the person making the amendment case Applicant name (679) RIKEN 4 , Agent 5, date of amendment order, amend the description of the invention specification as follows.

Claims (1)

[Scope of Claims] 0) Escherichia f4 containing deoxyliponucleus #I (DNA) i-incorporated grasmid, which carries the genetic information involved in the extracellular production of polymeric substances, is The macromolecular substance is inoculated into a medium containing the selected carbon source together with the inorganic salt required for the purpose, and after inoculation, from the time when the amount of microorganisms reaches the maximum, the polymer substance is substantially added to the medium. A method for culturing a microorganism, characterized in that the polymer substance is produced outside the microorganism by continuing the entire culture until production and accumulation cease. (2) The culture method according to item 11, wherein the inorganic salt is sodium chloride or potassium chloride. (3) The t@ culture method according to claim (2), wherein sodium chloride or potassium chloride accounts for 0.5 to 3.0% of the medium composition. (4) The culture method according to claim 1+, wherein the carbon source is wrinkles or xylan. (5) The culturing method according to claim (1), wherein the culturing time is 72 to 5 hours. (6) A microorganism belonging to the genus Escherichia is Escherichia coli HB10/ (pCX3//) (Imitation Koken Mayoyori No. 73'lS) (Escherlchla colt)
HB10/(ρCXJ'//)) The culture method according to claim (1). (7) pBR as a plasmid vector? Claim No. 12 (Culture method according to Claim 11) using plasmid DNA. (8) The polymeric substance is putty/I/X (Bacillus
s) and li. (9) Microorganisms belonging to Bacillus maple are Bacillus
illus)・C/25 ah (Microtechnical Laboratory No. 7
Claim No. 34(tg)), which is the culture method described in claim No. 81.
Method for culture of ridges described in Section 1.