JPH0256072B2 - - Google Patents

Description

[Detailed description of the invention]

(Technical Field) The present invention relates to a bacterium of the genus Estierichia transformed with a recombinant plasmid containing a cell wall lytic enzyme gene. (Prior Art) In order to utilize proteins from microorganisms such as yeast and chlorella as a food source or feed source, it is necessary to dissolve and remove the cell walls of these microorganisms. However, a technology that can effectively industrially produce enzymes capable of degrading these bacterial cell walls has not yet been established. (Objective of the Invention) An object of the present invention is to provide a bacterium belonging to the genus Estierichia into which a recombinant plasmid having a cell wall lytic enzyme gene has been introduced. (Structure of the invention) The recombinant plasmid used in the present invention is
It is obtained by introducing the cell wall lytic enzyme gene of Arthrobacter genus into the vector plasmid YRp7 of Escherichia coli trpC-9830. By introducing this into Escherichia genus bacteria such as Escherichia coli HB101 or Escherichia coli JA221, the cell wall lytic enzyme gene is expressed. Furthermore, the method for producing a cell wall lytic enzyme according to the present invention includes culturing a bacterium belonging to the genus S. coli containing the above recombinant plasmid and collecting the cell wall lytic enzyme accumulated within the bacterium or in the culture medium. The production of this cell wall lytic enzyme is
The parent strain Arthrobacter YCWD3 is inducible, whereas the transformed strain S. coli is constitutive. In the present invention, the cell wall lytic enzyme gene of Arthrobacter YCWD3 is cloned into Escherichia coli as follows: From logarithmically growing cells of Arthrobacter YCWD3 to J. Marmur's method (J. .
Chromosomal DNA is prepared by Marmur, J. Mol. Biol. 3 208 (1961)). Use this restriction enzyme Bam
Double-stranded DNA fragments are obtained by incomplete digestion with HI. On the other hand, the vector plasmid YRp7 contains the restriction enzyme Bam
It has a sequence that is cut at only one place on the tetracycline (Tc) resistance gene by HI. Cut this vector plasmid YRp7 into a straight line. Then,
Cut off the terminal phosphate with alkaline phosphatase. By doing this, the repair enzyme added later
_T4 DNA ligase prevents YRp7 from returning to its original circular DNA. Both DNAs are mixed and T ₄ DNA ligase is added to connect the broken phosphate bonds to obtain a mixture of various plasmids including the desired recombinant plasmid (named pX20).
This is then transformed into a host microorganism, Escherichia coli, which has been treated with CaCl ₂ to make it easier to accept foreign DNA.
Add to HB101 or Escherichia coli JA221. Recombinant plasmid pX20 is inserted into the chromosomal DNA into the Tc ^r of YRp7.
Since this fragment has been introduced into Escherichia coli HB101 (pX20) or JA221 (pX20), the tetracycline resistance gene has been isolated and the S. coli has become tetracycline sensitive (Tc ^s ).
becomes. Since the other ampicillin resistance gene (Am ^r ) of YRp7 is kept alive, S. coli HB101 (pX20) or JA222
(pX20) is ampicillin resistant (Am ^r ). In order to select transformants that express the cell wall lytic enzyme system gene derived from Arthrobacter YCWD3, colonies of Escherichia coli HB101 or JA221 of Am ^r are selected, and strains having cell wall lytic enzyme activity are screened among them.
Screening for S. coli HB101 (pX20) or JA221 (pX20) having cell wall lytic enzyme activity was performed by incubating ^Amr colonies with baker's yeast cell wall substrate and selecting strains that produced cell wall lytic plaques around the colonies. be exposed. By allowing ampicillin and cell walls to coexist in the selection medium, it is possible to simultaneously select strains that are both Am ^{r and} positive for cell wall lytic activity. The transformed strains Escherichia sp. coli HB101 (pX20 (accession number: FAIKEN BIKIYO NO. 7742) and JA221 thus obtained in the present invention
(pX20) (Accession number: FAIKEN BIGI no. 7744) has a cell wall lytic enzyme gene, in particular, a β-1,3 glucanase gene. This enzymatic activity is found primarily in the periplasmic compartment. Transformed strain Estieritia coli HB101
(pX20) and JA221 (pX20) produce β-
1.3 glucanase was determined by paper chromatograms of oligosaccharides obtained by acting on yeast glucans, adsorption properties by Avicel adsorption chromatography, and polyacrylamide gel electrophoresis.
It was confirmed that the glucanase was the same as the glucanase produced by the parent strain. (Example) The present invention will be described in detail below with reference to Examples. Example 1 (Preparation of recombinant plasmid) Parent strain: The parent strain Arthrobacter YCWD3 used in the present invention (accession number: Microtechnical Research Institute No. 7739) contains cell wall lytic enzyme β-1.3 glucanase (glucanase). and glucanase) was isolated and fixed from the surface of konnyaku leaves that had rotted in the air.
The identification data is shown in Table 1. Based on this identification data, this strain was identified by REBuchanan and N.
E. Gibbons, Bergey's Manual of
Determinative Bacteriology, 7th Edition,
The willams & Wilkins Company,
Baltimore. It was identified as a new bacterial species belonging to Arthrobacter and a mutant strain of Arthrobacter citreus. This strain is β-
It has a cell wall lytic enzyme gene containing 1,3 glucanase. Table 1 (1) Form: Liquid shaking culture (Difco nutrient
initially almost spherical (about 1 μm in diameter)
After 5 hours, the cells become rods (bent rod shape, total length about 1.0 μm), after 9 to 10 hours, spherical cells appear again, and after 24 hours, all the cells become almost spherical (1 μm in diameter). (2) Spores: Not formed. (3) Mobility: Spherical objects are non-motile;
The existence of motile individuals was observed only during a short period before and after the appearance of long rods. (4) Flagella: When the bacterial cells are observed under an electron microscope at the time when motile individuals appear during growth, the presence of long rod-shaped bacteria with peritial hairs is observed. (5) Pigment: Usually yellow in color. In young cultures, this dye does not diffuse into the medium, but when cultured with vigorous shaking for long periods of time, a small amount of the dye appears in the medium. (6) Liquid culture (Difco nutrient broth) Although the bacterial cells did not grow very well when incubated at 30°C, they grew well when cultured with shaking.
No film or ring is formed on the surface during incubation. (7) Difco nutrient agar
The colonies grew at 37°C, and on the first day, the entire edge of the colony formed a convex circular shape, and on the fifth day, the periphery of the colony became thread-like, and growth of pseudomycotic cells (each bacterial cell is a group of cocci) was observed. Colonies are always lemon-yellow in color and turn dark yellow-red as they age. (8) Meat juice culture Growth: Surface growth or turbidity: Slightly cloudy Aroma: Slight ammonia odor Sediment: Produces a slightly yellow sediment (9) Meat juice agar culture (30℃, 72 hours culture) Growth: Moderate; Diameter 0.8-1.0mm Shape: Circular Surface: Smooth Edge: Complete, but old culture (after 5 days)
Then it becomes radial. Uplift: Raised Viscosity: Butter-like Gloss: Yes Pigment: Yellow Deep settlement: Diameter directly below or in the center of the settlement
Hard settlements of less than 0.5 mm are observed. (10) Growth temperature: 20-40℃. (Optimum 30-37℃) (11) Growth PH: PH6.0-9.0. (Optimal PH7.0-8.0) (12) Anaerobic/Aerobic: Aerobic. (13) Gram staining: Positive (14) Acid-fastness: Negative (15) Methylred test: Positive (16) Fouges-Proskauer reaction: Negative (17) Indole production: Negative (Ehrlich method,
Nitrous acid method 30℃, 3 days) (18) Generation of hydrogen sulfide: Positive (Difco nutrient
broth, NaCl pbCO ₃ agar medium) (19) Ammonia production: Positive (20) Nitrate reduction: Positive (21) Catalase production: Positive (22) Urease production: Negative (30℃, 3 days of shaking culture) ( 23) Liquefaction of gelatin: Positive (20℃, 7 days) (24) Hydrolysis of starch: Negative (30℃, 3 days) (25) Utilization of citric acid: Not used (30℃,
(3 days shaking culture) (26) Milk coagulation: positive or negative (30℃,
(5 days) (27) Litmus reducibility: Negative (30℃, 5 days) (28) Nitrate availability: Negative (37℃, 13 days of shaking culture) (29) Cellulose decomposition: Negative (37℃, 10 days) (30) Salt tolerance: Grows up to 7% (30℃, shaking culture for 5 days) (31) Utilization of various carbon sources Glucose, mannose, fructose, galactose, maltose,
Produces acid from sucrose. No acid production is observed from lactose, mannitol, sorbitol, starch, or cellulose. Preparation of chromosomal DNA: Take a loopful of Arthrobacter YCWD3 from the L-broth agar slant,
Add this to 100ml of L-broth (1% Bactopeptone-
Planted in 0.5% Difco yeast extract − 0.5% NaCl)
The culture was incubated overnight at 37°C with shaking. 10ml of this culture solution
was added to 1000 ml of L-broth containing 2% glycine and shaken at 37°C for 2 hours. The resulting cells were collected by centrifugation. Add this to 0.15M NaCl−
Lysozyme (20 mg/ml, manufactured by Sigma, suspended in 0.1 M EDTA (PH8.0) and dissolved in the same solvent)
Add grade I) to 2 mg/ml at 37℃
It was kept warm for 65 minutes. Add 10% sodium dodecyl sulfate (SDS) to 2% (final concentration) and at 65°C.
A lysate was obtained by heating for 20 minutes. Add proteinase to this
K (manufactured by Merck) was added at a concentration of 120 μg/ml, and the mixture was kept at 37° C. for 2.5 hours. Final concentration of NaClO ₄
The mixture was added to a concentration of 1M and then shaken with an equal volume of chloroform-isoamyl alcohol (24:1v/v). The mixture was centrifuged (10,000 rpm, 10 minutes, 20°C) to remove the upper layer, layered with 2 volumes of ethanol, stirred, and the resulting filamentous precipitate was spread onto a glass rod. Dissolve this in 0.01M Tris-HCl (PH7.5) - 0.001MEDTA, add NaCl to 0.25M, and add EDTA.
Added to make it 0.02M. In addition to this,
100μ of RNAase A (Type-A manufactured by Sigma)
g/ml and kept at 37°C for 30 minutes. After adding an equal volume of chloroform-isoamyl alcohol and shaking, centrifuge (2400 rpm.
After 15 minutes at room temperature, the upper layer was removed and 2 volumes of ethanol were added. The resulting filamentous precipitate was spread onto a glass rod. This is again 0.01M Tris-HCl (PH7.5) −
Dissolved in 0.001M EDTA, added NaCl to 0.1M and EDTA to 0.01M. and α-amylase (Worthington)
It was added to 0.05 unit/ml and kept at 37°C for 1 hour. This was shaken with an equal volume of phenol (equilibrated with 0.05M Tris-HCl (PH8.0) - 0.1M NaCl), centrifuged (2400 rpm, 10 minutes, room temperature), and the upper layer was removed. After ether extraction to remove phenol, 2 volumes of ethanol were added. 0.01M of the resulting filamentous precipitate
Tris-HCl (PH7.5)-dissolved in 0.001M EDTA (390 μg/ml). Approximately 1 mg of chromosomal DNA preparation is obtained per 10 ml of glycine-treated steady-state culture. This chromosomal DNA is degraded better than BamHI and PstI, but hardly degraded by EcoRI, Hind and XbaI. Preparation of chromosomal DNA fragments: chromosomes obtained above
100 μg/ml of DNA was mixed with Bam HI buffer (0.01M Tris-HCl (PH8.0), 0.1M NaCl,
₁₅ , 30, 45 and
Treated for 60 minutes. Each treated product was combined and subjected to phenol extraction, and then the resulting precipitate was washed with ethanol and dried with 2 volumes of ethanol. The obtained chromosomal DNA fragment was dissolved in 0.01 M Tris-HCl (PH7.5) - 0.001 MEDTA (approx.
550 μg/ml). Vector plasmid: The vector plasmid used in the present invention is YRp7. This vector plasmid contains a fragment containing the yeast trpI gene.
It is a vector ligated with pBR332 and has a molecular weight of 3.8 x 10 ⁶ Daltons. Contains selectable markers for tetracycline, ampicillin, and tryptophan (trpI). 1 by restriction enzyme Bam HI
It is cut at several points, and when foreign DNA is inserted into this cut point, it loses tetracycline resistance (Tc ^r ). This vector plasmid YRp7 is well known and easily available, but in this example, Escherichia coli carrying it was used.
coli) trPC-9830 (YRp7) (Accession number: FAIKEN BIOLOGY NO. 7740). To obtain the vector plasmid YRp7, first, the above-mentioned S. coli trpC-9830 carrying this
(YRp7) to 100ml of L-broth (1%
Bactopeptone−0.5% Difco Yeastextract−0.5
% NaCl) and cultured at 37°C overnight. Add 10ml of this culture solution to L-broth from step 1 and heat at 37°C.
Incubate for 2.5 hours and add 170μ of chloramphenicol.
g/ml and further incubated overnight at 37°C. Collect the bacterial cells by centrifugation, suspend them in 15 ml of ice-cold 0.05 M Tris-HCl (PH8.0)-25% sucrose, and add lysozyme (Sigma, grade I) to 0.01 M Tris-HCl (PH8) to a concentration of 20 mg/ml. Add 3 ml of .0) to the mixture, mix and leave on ice for 5 minutes.
Next, 6 ml of 0.25M EDTA (PH 8.0) was added, mixed and kept on ice for 10 minutes, and then 7.5 ml of 5M NaCl was added and mixed, followed by 3 ml of 10% SDS and mixed.
After leaving on ice overnight, centrifuge (20,000 rpm., 45
minutes, 0°C), and the supernatant was removed. This was soaked in a 65°C water bath for 15 minutes. The resulting precipitate was removed by centrifugation, and the supernatant was shaken with an equal volume of phenol (equilibrated against 0.05M Tris-HCl (PH8.0) - 0.1M NaCl). Precipitated with ethanol. The precipitate was collected by centrifugation, washed with ethanol, and then air-dried. Add this to 0.01M Tris-HCl (PH7.5) - 0.001M
It was dissolved in EDTA and NaCl was added to make it 0.25M. Next, precipitation was performed with 2 volumes of ethanol, and the precipitate was washed with ethanol and air-dried. Add this again to 0.01M Tris-HCl (PH7.5) - 0.001M
Dissolved in EDTA. Add CsCl to this by weight at 48%
Add 400μ of dissolved ethidium bromide to
g/ml and left at room temperature for a while. The resulting precipitate was removed by centrifugation (3000 rpm, 20 minutes), and the supernatant was centrifuged (40000 rpm, 44 hours, 15°C). The centrifuge tube was observed under a Cypress line, and the plasmid band was removed with a syringe needle and extracted twice with equal volumes of n-butanol. Dilute the bottom layer 5 times with water,
Precipitated with 2 volumes of ethanol. The precipitate was collected by centrifugation, washed with ethanol, and then air-dried. Add this to 0.01M Tris-HCl (PH7.5) - 0.001M
It was dissolved in EDTA and NaCl was added to make it 0.25M. Then, it was precipitated again with 2 volumes of ethanol, and the precipitate was collected by centrifugation, washed with ethanol, and air-dried. This was finally dissolved in 0.01M Tris-HCl (PH7.5)-0.001M EDTA and stored at 4°C. In this way purified plasmid DNA
Got YRp7. Cleavage of vector plasmid YRp7: Purified vector plasmid DNA YRp7 50μg/ml
was treated with the restriction enzyme Bam HI (manufactured by Takara Shuzo Co., Ltd.) at 60 units/ml in Bam HI buffer at 30°C for 65 minutes. Heat the EDTA at 65°C for 15 min.
NaCl was added to 0.25M and precipitated with 2 volumes of ethanol. The precipitate was collected by centrifugation, washed with ethanol, and then dissolved in 0.05M Tris-HCl (PH8.0) (for YRp7).
100μg/ml). Next, calf small intestine alkaline phosphatase (manufactured by Boehringer, Grade []) was added.
0.1M Tris-HCl (PH8.5) − 0.001M MgCl ₂ −
Dialysis against 0.0001M Zn(OAc) ₂ followed by addition of equal volume of glycerin; 3.3 units/μ
) was added at 80 units/ml, kept at 37°C for 30 minutes, and then heated at 68°C for 15 minutes. Add EDTA to a concentration of 0.02M, extract with an equal volume of phenol (equilibrated against 0.05M Tris-HCl (PH8.0) - 0.1M NaCl), and add NaCl to the aqueous layer to a concentration of 0.25M. After that, it was precipitated with 2 volumes of ethanol. The precipitate was collected by centrifugation, washed with ethanol, and air-dried. Add this to 0.01M Tris-HCl (PH
7.5) - Dissolved in 0.001M EDTA. thus,
Alkaline phosphatase treated YRp7 was obtained. Preparation of a mixture of various plasmids containing recombinant plasmid pX20: 100 μg/ml of the chromosomal DNA fragment of the parent strain obtained above and 20 μg/ml of YRp7 that had been cut with Bam HI and treated with alkaline phosphatase were added to T4 DNA ligase buffer. Liquid (0.01M Tris-HCl (PH7.5), 0.05M
NaCl, 0.01M _MgCl2 , and 0.01M dithiothreitol) with 1mM ATP and 40 units/ml of _T4 DNA ligase (Takara Shuzo Co., Ltd.) at 4°C.
Recombinant plasmid was reacted for 24 hours at
A mixture of different plasmids containing pX20 was obtained.
After adding 1/25 volume of 0.5M EDTA (PH8.0) to this, it was heated at 65°C for 15 minutes, cooled on ice, and then stored at 4°C. Example 2 (Introduction of recombinant plasmid pX20 into a host microorganism) Host microorganisms: E.coli HB101 (accession number: Microtechnical Research Institute No. 7741) and JA221 (accession number: E. coli) were used as host microorganisms. Koken Bokuyori No. 7743) was used. Its genotypes are F ^- rk ^- mk ^- recA ^- pro leu thi, respectively.
lacY Str ^r endoI ^- and recA ^- leuB6 trp
ΔE5 hsdR ^- M ⁺ lacY thr thi. Pretreatment of host strain: A 1% culture of Escherichia coli HB101 cultured overnight in L-broth is inoculated into the same medium and cultured for 2.5 hours. Cells are collected by centrifugation, centrifugally washed with 100mM _MgCl2 , and suspended in 100mM _CaCl2 . Then,
Keep at 0°C for 20 minutes. Then, the bacterial cells are collected by centrifugation and resuspended in 100 mM CaCl ₂ to approximately 200×10 ⁸ cells/ml. Introduction of recombinant plasmid into host strain:
0.05 ml of a mixture of various plasmids containing the recombinant plasmid pX20 (Arthrobacter
(containing 5 μg of DNA fragments) was diluted with 0.02M Tris-HCl (PH8.0) - 0.001M EDTA - 0.02M NaCl to 0.2ml, and this was added to 0.4ml of the _CaCl2- treated suspension of S. coli HB101.
Mix and hold at 0℃ for 30 minutes. Then 42℃
After leaving it for 2 minutes, add 6ml of L-broth and heat at 37℃.
Cultured for 1.5 hours. Search for S. coli transformed strain having recombinant DNA plasmid pX20: S. coli transformed with the mixture of recombinant plasmids introduced above.
All cells of strain HB101 were treated with 100 μg/ml ampicillin and 0.5% baker's yeast cell wall (H.
Tanaka & H.J. Phaff, J.Bacteriol. 89 , 1570
(1965)) containing L-
It was plated on a broth agar plate and kept at 37°C. One out of about 3500 colonies that grew showed cell wall lysis plaques. All colonies produced by plating this transformed strain on the same agar medium showed cell wall lysis plaques. Host strain Estieritia coli
HB101 did not show such cell wall lytic plaques. Identification of recombinant plasmid pX20 containing the cell wall lytic enzyme gene: The above transformed strain was cultured in liquid in L-broth containing ampicillin, and a plasmid was prepared from the cells in the same manner as the vector plasmid YRp7. This recombinant plasmid was named pX20 as described above.
This recombinant plasmid pX20 is approximately 14.5Kb
Restriction enzymes in size (kilobase pairs)
By Bam HI, 5.8kb YRp7 part and about 8.5kb
The foreign DNA part was divided into two parts. This foreign DNA
Part of the chromosome of Arthrobacter YCWD3 that is not cut by EcoRI, Hind and XbaI
It had the characteristics of DNA. This recombinant plasmid pX20 has a concentration of approximately 3 x 10 ⁵ colonies/μg in the host strain S. coli HB101.
Alternatively, generate an ampicillin-resistant transformant in JA221. All of these transformed strains showed cell wall lysis spots on L-broth agar plates containing baker's yeast cell walls. By the way, YRp7
Estieritia collie introduced HB101
Both the strain and the S. coli JA221 strain introduced with pBR322 do not show cell wall lysis plaques on the same agar plate. Example 3 (Production of cell wall lytic enzyme by transformed strain) Culture conditions of S. coli transformant strain: Transformed strain was treated with ampicillin 100 μg/ml
Cultured overnight at 37℃ on an L-broth agar plate containing
The cells were inoculated into broth and cultured with shaking at 37°C for 24 hours. Cell wall lytic enzyme activity: Bacterial cells obtained by centrifugation at 0°C were treated with either of the following two methods, and cell wall lytic activity was examined. (i) Transfer the bacterial cells to cold PBS buffer (0.137M NaCl−
Wash with 0.01M sodium phosphate, PH7.2),
This was suspended in 5 ml of the same buffer and subjected to ultrasonication. 20 at 11000rpm at 0℃
The mixture was centrifuged for a minute and the supernatant was collected. (ii) Cool the bacterial cells with 0.01M Tris-HCl (PH7.3) −
Wash with 0.03M NaCl and add 40ml of 0.033M
Suspended in Tris-HCl (PH7.3). 40 of this
ml of 0.033M Tris-HCl (PH7.3) - mixed with 40% sucrose and added 0.16ml of 0.5M EDTA (PH7.5). This was gently stirred at 20-24°C for 15 minutes. Centrifugation (11000rpm., 15 minutes, 0℃)
Discard the supernatant and mix the bacterial cells with 80ml of ice water.
A stage fraction was prepared by stirring gently at ℃ for 15 minutes, centrifuging (11000 rpm, 15 minutes, 0 ℃) and removing the supernatant.
(NGNossal &L.A.Heppel; J.Biol.Chem.
Vo1241, P.3055 (1966)). The cell wall lytic activity obtained from the extract prepared in (i) above was
Shown in the table. To measure the activity, 2.5 ml of the reaction mixture having the composition of 0.01M phosphate buffer (PH6.25) - 0.1% baker's yeast cell wall was kept at 37°C, and the decrease in turbidity of the reaction mixture was measured using a Klett-Summerson photometer ( Using filter No.66; Klett MFG.CO.
Inc. NYUSA). The activity is defined as the difference from the control value with no enzyme added (ΔKlett
value). Regarding the parent strain Arthrobacter YCWD3 shown for comparison, this parent strain was added to 1%
Difco Tryptone - 0.1% Difco yeast
1 volume (600 ml) of 8% baker's yeast (manufactured by Oriental Yeast Co., Ltd.) - 1% K ₂ HPO ₄ -
Culture solution obtained by culturing in 0.01% MgSO ₄ 7H ₂ O at 35°C for 48 hours (K. Doi, A. Doi & T.
Fukui；Agr.Biol.Chem.Vol32, P.1261
(1963)) cell wall lytic activity of the supernatant.

[Table] In addition, the transformed strain Estieritia coli
When cultured cells of HB101 (pX20) were treated according to method (ii) above, almost no baker's yeast cell wall lytic activity was detected in the culture supernatant and cell washing solution collected at 0°C, indicating that the stage Approximately 70% of the total activity was detected in the fluid (the so-called periplasmic compartment). Production form of cell wall lytic enzyme: Transformed strain S. coli HB101 (pX20) was transformed into L-
broth or T-broth (Difco tryptone 1%,
Glucose 0.1%, NaCl 0.3%, _CaCl2 0.1mM,
The cells were grown overnight at 37° C. on an agar plate containing (MgCl ₂ 1 mM, KH ₂ PO ₄ 0.32 mM) and killed with chloroform vapor. This is layered with agar containing baker's yeast cell walls and kept at 37°C. Clear plaques of cell wall lysis were observed around colonies grown in either medium. The parent strain Arthrobacter YCWD3 was grown on an L-broth or T-broth agar plate and subjected to the same treatment as the transformed strain. Very few cell wall lysis plaques were observed in colonies grown on L-broth agar plates, but no cell wall lysis plaques were observed in colonies grown on T-broth agar plates. From the above facts, the parent strain Arthrobacter
YCWD3 produces cell wall lytic enzymes in an inducible manner, whereas the transformed strain S. coli
It is shown that HB101 (pX20) produces the enzyme constitutively. It was also confirmed that the transformed strain Escherichia coli JA221 (pX20) constitutively produces the enzyme. Therefore, the transformed strain obtained by the present invention does not require an inducer during culture. Purification of cell wall lytic enzyme: Cell extracts of transformed strains S. coli HB101 (pX20) and JA221 (pX20) prepared by the method in (i) in the previous section are treated with protamine sulfate, and then precipitated with ammonium sulfate. . Alternatively, the bacterial cell extract obtained by method (ii) was concentrated using a rotary evaporator or the like. The obtained flow precipitate or concentrate was dialyzed against 0.01M phosphate buffer (PH6.25) (which may contain 0.01M EDTA). Avicel TG101 (manufactured by Asahi Kasei Corporation) equilibrated with the same buffer solution was packed into a column with a volume 10 times that of the dialysis solution, and chromatography was performed at room temperature (K. Doi, A. Doi & S.
NaKamura；Agr.Biol.Chem.Vol40, P.1669
(1976)). Combine fractions with cell wall lytic activity,
This was concentrated on a rotary evaporator and dialyzed against water at 4°C overnight. The resulting enzyme was purified into a single protein. From the staining intensity of the glucanase protein band observed in electrophoresis (polyacrylamide gel and SDS-polyacrylamide gel) of the purified enzyme above, it was found that the stationary phase was reached after culturing in 100 ml of L-broth at 37°C for 24 hours. Approximately 50 μg of glucanase protein from the bacterial body (parent strain Arthrobacter
The sum of two bands corresponding to YCWD3 glucanases I-3 and I-4, respectively) is obtained.
Regarding the parent strain Arthrobacter YCWD3,
From 100 ml of culture supernatant obtained by culturing at 35°C for about 48 hours, a mixture of glucanase and glucanase was obtained by column chromatography using Avicel TG101, and of this, about 500 μg of glucanase was obtained by the above method. I understand. Identity of the cell wall lytic enzyme produced by the transformed strain and the cell wall lytic enzyme of the parent strain: After treating the bacterial cell extract obtained by the method (i) above with protamine sulfate,
It was concentrated by ammonium sulfate precipitation and dialyzed against 0.01M phosphate buffer (PH7.0). 0.2 ml of the resulting solution was added to yeast glucan (DJ Bell & D.H. Northcote, J.
Chem.Soc. 1950 1944) or Pachman (H.Saito, A.Misaki & T.
Prepared according to Harada, Agr. Biol. Chem. 32 1261 (1968). ) to make a total volume of 1 ml. This was kept warm at 37°C for 3 hours. The turbidity of the reaction solution decreased significantly. this
The reaction was stopped by heating at 100°C for 10 minutes and centrifuged. When the sugar contained in the obtained supernatant was subjected to paper chromatography, the chromatogram showed that yeast glucan or
Chromatogram of Pachyman's degradation products (K.
Doi, A.Doi, &T.Fukui; Agr.Biol.Chem
Vol37, 1619 (1973)). Similarly, the purified sample by Avicel adsorption chromatography was purified by yeast glucan or
The paper chromatogram of oligosaccharides produced when Pachyman was reacted with was essentially identical to that obtained with the glucanase of the parent strain Arthrobacter YCWD3. In addition, the purified sample was subjected to polyacrylamide gel electrophoresis and the protein was stained with amide black. As a result, e.g.
Regarding coli HB101 (pX20), as shown in the figure, two bands were observed, and although the quantitative ratios differ depending on the sample, these are the known glucanase molecular species (glucanase-a, - It matched with glucanase-3 and glucanase-4 of 1, -2, -3 and -4), respectively. Arrows in the figure indicate the direction of protein migration. In addition, we created a zymogram for yeast glucan and baker's yeast cell wall using one of the two gels run under the same conditions, and compared the results using the protein staining described above for the other gel. The bands were also found to have yeast glucan-degrading activity and baker's yeast cell wall-degrading activity, respectively. Furthermore, when the above purified preparations were subjected to SDS-polyacrylamide gel electrophoresis and protein staining was performed with Coomassie Blue R-250, a single band was observed in each preparation, and the mobility of the parent strain Arthrobacter
YCWD3 glucanase-4 (molecular weight 55000)
was consistent with the mobility of Estieritia collie
Similar results were obtained for JA221 (pX20). (Effect of the invention) Recombinant plasmid obtained by the present invention
pX20 and Escherichia coli HB101 (pX20) and Escherichia coli JA221 (pX20) transformed with it contain a β-1.3 glucanase gene having cell wall lytic activity. Since the transformed strain constitutively produces β-1.3 glucanase, it can be cultured easily and inexpensively.

[Brief explanation of the drawing]

Figures 1a and 1b show protein bands of cell wall lytic enzymes produced by the parent strain Arthrobacter YCWD3 and the transformed strain of the present invention, Escherichia coli HB101 (pX20), respectively, by polyacrylamide gel electrophoresis. -a, -b, -1, -2, -3 and -4...Glucanase molecular species of the parent strain.

Claims (1)

[Scope of Claims] 1. A microorganism belonging to the genus Estieritia that constitutively produces β-1,3 glucanase and is transformed with a recombinant plasmid containing β-1,3 glucanase gene DNA derived from the genus Arthrobacter. The recombinant plasmid contains the β-1.3 glucanase gene DNA as a vector plasmid.
The β-1,3 glucanase gene is obtained by linking to YRp7, and the chromosomal gene derived from the Arthrobacter genus is ligated with a restriction enzyme.
Microorganisms obtained by cutting with BamHI. 2. The microorganism according to claim 1, wherein the β-1.3 glucanase gene DNA is derived from Arthrobacter YCWD3.