JPH0458889A - Alpha-1,3-glucanase gene - Google Patents

Abstract

NEW MATERIAL:DNA coding an alpha-1,3-glucan 3-glucanohydrolase enzyme protein produced by Trichoderma harzianum. USE:Mass-production of alpha-1,3-glucanase, etc. PREPARATION:The amino acid composition and the N-terminal amino acid sequence of alpha-1,3-glucanase are analyzed and an anti-alpha-1,3-glucanase serum is prepared. A cDNA library is prepared from the microbial cell, alpha-1,3-glucanase cDNA is screened, the longest cDNA among the cloned cDNA is subjected to sub-cloning and a transformant cloned with the cDNA fragment is separated. The objective alpha-1,3-glucanase cDNA is produced from a plasmid DNA separated from the transformant.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention is directed to the use of Trichoderma harzianum (Trich).
oderma harzianum: IFo 3
1292) and has the function of degrading insoluble glucan, which is a causative agent of dental caries.
DNA encoding a glucanohydrolase enzyme (hereinafter referred to as α-1,3 glucanase) protein and a signal peptide that functions to secrete the protein outside the bacterial body
, a protein or polypeptide having the amino acid sequence encoded by the DNA 8, a DNA having a nucleotide sequence that controls the expression of the protein and signal peptide, and a recombinant DNA in which these DNAs are linked to heterologous DNA, and relates to alpha-1,3 glucanase enzymes with unique N-terminal amino acid sequences.

(Prior art) α-1,3 glucanase hydrolyzes the α-1,3 glucoside bond portion of α-1,3 glucan, a polysaccharide in which glucose is linearly linked through α-1,3 glucoside bonds. This enzyme is widely distributed in microorganisms such as molds and bacteria. The enzyme is also used in Streptococcus mutans, a bacterium that causes dental caries.
) Bacteria also effectively decompose insoluble glucan synthesized from sucrose contained in food in the oral cavity, and the use of this enzyme as a caries preventive agent is attracting attention.

The above-mentioned insoluble glucan is a major component of dental plaque, which causes dental caries, and consists of glucose chains linked by α-1,3 glucosidic bonds and glucose chains linked by α-1,6 glucosidic bonds, which are complexly interconnected. It has a branched structure, α
The -1,3-linked portion of the glucose chain gives it water-insoluble properties (see Figure 1). α-1,3 glucanase acts on the insoluble glucan, and the insoluble glucan becomes water-insoluble. If its properties are weakened, insoluble glucan will dissolve in the saliva in the oral cavity, and as a result, plaque will fall off the tooth surface. In this way, α
-1,3 glucanase is an extremely effective anti-caries agent for removing dental plaque that causes tooth decay, and dextrin is also an enzyme that cleaves the α-1,6 glucoside-linked sugar chain moiety contained in insoluble glucan. When used in combination with Stranase,
It is thought that dental plaque can be removed even more efficiently, and from this perspective, much research and development has been conducted on methods for separating α-1,3 glucanase and dextranase from microbial culture fluids (particularly Publication No. 56-45587, Special Publication No. 4
No. 6-42955, JP-A No. 63-301788, etc.).

In addition, on the other hand, α that has the ability to decompose dental plaque as mentioned above
- Many studies have been conducted on incorporating 1,3 glucanase into toothpastes, and it has been recognized that the ability to remove plaque is higher than that of conventional toothpastes (Special Publication No. 55-50006, Japanese Patent Publication No. 12254-1982). .

As mentioned above, it is clear that α-1,3 glucanase is extremely effective in removing dental plaque, but when the enzyme itself is added to a toothpaste, it is difficult to clean the teeth by cleaning the oral cavity after using the toothpaste. There was a drawback that the α-1,3 glucanase contained in the agent itself was also removed from the oral cavity.

As a way to eliminate these drawbacks and more effectively remove plaque and inhibit plaque formation, we cloned the α-1,3 glucanase gene into oral bacteria and used it as a caries preventive agent. A method has also been developed in which it is administered into the oral cavity to constantly express α-1,3 glucanase in the oral cavity and remove dental plaque (Japanese Patent Application Laid-open No. 62-25).

(Problems to be Solved by the Invention) As mentioned above, α-1,3 glucanase is produced by various microorganisms, but in order to produce α-1,3 glucanase using these microorganisms, Expensive insoluble glucan must be added to the medium as an enzyme production inducer, and on the other hand, purification of the α-1,3 glucanase produced is complicated due to the insoluble glucan remaining in the medium after the completion of culture. It was becoming difficult.

In addition, the production amount of the enzyme is small (for this reason, α-1,3 glucanase has not been industrially produced in large quantities, although it is extremely useful for removing dental plaque. There was no such problem.We solved the above problems and made it inexpensively.
Moreover, genetic engineering methods could be used as a means to obtain large amounts of α-1,3 glucanase, but until now very little research has been done on the gene of this enzyme, and the base sequence of the gene is completely unknown. It remained as it was. In addition, the enzymatic properties of α-1,3 glucanase can be modified, and α-1,3 glucanase can be
In the production of 1,3 glucanase, it was necessary to clarify the base sequence of the gene DNA and design the structure of the plasmid to be introduced in order to secrete a large amount of enzyme protein outside the bacterial cell.

(Means for Solving the Problems) The present invention has been made to solve the above-mentioned conventional problems, and its gist is that Trichoderma harzianum (Trichoderma harzianum)
um) DNA encoding an α-1,3 glucan 3-glucanohydrolase enzyme protein produced by the bacterium and a signal peptide having the function of secreting α-1,3 glucanase to the outside of the bacterium.

Proteins and signal peptides having amino acid sequences encoded by these DNAs, DNAs encoding nucleotide sequences that control the expression of said proteins and signal peptides, recombinant DNAs in which said DNAs are linked to heterologous DNAs, unique N It provides information regarding α-1,3 glucanase enzyme proteins having terminal amino acid sequences, and their structures and functions.

That is, the present invention utilizes the above-mentioned DNA, enzyme protein, signal peptide, and information regarding their structures and functions to develop an inexpensive mass production method for α-1,3 glucanase, which has been difficult in the past, and to develop a method for gene production. This was done with the aim of making it possible to improve enzymatic functions through modification, to develop new and more effective anti-caries agents, and to put these results into practical use.

(Example) Examples of the DNA separation procedure, cloning procedure, base sequence analysis method, etc. of the present invention will be described in detail below.

Trichoderma harzianum bacteria were inoculated into a medium having the composition shown in Table 1 below, and cultured with shaking at 30°C for 8 days.

Table 1 *1) The trace metal solution is FeSO4”7! (zo 5
00mg.

Mn5Oa・HzO156mg5ZnC1z 167
B.

CoC1z 200mg, and 19% flcl 1
A 100 ml aqueous solution was used.

*2) α-1,3 glucan is derived from Streptococcus mutans.
s) α-1,3-1,6 glucan produced by the bacteria was digested with dextranase and the α-1,6 glucan moiety was removed.

The culture solution was collected and the bacterial cells were removed by centrifugation, and the resulting culture supernatant was concentrated by ultrafiltration.

This concentrated solution was salted out with 80% saturation of ammonium sulfate, and the precipitate was collected by centrifugation and dissolved in 50 mM acetate buffer (pH 5,0). Then, it was dialyzed with the same buffer, and the dialysis supernatant was equilibrated with the same buffer.
Pharmacia) column. The flow-through fraction eluted without being adsorbed with 50 mM acetate buffer (pH 5.0) was collected and dialyzed against 50 mM acetate buffer (PTL 4.0) as an external solution.

Further, this was adsorbed onto a CM-Sepharose CL-6B column equilibrated with the same buffer, and eluted using a linear gradient end of sodium chloride. Both fractions showing α-1,3 glucanase activity were collected, concentrated by ultrafiltration, and then subjected to gel filtration using a Biogel P-200 (Bioland) column.

By the above operations, 242
．． It was possible to obtain 4 mg of electrophoretically homogeneous α-1,3 glucanase.

In addition, the molecular weight of this α-1,3 glucanase was measured by 5O5-polyacrylamide gel electrophoresis.
It was found to have a molecular weight of 6,000 Daltons.

Table 2 The α-1,3 glucanase purified in Example 1 above was hydrolyzed with hydrochloric acid (at a temperature of 110°C for 24 to 72 hours).
The amino acid composition was investigated using an amino acid analyzer (Beckman Model 6300). The results are shown in Table 2 above along with the amino acid composition predicted from the cDNA base sequence examined later. In the determination of the amino acid composition by amino acid analysis, the total content of asparagine and aspartic acid was expressed as Asx, and similarly, glutamine and glutamic acid were expressed as Glx. Furthermore, cysteine and tryptophan residues were not quantified.

Furthermore, the N-terminal amino acid sequence of α-1,3 glucanase was analyzed using a peptide sequencer (Applied Biosystems Model 477A/120A), and it was found to have the following N-terminal amino acid sequence.

(N-terminus) Ala-Ser-Ser-Ala-Asp
-Arg-Leu-Val-Phe-Cys-His-
Phe-Met-11e--3: α-1,3 glucanase Rabbit anti-α-1 against purified α-1,3 glucanase from blood
, 3 glucanase serum was prepared by the following method.

α-1,3 glucanase purified as primary immunization 0.5
mg and complete Freund's adjuvant until it becomes colloidal, and add rabbit (Japanese White
Rabbtt) was injected subcutaneously.

After 3 weeks, α-1,3 glucanase 0.0.
5 mg and incomplete Freund's adjuvant were mixed and subcutaneously injected in the same manner as before.

One week later, blood was collected and the antibody titer of the serum obtained was determined by EL.
ISA method (Davisl, G., et al., B.
asicMethods in molecular
biology, 348-3541986+ Els
I! clumsy. After confirming that antibodies against α-1,3 glucanase were clearly produced, whole blood was collected. The blood thus obtained was left at 4°C overnight and then centrifuged, and the supernatant anti-α-1,3 glucanase serum was collected.

4: A medium with the same composition as the medium used in Example 1 for Trichoderma harzianum, at a temperature of 3.
Trichoderma herdianum cells cultured at 0°C for 3 to 6 days were collected and frozen in dry ice-acetone. 6g of frozen bacterial cells and glass peas (diameter 0.4
mm) were ground together using a pestle on a mortar cooled with dry ice. To this was added a guanidine isothiocyanate modified solution (5-leaf sodium citrate, 0.5% N-
25 ml of a 5M guanidine isothiocyanate solution containing sodium lauroyl sarcosine and 0.1M 2-mercaptoethanol was added and homogenized while dissolving at room temperature (Chirgtvin, J, M,...
et al, + Bio-chemistry, 1
8.5294-5299.1979). Next, 10.0
The mixture was centrifuged at 00×g for 10 minutes to remove the residue, and cesium chloride was added at a ratio of 0.2 g to 1 ml of the supernatant. 0.75 in a 2.1 ml centrifuge tube
1Il of 0. IM EDTA and 5.7M cesium chloride solution containing 0.2% diethylpyrocarbonate was placed in advance, and 1.45n+1 portions of the above supernatant were overlaid. Then, an ultracentrifuge (Beckman, TL-
100 type, TLA 100.20-ter)
Ultracentrifugation was performed at 20 °C for 3 hours with o, ooo rotation to precipitate RNA (Glisin, V, et al.
al,, Biochemistry, 13+26
33-2637.1974). The RNA was dissolved in distilled water and further purified by phenol extraction and ethanol precipitation to yield 1.4 mg of purified total RN^.

Furthermore, this total RNA was subjected to Oligo dT nacellous column chromatography (Pharmacia, Oli
go dT cellulose type 3) twice and poly(
8) RNA was purified (8 viv, h,, et a
l.

Proc, Natl, Acad, Sci, u,
s, x, l fallen + 1408-1412, 1972).

Through the above operations, we finally obtained approximately 20ug of poly(8)
RNA was purified.

cDNA was synthesized from this poly(A) RNA using a cDNA synthesis system kit (Amarjam), and approximately 5
00 ng of double-stranded cDNA could be obtained.

This was subjected to methylation of the Eco RI site, ligation of an Eco RI linker, and cleavage with the restriction enzyme Eco R1 using the cDNA cloning system λgtlo kit (Amerjam), followed by phenol extraction and ethanol precipitation. Electrophoresis was performed using a % agarose gel. A portion corresponding to a size of 2 to 4 kb was cut out from the gel and subjected to electroelution method (Life on
theEdge, vol, II+ 41-42+
International Bio-technology
ogies Inc, +86/87 catalog
) was used to recover cDN8 from the gel. Recovered cDN
A to λgtll/EcoRI digested ve
ctor kit (Stratagene) and Gig
λ using apack Gold (Stratagene)
Ligation to gtll phage vector
) and packaging to finally create a λgtll cDNA library equivalent to 500,000 plaques.

5: Screening example 4 of α-1,3 glucanase cDNA
An immunoscreening method using anti-α-1,3 glucanase rabbit serum (
Young, R.A., + Davis.

R, W, ONA Cloning 1.49-78
．． 1985. a-1,3 by IRLPRESSS)
Screening was performed for phages carrying glucanase cDN8.

First, E. coli strain Y1090 was sterilized in LB medium (1% tryptone, 0.5% yeast extract, 0.5%
% sodium chloride, 50 μg/ml ampicillin aqueous solution) and cultured with shaking at 30°C overnight. Add a cDNA library phage suspension (5,000-10,000 plaques) to 0.2 ml of the culture solution.
Leave for 2.5 ml of NZY medium (0,5
% Sodium Chloride, 0.2% Magnesium Sulfate, 0.5
% yeast extract, 1% NZ amine aqueous solution)
NZY plate (containing 1,5% Batato agar) with top agar solution (containing 0,6% Batato agar)
spread over the top layer. After incubation for 2-3 hours at 42 °C
A nitrocellulose filter impregnated with an mM IPTG (isopropyl β-lowthiogalactopyranoside) solution was placed in close contact with this, and the cells were further cultured at 37°C for 3 hours. After leaving it at 4°C for 1 hour, remove the filter and use TB.
The plate was washed with S (50mM Tris buffer y-(pH 8,0) containing 150mM NaCl), and further immersed in 5% skim milk-TBS for blocking. Next, the filter was soaked in a primary antibody solution (anti-α-1,3 glucanase rabbit serum diluted 1.000 times with TBS containing 1% skim milk and 0.05% Tween 20) and shaken at room temperature for 18 hours. . T containing 0.05% Tween 20
After washing the filter with BS, peroxidase-labeled anti-heron IgG to goat IgG solution (Bioland, 3,0
00 times diluted with the same buffer as the primary antibody dilution solution) and shaken at room temperature for 1 hour. After washing the filter, it was immersed in a coloring solution (0.015x hydrogen peroxide solution containing 0.01x diaminobenzideine), and the colored positive plaques were separated and purified in the same manner. In this way, the cDNA library was screened,
Finally, phage plaques that reacted with 16 anti-α-1,3 glucanase sera and were thought to contain α-1,3 glucanase cDNA were isolated.

Phage DNA from these plaques was prepared using Lambdasorb (Promega), cut with restriction enzyme Eco R1, and subjected to agarose electrophoresis to examine the size of the cloned cDNA. The longest c
To subclon DNA (017), 8 μg of Eco RI digest of this phage DNA was preliminarily mixed with Eco RI digest of this phage DNA.
o R1-cleaved and phosphatase-treated pUc11
B cloning vector (0.1 μg) was ligated using a DNA ligation kit (Takara Shuzo Co., Ltd.), and E. coli J
The cells were introduced into M109 competent cells (Takara Shuzo).

This was combined with 50μs/ml ampicillin, 40ug
/ml X-gal (5-bromo-4-chloro-3-indolyl-β-galactopyranoside), 0.5mM I
The cells were inoculated onto a PTG-containing LB-plate (containing 1,5x Bacto agar) and cultured overnight at 37°C to form colonies. White colonies with no β-galactosidase activity (if there is β-galactosidase activity, χ
-gal is decomposed and becomes a blue colony).
Among them, a transformant in which the D17 cDNA fragment was cloned was isolated.

Plasmid DNA (017 and ρU
C11B bound at Eco R1 site)
Separate pD1'? It was named as -1 (see Figure 2) and used in the following experiments.

Transformed E. coli carrying the Terukaji plasmid pD17-1 was cultured and subjected to conventional alkaline 5OS-cesium chloride density gradient ultracentrifugation (Molecular Cloning+ 90-9).
4+1982, Co1d Spring Harbo
plasmid pD17 by
-1 was purified. This was cut with an appropriate restriction enzyme and subjected to agarose gel electrophoresis to examine the size of the cut DNA fragment, and the restriction enzyme map shown in FIG. 3 was prepared.

Next, the entire base sequence of cDNA (017) was determined using the dideoxy base sequencing method using M13 phage (Sanger, F., et al., Proc.
Natl.

Δcad, Sci, [J, S, ^,, 74.546
3-5467, 1977).

According to the restriction enzyme map of cDNA (017), combine restriction enzymes to obtain a DNA fragment of an appropriate size.
Cut N8 and combine them with vector M13mp18 and mp
19 and was subcloned. -Heavy chain DNA was prepared from these Davis Davis.

L,Get al,Ba5ic methods
in molecular biology, 258
-260+ 1986t Elsevier) ,,
The reaction was carried out using a DNA sequencing kit (Takara Shuzo Co., Ltd.), and the base sequence was determined using a polyacrylamide gel electrophoresis device for determining base sequences. For regions where it was difficult to determine the nucleotide sequence using the above method, deletion mutants were created using Kirosea Fence deletion kit (Takara Shuzo Co., Ltd.), and then the nucleotide sequence was determined using the same method as above.

As a result of the above experiment, the size of cDNA (017) is 20
It was 96 bp and had 14 poly(^) on the 3° side. In addition, when the amino acid sequence was deduced from this base sequence, it was found that 2016b encodes 672 amino acid residues.
It was revealed that it has an open reading frame of p. The 38th amino acid (counting from the first amino acid (methionine) of this open reading frame)
The sequence from the 51st amino acid (alanine) to the 51st amino acid (isoleucine) is the N of the purified α-1,3 glucanase described above.
It matched the terminal amino acid sequence (see Example 2 above). Furthermore, the amino acid composition of the open reading frame after the 38th amino acid is different from that of the purified α-1,3
It matched well with the amino acid composition of glucanase (the second
(See table), the 37 amino acid sequence starting from methionine upstream of the part that matches the N-terminal amino acid sequence of α-1,3 glucanase has many highly hydrophobic amino acids, and considering its characteristics, this This cDNA (017) was confirmed to contain the full length of the structural gene of α-1,3 glucanase.

The gene structure of α-1,3 glucanase cDNA revealed from the above experiments is shown in FIG.

Step 7: Cloning of genomic DNA The method of Tilburn et al. (Tilburn, J.,
et al, +Gene, 26.205-221.1
Genomic DNA was purified from frozen bacterial cells according to 983). In the same manner as in Example 4, 10 g of frozen bacterial cells were crushed together with glass peas in a mortar, and 25 ml of DNA extraction solution [251 Tris buffer (pH 7,5) containing 0, sucrose, and 20 mM EDTA] was added. This was transferred to another tube, 2.2 ml of a DNA extraction solution containing 35% N-lauroylsarcosine sodium was added thereto, and the mixture was kept at 60°C for 1 hour with shaking.

Next, 4 mg of proteinase K (Boehringer Mannheim) was added and reacted at 37°C for 5 hours.

Centrifugation was performed at 3.000×g for 10 minutes, and the supernatant was subjected to phenol extraction and ethanol precipitation. Collect the precipitated DNA with a glass rod and add it to 6.5ml of 100mM Tris (pH 8,0)-10mMED.
Dissolved in TA solution. Add 6.5 g of cesium chloride and 420 μl of 1χ ethidium bromide, and perform density gradient ultracentrifugation (100,000 revolutions, 5 hours) using an ultracentrifuge (Beckman, Model TL-100, TL^, 20-ter). The fraction containing the separated genomic DNA was extracted from the tube, the ethium bromide was removed with butanol, and the genome DNA was separated by ethanol precipitation.
NA was purified. Approximately 600 μg of genomic DNA was obtained in the manner described above. After cutting this genomic DN^ with several restriction enzymes and separating it by agarose gel electrophoresis,
Southern blotting method (Davts, L, G, et al.
al. Ba5ic methods inmole-
cular biology, 61-65+ 198
6. DNA was transferred to a nitrocellulose (NG) membrane plate using a B15evier). Hybridization (Davis,
L.G.

et al., Ba5ic methods in
molecular bio-1ogy+ 84-87
．． 1986. Elsevier) was performed. The results are shown in FIG. In the figure, lane 1 is marker DNA, and its size is indicated by an arrow on the left axis. lane 2
is genomic DNA, and a hybridized band can be seen at a position of extremely high molecular weight. Lanes 3 to 5 are restriction enzymes Eco R1, old ndnl, and Sal.
This figure shows the results of Southern hybridization using a sample in which genomic DNA was completely cleaved with I.

From Figure 4, a band that hybridizes around 4 to 5 kb of the DNA cut with the restriction enzyme Eco RI was observed, and the DNA around this area was recovered from the gel and the recovered DNA
λgtlO/EcoRI digested vec
tor (Stratagene) and Gigapackgol
A genomic DNA library was created using d (Stratagene). After purifying this genomic DNA library, screening was performed by plaque hybridization using cDNA (017) as a probe (Gene Research Methods II).

Biochemistry Experiment Course 1.83-99.1986. Tokyo Kagaku Doujin). As a result, we were able to obtain positive plaques that hybridized at a rate of about 1 in 800. The phage was purified, phage DNA was prepared, and 4 μg of the phage DNA was digested with the restriction enzyme IEco R1, and further separated by agarose gel electrophoresis, and the inserted genomic DNA was recovered from the gel. The size of the recovered DNA was 4.7 kb, which was converted into pUc19 E.
co R1 site and subcloned into E. coli JM109 strain.

The plasmid DNA retained from E. coli into which the genomic DNA was subcloned in Example 7 was extracted and purified.
After cutting with an appropriate restriction enzyme, the mixture was separated by agarose gel electrophoresis. Examine the cutting pattern of the
．． A restriction enzyme map of the 7kb genomic DN^ was created. In FIG. 5, the portion where α-1,3 glucanase is encoded is represented by a black square. cDNA (0
1'? ) Comparison of this with the restriction enzyme map of 4.
1iindI in the 7kb Eco RI DNA fragment [
It was confirmed that cDNA (017) was contained in the I DNA fragment (see Figure 5).

Therefore, the base sequence of this Hind m fragment was investigated using the method shown in Example 6. The results are shown in Figure 6, and the size of this HindlII fragment is 2877 bp;
Comparison of this with the nucleotide sequence of cDNA (017) reveals that the α-1,3 glucanase gene is separated by three introns (indicated by lowercase letters in Figure 6) in the genomic DNA. It became clear. This intron also contains splicing signals of filamentous fungal introns (Kinnaird J,) 1. et al.
.

Gene, 26.253-260.1983) was observed. The boxed area is a typical TA present in the promoter region.
It is a TA box, and the black star is 5 of cDNA (017).
° Shows the starting point for the example. In FIG. 6, the single underline indicates the signal sequence, and the double underline indicates the portion that corresponds to the N-terminal amino acid sequence of the purified α-1,3 glucanase protein. The sequence represented by the wavy underline is a typical poly(A) RNA addition signal sequence, and the position indicated by the black triangle is the actual location where poly(A) RNA is added.

Furthermore, the region 3' from this region including this region is considered to be an important part as a terminator for terminating transcription.

(Effect) As explained above, the present invention is effective against Streptococcus mutans 4-coccus muta, a bacterium resident in the oral cavity.
Trichoderma herzianum (ns) encodes α-1,3 glucanase, which is produced by bacteria and has the effect of effectively decomposing insoluble glucans that cause dental caries.
erma hary, ianum), DNA encoding the signal peptide of the enzyme, information on the primary structure of proteins and peptides encoded by these DNA, and nucleotide sequences that control the expression of these proteins and peptides. It provides DNA that has

For example, by ligating the above DNA to an appropriate vector DNA and introducing it into an appropriate host (particularly bacteria resident in the oral cavity) to create a transformed strain, this can be administered into the oral cavity. It is also possible to produce a caries preventive agent that secretes and produces α-1,3 glucanase and permanently degrades and removes insoluble glucan in the oral cavity.

Furthermore, we have improved the enzymatic properties of the α-1,3 glucanase by modifying its amino acid sequence, created hybrid proteins with heterologous proteins, and used chemical methods to improve the enzymatic properties of α-1,3 glucanase.
It also paved the way for the synthesis of 1,3-glucanases.

In this way, the present invention opens the foundation for future improvements in protein engineering of α-1,3 glucanases and the development of methods for their use in a wide range of fields.

[Brief explanation of drawings]

Figure 1 shows the structure of insoluble glucan, Figure 2 shows the structure of insoluble glucan.
Figure 3 shows the restriction enzyme map of plasmid pD17-1.
Figure 4 shows the restriction enzyme map and gene structure of 7), size marker (lane 1), genomic DNA (lane 2), genomic DNA completely digested with restriction enzyme Eco R1 (lane 3), restriction enzyme Figure 5 shows the results of Southern blot hybridization of genomic DNA completely digested with the enzyme Hindl [I (lane 4) and genomic DNA completely digested with the restriction enzyme ■ (lane 5). Figure 6 (A) (B) (C) shows the restriction enzyme map and gene structure of the genomic DNA fragment containing the α-1,3 glucanase gene, and the base sequence of the genomic DNA fragment containing the α-1,3 glucanase gene. , and a diagram showing the gene structure of α-1,3 glucanase.

Claims (9)

[Claims] (1) Trichoderma hazianum (￥Trichod)
α-1, produced by the bacterium ermaharzianum)
DNA encoding the 3-glucan 3-glucanohydrolase enzyme protein. (2) Trichoderma hazianum (￥Trichod)
α-1, produced by the bacterium ermaharzianum)
DNA encoding the signal peptide of the 3-glucan 3-glucanohydrolase enzyme protein. (3) DNA having a nucleotide sequence that controls the expression of the protein according to claim 1 and the signal peptide according to claim 2. (4) DNA that hybridizes with the DNA according to claim 1 and encodes an α-1,3 glucan 3-glucanohydrolase enzyme protein. (5) DNA that hybridizes with the DNA according to claim 2 and encodes a signal peptide. (6) A protein having an amino acid sequence encoded by the DNA according to claim 1 and the DNA according to claim 4. (7) A polypeptide having an amino acid sequence encoded by the DNA according to claim 2 and the DNA according to claim 5. (8) A recombinant DNA in which the DNA according to any one of claims 1 to 5 is linked to a heterologous DNA. (9) α-1,3 glucan 3-glucanohydrolase having the following N-terminal amino acid sequence. (N-terminus) [There is a gene sequence. ]