JPH069513B2 - α-1,3 glucanase gene - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention provides an α-produced by Trichoderma harzianum (IFO 31292) and having a function of degrading insoluble glucan which is a causative agent of caries. DNA encoding 1,3 glucan 3-glucanohydrolase enzyme (hereinafter referred to as α-1,3 glucanaase) protein and a signal peptide that functions to secrete the protein outside the bacterial cell, and is encoded by the DNA A protein or polypeptide having an amino acid sequence, a DNA having a nucleotide sequence that controls the expression of the protein and the signal peptide, a recombinant DNA obtained by binding these DNAs to a heterologous DNA, and a unique N-terminal amino acid sequence It has an α-1,3 glucanase enzyme.

(Prior art) α-1,3 glucanase is a polysaccharide in which glucose is bound linearly by α-1,3 glucoside, and hydrolysis of α-1,3 glucoside binding part of α-1,3 glucan. An enzyme that does
Widely distributed in microorganisms such as mold and bacteria. The enzyme also
Streptococcus mutans, the causative agent of tooth decay
The (Streptococcus mutans) bacterium also has a function of well decomposing insoluble glucan synthesized from sucrose contained in food in the oral cavity, and the use of the enzyme as a dental caries preventive agent has been drawing attention.

The above-mentioned insoluble glucan is a major constituent of dental plaque that causes tooth decay, and glucose chains bound by α-1,3 glucoside bonds and glucose chains bound by α-1,6 glucoside bonds are intricately linked to each other. It has a branched structure, α-1,
The part of the three-linked glucose chain gives it the insoluble nature of water (see Figure 1). If α-1,3 glucanase is allowed to act on insoluble glucan to reduce the water-insoluble property of insoluble glucan, insoluble glucan will be dissolved in saliva in the oral cavity, resulting in plaque It will come off from the surface. Thus, α-1,3 glucanase is an extremely effective anti-cavity agent for the removal of dental plaque, which causes dental caries, and also cleaves the α-1,6 glucoside-linked sugar chain portion contained in insoluble glucan. It is thought that plaque can be removed even more efficiently by using it in combination with dextranase, which is an enzyme that does this.From such a viewpoint, α-1,3 glucanase and dextranase can be collected from the microbial culture solution. There have been many researches and developments of the method (Japanese Patent Publication No. 56-45587, Japanese Patent Publication No. 46-42955, and Japanese Patent Laid-Open No. 63-42955).
No. 301788).

On the other hand, it has the ability to decompose plaque as described above.
Many studies have been conducted on the incorporation of -1,3 glucanase into dentifrices, and it has been confirmed that the ability to remove plaque is higher than that of conventional dentifrices (Japanese Patent Publication Nos. 55-50006 and 58-1).
No. 2254).

As mentioned above, it is clear that α-1,3 glucanase is extremely effective in removing dental plaque, but when the enzyme itself is blended in a dentifrice, the toothpaste is washed by cleaning the oral cavity after use. There was a drawback that the α-1,3 glucanase itself contained in the agent was also removed from the oral cavity.
As a method of eliminating such defects and more effectively removing plaque and inhibiting the production of plaque, the α-1,3 glucanase gene was cloned into an oral indigenous bacterium and used as a dental caries preventive agent. A method has also been developed in which it is administered into the oral cavity and α-1, 3 glucanase is constantly expressed in the oral cavity to remove dental plaque (JP-A-62-25).

(Problems to be Solved by the Invention) As described above, α-1,3 glucanase is produced by various microorganisms, but in order to produce α-1,3 glucanase using these microorganisms, As an enzyme production inducer in the medium, expensive insoluble glucan must be added, on the other hand, after the completion of the culture, the insoluble glucan remaining in the medium, purification of the produced α-1,3 glucanase is complicated, It was difficult. In addition, the enzyme production is low, and for this reason, although it is extremely useful for removing plaque, it has not been industrially possible to mass-produce α-1,3 glucanase. It was By solving the problems described above, α-1,
As a means for obtaining 3 glucanase, it is possible to use a genetic engineering method, but research on the gene of the enzyme has hardly been conducted until now, and the nucleotide sequence of the gene has remained completely unknown. Also, α-
In order to modify the enzyme properties of 1,3 glucanase or to secrete a large amount of enzyme protein outside the bacterial cells in the production of α-1,3 glucanase by the above-mentioned resident bacteria in the oral cavity, the nucleotide sequence of gene DNA It was necessary to clarify and introduce the structure of the plasmid to be introduced.

(Means for Solving the Problems) The present invention has been made to solve the above-described conventional problems, and the gist thereof is to provide α-produced by Trichoderma harzianum bacteria.
1,3 glucan 3-glucanohydrolase enzyme protein, and DNA encoding a signal peptide having a function of secreting α-1,3 glucanase outside the cells, a protein having an amino acid sequence encoded by these DNAs, and Encodes a signal peptide, a nucleotide sequence that controls the expression of said protein and signal peptide
It provides information on DNA, recombinant DNA obtained by binding the DNA to heterologous DNA, α-1,3 glucanase enzyme protein having a unique N-terminal amino acid sequence, and structures and functions thereof.

That is, the present invention uses the above-mentioned DNA, enzyme protein, signal peptide, and information about their structure and function to develop an inexpensive mass production method of α-1,3 glucanase, which has been difficult in the past, and The purpose was to improve the enzymatic function by modification, to develop new and more effective anti-cavity agents, and to put the results to practical use.

(Examples) Examples of the DNA separation procedure, cloning procedure, nucleotide sequence analysis method and the like of the present invention will be described in detail below.

Example 1: Trichoderma h
Purification of α-1,3 glucanase produced by Bacillus arzianum) Trichoderma harzianum was inoculated into a medium having the composition shown in Table 1 below and shake-cultured at 30 ° C for 8 days.

The culture solution was collected and centrifuged to remove the cells, and the obtained culture supernatant was concentrated by ultrafiltration. Ammonium sulfate 80
Salt out at% saturation, collect the precipitate by centrifugation, 50 mM
It was dissolved in an acetate buffer (pH 5.0). Then, the solution was dialyzed against the same buffer solution, and the dialyzed supernatant was added to a CM-Sepharose CL-6B (Pharmacia) column equilibrated with the same buffer solution. The flow-through fraction that was eluted without being adsorbed with 50 mM acetate buffer (pH 5.0) was collected, and dialyzed with 10 mM acetate buffer (pH 4.0) as an external solution. Further, this was adsorbed on a CM-Sepharose CL-6B column equilibrated with the same buffer and eluted using a linear gradient of sodium chloride. Fractions showing α-1,3 glucanase activity were collected and concentrated by ultrafiltration, then Biogel P-20
This was subjected to gel filtration using a 0 (Bio-Rad) column.

By the above operation, 242.4 mg of electrophoretically uniform α-1,3 glucanase could be obtained from the culture supernatant of 11.5.

The molecular weight of this α-1,3 glucanase was measured by SDS-polyacrylamide gel electrophoresis, and it was found to have a molecular weight of 76,000 daltons.

Example 2: Amino acid composition and N of α-1,3 glucanase
Analysis of Terminal Amino Acid Sequence 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) to give an amino acid analyzer (Beckman). 6300) was used to examine the amino acid composition. The results are shown in Table 2 above together with the amino acid composition predicted from the cDNA nucleotide sequence examined later. In the quantification of amino acid composition by amino acid analysis, the total content of asparagine and aspartic acid was shown as Asx, and similarly glutamine and glutamic acid were shown as Glx. In addition, cysteine and tryptophan residues were not quantified.

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

Example 3: Preparation of anti-α-1,3 glucanase serum Rabbit anti-α-1,3 glucanase serum against purified α-1,3 glucanase was prepared by the following method.

As a primary immunization, 0.5 mg of purified α-1,3 glucanase and complete Freund's adjuvant were mixed until they became colloidal, and subcutaneously injected into rabbits (Japanese White Rabbit). Three weeks later, as a secondary immunization, 0.5 mg of α-1,3 glucanase and incomplete Freund's adjuvant were mixed in the same manner as the previous time and subcutaneous injection was performed. One week later, blood was collected and the antibody titer of the obtained serum was measured by ELISA (Davis, LG, et a
l., Basic Methods in molecular biology, 348-354, 1
986, Elsevier). After confirming that the antibody against α-1,3 glucanase was clearly formed, whole blood was collected. The blood thus obtained was allowed to stand overnight at 4 ° C. and then centrifuged to collect the supernatant anti-α-1,3 glucanase serum.

Example 4: Preparation of Trichoderma harzianum cDNA library A medium having the same composition as the medium used in Example 1 above, at a temperature of 30
The cells of Trichoderma harzianum cultivated at 3 ° C. for 3 to 6 days were collected and frozen in dry ice-acetone. 6g of these frozen cells and glass beads (diameter 0.4mm) 6
g was ground together with a pestle on a mortar cooled with dry ice. To this, 25 ml of a guanidine isothiocyanate-denaturing solution (5 M guanidine isothiocyanate solution containing 5 mM sodium citrate, 0.5% sodium N-lauroylsarcosine, 0.1 M 2-mercaptoethanol) was added,
Homogenized while melting at room temperature (Chirgwin, J.
M., et al., Biochemistry, 18 , 5294-5299, 1979). Next, the mixture was centrifuged at 10,000 × g for 10 minutes to remove the residue, and cesium chloride was added so that the ratio was 0.2 g per 1 ml of the supernatant. 0.75 ml 0.1M EDT in 2.1 ml centrifuge tube
A, 1.45 ml of the above supernatant was added to a 5.7 M cesium chloride solution containing 0.2% diethylpyrocarbonate.
Each was overlaid. Then, using an ultracentrifuge (TL-100 type rotor, TL-100 type, manufactured by Beckman), ultracentrifugation was performed at 100,000 rpm for 3 hours at 20 ° C. to precipitate RNA.
(Glisin, V., et al., Biochemistry, 13 , 2633-2637,
1974). Dissolve RNA in distilled water, extract with phenol,
Purification by ethanol precipitation yielded 1.4 mg of purified total RNA.

Furthermore, Oligo dT cellulose column chromatography (Pharmacia, Oligo dT cellulose type 3) was performed twice on this total RNA to purify poly (A) RNA in Aviv,
h., et al., Proc. Natl. Acad. Sci. USA, 69 , 1408
-1412, 1972).

By the above operation, about 20 μg of poly (A) RNA was finally purified.

CDNA was synthesized from this poly (A) RNA using a cDNA synthesis system kit (Amersham), and about 500 ng of double-stranded cDNA was synthesized.
I was able to get Using the cDNA cloning system λgt1 kit (Amersham), methylation of Eco RI site, ligation of Eco RI linker, and restriction enzyme Ec
o Cleavage with RI, phenol extraction and ethanol precipitation were performed, and then electrophoresis was performed using a 2% agarose gel. A portion corresponding to 2-4 kb in size was cut out from the gel and electroeluted (Life on the Edge, vol.
I, 41-42, International Biotechnologies., 86/87 ca
The cDNA was recovered from the gel by talog). Recovered cDNA
Was ligated and packaged with the λgt11 phage vector using λgt11 / EcoRI digested vector kit (Stratagene) and Gigapack Gold (Stratagene), and finally the equivalent of 500,000 plaques was obtained. A λgt11 cDNA library was prepared.

Example 5: Screening and subcloning of α-1,3 glucanase cDNA From the cDNA library prepared in Example 4, anti-α-1,3
Immunoscreening method using glucanase rabbit serum
(Young, RA, Davis, RW, DNA Cloning I, 49-78,19
85, IRL PRESSS) were used to screen phages carrying α-1,3 glucanase cDNA.

First, the Escherichia coli Y1090 strain was inoculated into a sterilized LB medium (1% tryptone, 0.5% yeast extract, 0.5% sodium chloride, 50 μg / ml ampicillin in water) and cultured at 30 ° C. overnight with shaking. The phage suspension of the cDNA library (5,000-10,000 plaques) was added to 0.2 ml of the culture solution, and the mixture was allowed to stand at 37 ° C for 20 minutes, and then 2.5 ml of NZY medium (0.5% sodium chloride, 0.2% magnesium sulfate, 0.5%). Yeast extract, 1% NZ amine in water) was spread on top of an NZY plate (containing 1.5% Bacto agar) with a solution of top agar (containing 0.6% Bacto agar). After culturing at 42 ° C for 2-3 hours, 10 mM IPTG (isopropyl β-D-
A nitrocellulose filter impregnated with a thiogalactopyranoside) solution is adhered to this, and further at 37 ° C for 3 more times.
Incubated for hours. After standing at 4 ° C for 1 hour, the filter was removed and TBS [50 mM Tris buffer containing 150 mM NaCl (pH 8.
0)] and then soaked in 5% skim milk-TBS for blocking. Next, the filter is placed in the primary antibody solution (anti-α-1,
3 Glucanase rabbit serum 1% skim milk, 0.05%
Diluted 1,000 times with TBS containing Tween 20)
Shake for 18 hours at room temperature. TBS containing 0.05% Tween 20
After washing the filter with a peroxidase-labeled anti-rabbit
The sample was immersed in an IgG-goat IgG solution (Bio-Rad, diluted 3000 times with the same buffer as the primary antibody diluent) and shaken at room temperature for 1 hour. After washing the filter, the coloring solution (0.0
It was soaked in 0.015% hydrogen peroxide solution containing 1% diaminobenzidine), and the positive plaques that developed color were separated and purified by the same procedure. In this way, screening of the cDNA library was performed, and phage plaques that seemed to have α-1,3 glucanase cDNA incorporated therein were finally isolated, which reacted with 16 anti-α-1,3 glucanase sera. .

The phage DNA of these plaques was prepared using Lambda Soap (Promega), cleaved with the restriction enzyme Eco RI, and then subjected to agarose electrophoresis to obtain the cloned cD.
The size of NA was investigated. In order to subclone the longest cDNA (D17), 8 µg of Eco RI digest of this phage DNA was ligated with 0.1 µg of pUC118 cloning vector pre-cut with Eco RI and treated with phosphatase using a DNA ligation kit (Takara Shuzo). And E. coli JM
It was introduced into 109 Competent Cell (Takara Shuzo).

50 μg / ml ampicillin, 40 μg / ml X-ga
1 (5-bromo-4-chloro-3-indolyl-β-galactopyranoside), 0.5 mM IPTG containing LB-plate (containing 1.5% bactoagar) was inoculated, and cultured overnight at 37 ℃ Colonies were formed. White colonies with no β-galactosidase activity (if β-galactosidase activity is present, X-
gal was decomposed into a blue colony), and a transformant in which the D17 cDNA fragment was cloned was isolated from them.

From this transformant, plasmid DNA (D17 and pUC118
Separated at the RI site) to separate pD17-1 (second
(See the figure) and used in the following experiments.

Example 6: Determination of nucleotide sequence of α-1,3 glucanase cDNA (D17) and analysis of its gene structure Transformed Escherichia coli harboring plasmid pD17-1 was cultivated and subjected to conventional alkaline SDS-cesium chloride density gradient ultracentrifugation. Law (Molecul
ar Cloning, 90-94,1982, Cold Spring Harbor Laborat
ory) to purify the plasmid pD17-1. This was cleaved with an appropriate restriction enzyme and subjected to agarose gel electrophoresis, the size of the cleaved DNA fragment was examined, and the restriction enzyme map shown in FIG. 3 was prepared. Next, the determination of the entire nucleotide sequence of cDNA (D17) is shown below using the dideoxy nucleotide sequencing method using M13 phage (Sanger, F., et al., Proc. Natl. Acad. Sci.
USA, 74 , 5463-5467, 1977).

According to the restriction enzyme map of cDNA (D17), a control enzyme was combined to obtain a DNA fragment of an appropriate size, the DNA was cleaved, and these were incorporated into vectors M13mp18 and mp19 and subcloned. Single-stranded DNA was prepared from these (Davis, LG,
et al., Basic methods in molecular biology, 258-26
0, 1986, Elsevier), a DNA sequencing kit (Takara Shuzo) was used to carry out the reaction, and the nucleotide sequence was determined using a polyacrylamide gel electrophoresis apparatus for nucleotide sequence determination. In the region where it was difficult to determine the nucleotide sequence by the above method,
After a deletion mutant was prepared using a Kilo-sequencing deletion kit (Takara Shuzo), the nucleotide sequence was determined by the same method as described above.

As a result of the above experiment, the size of cDNA (D17) was 2096 bp, and 14 poly (A) s were on the 3'side. In addition, when the amino acid sequence was deduced from this nucleotide sequence, it was revealed that it had an open reading frame of 1905 bp encoding 635 amino acid residues. 38 counting from the first amino acid (methionine) of this open reading frame
The sequence from the 1st amino acid (alanine) to the 51st amino acid (isoleucine) matched the N-terminal amino acid sequence of the purified α-1,3 glucanase described above (see Example 2 above). Furthermore, the amino acid composition of the open reading frame after the 38th amino acid was in good agreement with the amino acid composition of the purified α-1,3 glucanase (see Table 2 above). The 37 amino acid sequence starting from methionine upstream from the part corresponding to the N-terminal amino acid sequence of α-1,3 glucanase has many highly hydrophobic amino acids. It is considered to correspond to the region of the signal peptide for secretion to the outside, and this cDNA (D17) was confirmed to contain the entire length of the α-1,3 glucanase structural gene.

Α-1,3 glucanase cDN revealed by the above experiments
The gene structure of A is shown in FIG.

Example 7: Cloning of genomic DNA Tilburn et al. (Tilburn. J., et al., Gene, 26 , 205-
221, 1983), the genomic DNA was purified from frozen cells. In the same manner as in Example 4, 10 g of frozen bacterial cells were crushed together with Zarath beads in a mortar, and a DNA extraction solution [0.5 M sucrose, 2
25 ml of 25 mM Tris buffer (pH 7.5) containing 0 mM EDTA] was added. Transfer this to another tube, and add the DNA extraction solution containing 35% N-lauroyl sarcosine sodium to it.
2.2 ml was added, and the mixture was kept warm while shaking at 60 ° C for 1 hour. Next, 4 mg of proteinase K (Boehringer Mannheim) was added, and the mixture was 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 deposited DNA with a glass rod,
This was dissolved in 6.5 ml of 100 mM Tris (pH8.0) -10 mM EDTA solution. 6.5g cesium chloride and 1% ethidium bromide 42
Ultracentrifuge (Beckman, TL-100)
Type, TLA.2 rotor) density gradient ultracentrifugation process (100,0
00 rotation, 5 hours, 20 ° C). Isolated genomic DNA
Was removed from the tube, ethidium bromide was removed with butanol, and the genomic DNA was purified by ethanol precipitation. As described above, about 600 μg of genomic DNA was obtained. This genomic DNA is cleaved with several restriction enzymes and separated by agarose gel electrophoresis, followed by Southern blotting (Davis, LG, et al., Basic methods
in molecular biology, 61-65, 1986, Elsevier) D
NA was transferred to a nitrocellulose (NC) membrane. This NC membrane was hybridized with cDNA (D17) labeled with Nick Translation Kit (Takara Shuzo) as a probe (Davis, LG, et a
l., Basic methods in molecular biology, 84-87,198
6, Elsevier). The results are shown in FIG. In the figure, lane 1 shows the size of the marker DNA with an arrow on the left vertical axis. Lane 2 is genomic DNA, and a hybridized band is seen at a very high molecular weight position. Lanes 3 to 5 are restriction enzymes Eco RI,
The result of Southern hybridization when the sample in which the genomic DNA was completely cleaved with HindIII and SalI was used is shown.

From FIG. 4, 4-5 kb of DNA cleaved with restriction enzyme Eco RI
A hybridizing band was observed in the vicinity, and the DNA in the vicinity was recovered from the gel, and the recovered DNA was λgt10 / EcoRI d
igested vector (Stratagene) and Gigapackgold
(Stratagene) was used to prepare a genomic DNA library. For this genomic DNA library, cDN
Screening was carried out by plaque hybridization using A (D17) as a probe (Gene Research Method II, Second Biochemistry Laboratory, 1, 83-99, 1986, Tokyo Kagaku Dojin). As a result, positive plaques that hybridized at a ratio of about 1 in 800 could be obtained. The phages were purely separated, phage DNAs were prepared, 4 μg of the phage DNAs were cleaved with restriction enzyme Eco RI, and further separated by agarose gel electrophoresis to recover the inserted genomic DNAs from the gel. The size of the recovered DNA was 4.7 kb, which was ligated to the Eco RI site of pUC19 and subcloned into Escherichia coli JM109 strain.

Example 8: Determination of nucleotide sequence of genomic DNA and analysis of gene structure of α-1,3 glucanase The plasmid DNA retained was extracted and purified from Escherichia coli subcloned in Example 7 and cleaved with an appropriate restriction enzyme. After that, they were separated by agarose gel electrophoresis. The cutting pattern was examined and 4.7k as shown in Fig. 5
A restriction map of the genomic DNA of b was constructed. In FIG. 5, the part in which α-1,3 glucanase is coded is represented by a black square. Comparing this with the restriction enzyme map of cDNA (D17), H in the 4.7 kb Eco RI DNA fragment was compared.
It was confirmed that the indIII DNA fragment contained cDNA (D17) (see FIG. 5).

Therefore, the nucleotide sequence of this HindIII fragment was determined in Example 6
It investigated by the method shown by. The results are shown in Fig. 6. The size of this HindIII fragment is 2877 bp, and by comparing this with the nucleotide sequence of cDNA (D17), the gene of α-1,3 glucanase has three introns in genomic DNA ( It has been clarified that it is divided by (indicated by English lowercase letters in FIG. 6). In addition, the splicing signal of the filamentous fungal intron (Kinnaird JH et al., Ge
ne, 26 , 253-260, 1983) and a common sequence (dotted underline) was recognized. Also, the boxed area is a typical TATA box existing in the promoter region, and the black asterisk indicates the 5'side start point of cDNA (D17). In FIG. 6, the single underline shows the signal sequence, and the double underline shows the purified α.
The portion corresponding to the N-terminal amino acid sequence of the -1,3 glucanase protein is shown. The sequence underlined is a typical poly (A) RNA addition signal sequence, and the position of the black triangle is the position where poly (A) RNA is actually added.
In addition, the region on the 3 ′ side including this region is considered to be an important part as a terminator for termination of transcription.

(Effect) As described above, the present invention produces α-1,3 which effectively decomposes insoluble glucan that causes tooth decay, which is produced by Streptococcus mutans bacteria which is a resident bacteria in the oral cavity. DNA from Trichoderma harzianum bacterium encoding glucanase, DNA encoding a signal peptide of the enzyme,
It provides information on the primary structures of proteins and peptides encoded by these DNAs, and DNAs having nucleotide sequences that control the expression of these proteins and peptides.

For example, by ligating the above DNA to an appropriate vector DNA,
By introducing into a suitable host (particularly, bacteria resident in the oral cavity) to prepare a transformant and administering this into the oral cavity, the α-1,3 glucanase is secreted and produced in the oral cavity, and is permanently produced. It would also be possible to prepare an agent for preventing dental caries that decomposes and removes insoluble glucan in the oral cavity.

Furthermore, improvement of enzymatic properties by modification of the amino acid sequence of α-1,3 glucanase described above, preparation of a hybrid protein with a heterologous protein, α-1, by a chemical method,
3 It also opened the way for the synthesis of glucanase.

Thus, the present invention opens the basis for future protein engineering improvement of α-1,3 glucanase and development of its usage in a wide field.

[Brief description of drawings]

FIG. 1 shows the structure of insoluble glucan, FIG. 2 shows the restriction enzyme map of plasmid pD17-1, and FIG. 3 shows the restriction enzyme map of cDNA of α-1,3 glucanase (D17). , And a diagram showing the gene structure. FIG. 4 shows a size marker (lane 1) and genomic DNA.
(Lane 2), genome completely digested with restriction enzyme Eco RI
Fig. 5 shows the results of Southern blot hybridization of DNA (lane 3), genomic DNA completely digested with restriction enzyme HindIII (lane 4), and genomic DNA completely digested with restriction enzyme SalI (lane 5). Is genomic DNA containing the α-1,3 glucanase gene
FIG. 6 (A) (B) (C) is a diagram showing the restriction enzyme map of the fragment and the gene structure, and FIG. 6 (A) (B) (C) shows the nucleotide sequence of the genomic DNA fragment containing the α-1,3 glucanase gene and α-1,3 glucanase. It is a figure which shows the gene structure of.

Front page continuation (72) Inventor Soichiro Matsushiro 4-1-1 Nishinakajima, Yodogawa-ku, Osaka-shi, Nissin Foods Co., Ltd. (72) Inventor Junichi Miyahara 4-1-1 Nishinakajima, Yonagawa-ku, Osaka No. 1 within Nissin Foods Co., Ltd. (72) Inventor Minoru Okushima 4-1-1 Nishinakajima, Yodogawa-ku, Osaka City, Osaka Prefecture Within Nisshin Foods Co., Ltd.

Claims (4)

[Claims] 1. A Trichoderm
a harzianum) α containing the following amino acid sequence
DNA encoding 1,3 glucan 3-glucanohydrolase enzyme protein. ASSADRLVFC HFMIGIVGDR GSSADYDDDM QRAKAAGIDA FALNIGVDGY TDQQLGYAYD SADRNGMKVF ISFDFNWWSP GNAVGVGQKI AQYANRPAQL YVDNRPFASS FAGDGLDVNA LRSAAGSNVY FVPNFHPGQS SPSNIDGALN WMAWDNDGNN KAPKPGQTVT VADGDNAYKN WLGGKPYLAP VSPWFFTHFG PEVSYSKNWV FPGGPLIYNR WQQVLQQGFP MVEIVTWNDY GESHYVGPLK SKHFDDGNSK WVNDMPHDGF LDLSKPFIAA YKNRDTDISK YVQNEQLVYW YRRNLKALDC DATDTTSNRP ANNGSGNYFE GRPDGWQTMD DAVYVAALLK TAGSVTITSG GTTQTFQANA GANLFQIPAS IGQQKFALTR NGQTIFSGTS LMDITNVCSC GIYNFNPYVG TIPAGFDDPL QADGLFSLTI GLHVTTCQAK PSLGTNPPVT SGPVSSLPAS STTRASSPPP VSSTRVSSPP VSSPPVSRTS SPPPPPASST PPSGQVCVAG TVADGESGNY IGLCQFSCNY GYCPPGPCKC TAFGAPISPP ASNGRNGCPL PGEGDGYLGL CSFSCNHNYC PPTACQYC 2. The DNA according to claim 1, wherein the amino acid sequence is encoded by the following base sequence. GCTTCTTCTG CTGACCGTCT CGTATTCTGT CACTTCATGA TTGGTATCGT TGGTGACCGT GGCAGCTCGG CAGACTATGA TGACGATATG CAACGTGCCA AAGCCGCTGG CATTGACGCC TTCGCTCTGA ACATTGGCGT TGACGGCTAT ACCGACCAGC AGCTCGGGTA TGCCTATGAC TCTGCCGATC GTAATGGAAT GAAAGTCTTC ATTTCATTCG ATTTCAACTG GTGGAGCCCC GGTAATGCAG TTGGTGTTGG CCAGAAGATT GCGCAGTATG CCAACCGTCC CGCCCAGCTG TATGTCGATA ACCGGCCATT CGCCTCTTCC TTCGCTGGTG ACGGTCTGGA TGTAAATGCG TTGCGCTCTG CTGCAGGCTC CAACGTTTAC TTTGTGCCCA ACTTCCACCC TGGTCAATCT TCCCCCTCCA ACATTGATGG CGCTCTTAAC TGGATGGCCT GGGATAATGA TGGAAACAAC AAGGCACCCA AGCCGGGCCA AACCGTCACA GTGGCAGACG GTGATAACGC TTACAAGAAC TGGTTGGGTG GCAAGCCTTA CCTGGCGCCT GTCTCACCTT GGTTTTTCAC CCATTTCGGC CCTGAAGTTT CATATTCCAA GAACTGGGTC TTCCCAGGTG GTCCTCTGAT CTATAACCGG TGGCAACAGG TCTTGCAGCA GGGCTTCCCC ATGGTTGAGA TCGTTACCTG GAATGACTAC GGCGAGTCTC ACTACGTCGG TCCACTAAAG TCTAAGCATT TCGATGATGG CAACTCCAAA TGGGTCAATG ATATGCCCCA CGATGGATTC CTGGATCTTT CGAAGCCGTT CATAGCCGCA TATAAAAACA GGGATACCGA CATCTCCAAG TATGTTCAAA ATGAGCAGCT TGTTTACTGG TACCGCCGCA ACTTAAAGGC ACTGGACTGT GACGCCACCG ACACAACCTC TAACCGCCCG GCTAACAATG GAAGCGGCAA TTACTTTGAG GGACGCCCCG ATGGTTGGCA AACTATGGAT GATGCCGTTT ATGTTGCCGC ACTTCTCAAG ACTGCTGGCT CTGTTACGAT CACCTCTGGC GGCACCACTC AAACGTTCCA GGCTAACGCC GGAGCCAACC TCTTCCAAAT CCCAGCCAGC ATCGGCCAAC AAAAGTTTGC TCTCACTCGC AACGGTCAGA CCATCTTTAG CGGAACTTCA TTGATGGATA TCACCAACGT TTGCTCTTGC GGTATCTACA ACTTCAACCC ATATGTTGGC ACCATTCCTG CCGGCTTTGA CGACCCCCTA CAGGCTGACG GTCTTTTCTC TTTGACCATT GGATTGCATG TCACAACTTG TCAGGCCAAG CCATCTCTTG GAACCAACCC TCCTGTCACT TCCGGCCCTG TGTCCTCACT TCCAGCTTCC TCCACCACCC GCGCATCCTC GCCGCCTCCT GTTTCTTCAA CTCGTGTCTC TTCTCCCCCT GTCTCTTCCC CTCCAGTTTC TCGCACCTCT TCTCCCCCTC CCCCTCCGGC TAGCAGCACG CCGCCATCGG GTCAGGTTTG CGTTGCCGGT ACCGTTGCTG ATGGCGAGTC TGGCAACTAC ATCGGCCTGT GCCAATTCAG CTGCAACTAC GGTTACTGCC CACCAGGACC GTGCAAGTGC ACCGCCTTTG GCGCTCCTAT CTCGCCACCG GCATCCAATG GCCGCAACGG CTGCCCTCTA CCGGGAGAAG GCGATGGTTA TCTGGGCCTG TGCAGTTTCA GTTGTAACCA TAATTACTGC CCCCCAACGG CATGTCAATA CTGC 3. The DNA according to claim 1, wherein the α-1,3 glucan 3-glucanohydrolase enzyme protein further comprises the following amino acid sequence. MLGVFRRLRL GALAAAALSS LGSAAPANVA IRSLEER 4. The DNA according to claim 3, wherein the amino acid sequence is encoded by the following base sequence. ATGTTGGGCG TTTTCCGCCG CCTCAGGCTC GGCGCCCTTG CCGCCGCAGC TCTGTCTTCT CTCGGCAGTG CCGCTCCCGC CAATGTTGCT ATTCGGTCTC TCGAGGAACG T