Protein having alpha-glucosidase activity, DNA having genetic information thereof, and production of alpha-glucosidase

Abstract: A protein having an α-glucosidase activity, wherein its N-terminal amino acid sequence is the sequence depicted in the Sequence Listing of the present invention at Sequence No. 1; a DNA having the genetic information of the protein having an α-glucosidase activity; a recombinant vector containing said DNA; a transformant transformed with said vector; and production of α-glucosidase comprising culture of said transformant in a medium so as to grow α-glucosidase, and harvesting said α-glucosidase. According to the present invention, α-glucosidase without contamination of amylase can be easily produced in high yields and in large amounts by genetic engineering.

Description: FIELD OF THE INVENTION The present invention relates to an α-glucosidase usable for the production of starches, as well as for the determination of α-amylase present in body fluids which is clinically used as an index for the diagnoses of pancreatic diseases and-salivary gland diseases; a DNA having the genetic information of the protein having an α-glucosidase activity; a recombinant vector containing said DNA; a transformant transformed with said vector; and production of α-glucosidase using said transformant. BACKGROUND OF THE INVENTION The organisms which produce α-glucosidase (α-D-glucoside glucohydrolase, EC 3. 2. 1. 20) can be found widely in animals, plants, and microorganisms, and the α-glucosidases produced thereby show various characteristic properties depending on their source of origin. For example, the α-glucosidase originated from yeast is an enzyme scarcely affected by the sugar linked to non-reducing terminal glucose or aglycon of maltooligoglucoside derivatives as substrates, and is mainly used for the determination of α-amylase. The α-glucosidase in human urine or hog blood is known to scarcely show activity unless substrates have a glucose linked to non-reducing terminal glucose and said linkage is α-1,4-linkage. With regard to the DNA sequence and the amino acid sequence of α-glucosidase gene, there have been known those of human lisosomal α-glucosidase [Biochem. J. 272 : 493-497 (1990)], fly α-glucosidase [J. Mol. Biol. 166 101-118 (1983)], mosquito α-glucosidase [Gene 75 : 73-83 (1989)], yeast α-glucosidase [Gene 41 : 75-84 (1986)], and so on. They are all derived from eukaryotes, and a prokaryote-originated gene useful for industrial production has not been found. The α-glucosidase derived from the genus Bacillus is different from the one derived from yeast or animals hitherto known, and has superior heat resistance and wide substrate specificity permitting substantial reaction on maltotetraose, maltopentaose, etc. [Biochimica et Biophysica Acta: 787 281-289 (1984)]. Among strains belonging to the genus Bacillus , however, are those capable of producing α-amylase concurrently with α-glucosidase. The α-glucosidases obtained from those strains contain α-amylase even after purification, and occasionally cause error in determining amylase in body fluids. SUMMARY OF THE INVENTION Accordingly, it is an object of the invention to clarify molecular structure of α-glucosidase by identifying the N-terminal amino acid sequence of α-glucosidase derived from the genus Bacillus , and to provide a means for producing said α-glucosidase in a pure form without contamination of α-amylase, at low costs and in large amounts by genetic engineering. So as to achieve the object mentioned above, the present inventors selected Bacillus stearothermophilus ATCC 12016 as an α-glucosidase producing cell, and, by the purification of its culture, successfully obtained an α-glucosidase preparation having a purity superior to the one reported in Biochimica et Biophysica Acta: 787 281-289 (1984) wherein an α-glucosidase was obtained by purifying the culture of a known strain, and the inventors identified its N-terminal amino acid sequence. Furthermore, the present inventors have succeeded in the isolation of α-glucosidase gene from chromosome DNA extracted from Bacillus stearothermophilus ATCC 12016, and identified the entire structure of its DNA. Additionally, they have succeeded in producing said α-glucosidase in high yields by genetic engineering from a transformant which produces substantially no α-amylase, and made it possible to produce high purity α-glucosidase in large amounts at low costs. BRIEF DESCRIPTION OF THE DRAWING Fig. 1 is a restriction enzyme map of a DNA insert in pSAGH2-9. DETAILED DESCRIPTION OF THE INVENTION The present invention provides (1) a purified protein having an α-glucosidase activity, the protein being characterized by (a) an amino acid sequence as depicted at Sequence No. 3 in the Sequence Listing, (b) a molecular weight of 65,000 (SDS-PAGE), (c) an optimal temperature of 60 °C, and (d) being essentially free of contamination by α-amylase; (2) a DNA encoding the protein according to (1) above; (3) a recombinant vector containing the DNA according to (2) above; (4) a transformant transformed with the vector according to (3) above; and (5) a method for producing the α-glucosidase as defined in (1) above, which method comprises culturing the transformant according to (4) above in a medium so as to grow α-glucosidase, and harvesting said α-glucosidase. The protein as defined in (1) above has an N-terminal amino acid sequence depicted in the Sequence Listing at Sequence No. 1 which is different from that of known α-glucosidase (e.g., those derived from human lisosomal, fly, mosquito, or yeast). The protein as defined in (1) above preferably is an enzyme produced by Bacillus stearothermophilus ATCC 12016, and is different from known α-glucosidases in purity. The microorganism belonging to the genus Bacillus stearothermophilus which is exemplified by Bacillus stearothermophilus ATCC 12016. The microorganisms belonging to the genus Bacillus may be cultured in a medium containing carbon source, nitrogen source, and inorganic ion, which may be further supplemented with nitrate or phosphate on demand. As the carbon source, usable are starch, hydrolyzed starch and molasses. As the nitrogen source, usable are polypepton, trypton, meat extract and yeast extract. It is preferred, although not required, that the culture is conducted under the aerobic conditions using pH and temperature of medium appropriately adjusted. Culture should be continued until the α-glucosidase activity of the culture reaches its maximum Level. Harvesting of α-glucosidase from a culture comprises the following steps. The culture solution is centrifuged to collect cells, and then, an α-glucosidase-containing lysate is prepared by lysis. The lysis is suitably conducted by the treatment with a cell wall lytic enzyme such as lysozyme or β-glucanase, or physical lysis by ultrasonication or French press treatment. The lysate thus obtained is subjected to, for example, ammonium sulfate precipitation fractionation, and after desalting, subjected to fractionation using a DEAE Sepharose® CL-6B column (Pharmacia LKB). Other columns may be used insofar as separation by ion exchange can be performed. Otherwise, this step may be skipped. The fractionated sample is subjected to High Performance Liquid Chromatography (HPLC, Pharmacia LKB) using Q-Sepharose® HiLoad 16/10 (Pharmacia LKB), and then TSKgel Phenyl-5PW (Toso) for further purification. The purified product migrated as a single band on SDS polyacrylamide gel electrophoresis (SDS-PAGE). Note that the combination of column chromatographys is not limited to the one exemplified above, but may be altered optionally including the kind of the column to be used. In the present invention, the purified standard α-glucosidase product was then applied to an amino acid sequencer, and the following N-terminal amino acid sequence (Sequence No. 1) was determined. Met Lys Lys Thr Trp Trp Lys Glu Gly Val Ala Tyr Gln Ile Tyr The protein of the present invention as defined in (1) above has the amino acid sequence as depicted in the Sequence Listing at Sequence No. 3. The DNA of the present invention as defined in (2) above (hereinafter also referred to as α-glucosidase gene) may be extracted from Bacillus stearothermophilus , or may be synthesized. The DNA sequence is, for example, a DNA sequence encoding the amino acid sequence as depicted in the Sequence Listing at Sequence No. 3, or a DNA having the nucleotide sequence as depicted in the Sequence Listing at Sequence No. 2. The recombinant vector for the DNA of the present invention is prepared by, for example, separating and purifying the DNA of Bacillus stearothermophilus , cleaving out DNA using ultrasonic waves or restriction enzyme, ligating said DNA with a linear expression vector by cyclization with DNA ligase at the blunt or cohesive ends of the both DNAs. The thus obtained recombinant vector is transfected into a replicable host, from which a microorganism carrying said recombinant vector is obtained by screening with the use of a marker of the vector and α-glucosidase activity as indices. The obtained microorganism is cultured, and from the cultured cells, a recombinant DNA vector is isolated and purified. Then, α-glucosidase gene can be harvested from said recombinant vector. A detailed explanation of DNA harvesting follows hereunder. The DNA derived from a gene donor microorganism is harvested as follows. A bacterium as mentioned above which is the donor microorganism is cultured, for example, in a liquid medium with aeration and agitation for 1-3 days, after which the obtained culture is centrifuged, and cells are collected. By lysis, an α-glucosidase gene-containing lysate can be prepared. The lysis can be conducted by, for example, a treatment with a cell wall lytic enzyme such as lysozyme or β-glucanase, with, where necessary, a concurrent use of other enzyme such as protease or a surfactant such as sodium lauryl sulfate. A physical lysis by freeze-thawing or French press treatment may be further combined with the lysis as described. From the lysate thus obtained, DNA is isolated and purified by conventional methods such as protein removal by phenol extraction, protease treatment, ribonuclease treatment, alcohol precipitation centrifugation, which may be combined as appropriate. DNA isolated from microorganism and then purified is cleaved by, for example, ultrasonic treatment or restriction enzyme treatment, with preference given to restriction enzyme treatment using a restriction enzyme which acts on a specific nucleotide sequence, such as type II restriction enzymes (e.g. Eco RI , Hin dIII , Bam HI ) such that the ligation of a DNA fragment obtained with a vector is facilitated. As the vector, suitable is the one constructed for use in gene recombination from a phage or a plasmid capable of autonomic proliferation in a host. As the phage, usable is, for example, λgt 10 or λgt 11 when Escherichia coli is used as a host. As the plasmid, usable is, for example, pBR322 or pUC19 when Escherichia coli is used as a host, and pBU110 or pC194 when Bacillus subtilis is used as a host. In addition, a shuttle vector capable of autonomic proliferation in two or more hosts including both Gram-negative hosts such as Escherichia coli and Gram-positive hosts such as Bacillus subtilis may be used. Such a vector is preferably cleaved by the same restriction enzyme as used for the cleavage of DNA of the microorganism which is the α-glucosidase gene donor as mentioned above, to give a vector fragment. The method for ligating the microorganism DNA fragment with a vector fragment may be any method using a known DNA ligase, and is exemplified by a method comprising annealing cohesive ends of a microorganism DNA fragment and a vector fragment, and constructing a recombinant vector from the microorganism DNA fragment and the vector fragment with the use of a suitable DNA ligase. Where necessary, a recombinant vector may be prepared by a transfection into a host after annealing so that a DNA ligase in the host may be used. As the host, any microorganism can be used as long as a recombinant vector is stable and capable of autonomic proliferation, and permits expression of the character of extraneous DNA, and usable are, for example, Escherichia coli W3110, Escherichia coli C600, or Escherichia coli JM109. The recombinant vector is transfected into a host by, for example, when the host is a microorganism belonging to the genus Escherichia , transfection in the presence of a calcium ion, and when the host is a microorganism belonging to the genus Bacillus , by competent cell method or protoplast method, additionally with or without microinjection. It has been found that the microorganism (transformant) thus obtained can produce α-glucosidase stably and in a large amount by the culture thereof in a nutrient medium. The host microorganism to which the objective recombinant vector can be transfected is selected from the microorganisms capable of simultaneous expression of α-glucosidase and the drug tolerance marker of the vector carrying the objective DNA, and for example, a microorganism which can grow in a selective medium prepared according to the drug tolerance marker and produce α-glucosidase may be selected. Preferably, 5-bromo-4-chloro-3-indolyl-α-D-glucopyranoside is added to a selective medium as a substrate for α-glucosidase, and a recombinant vector containing an α-glucosidase gene is screened using blue colony as an index. The nucleotide sequence of the α-glucosidase gene obtained by the aforementioned steps was analyzed by the dideoxy method described in Science 214 , 1205-1210 (1981), and the amino acid sequence of α-glucosidase was deduced from the nucleotide sequence. The N-terminal amino acid sequence of the α-glucosidase deduced from the nucleotide sequence was in perfect coincidence with the result of the analysis of the N-terminal amino acid sequence of the α-glucosidase derived from Bacillus stearothermophilus mentioned above. In this way, the recombinant vector containing the α-glucosidase gene once selected may be cleaved out from the transformed microorganism and transfected into other host with ease. Also, the DNA which is an α-glucosidase gene may be cleaved out from the recombinant vector containing the α-glucosidase gene with the use of a restriction enzyme, ligated with a vector fragment obtained by cleavage in a similar manner, and transfected into a host with ease. Culture of a host microorganism may be conducted under the conditions selected in consideration of nutritional-physiological characteristics of the host. Generally, a liquid culture is selected, though culture with aeration and agitaion is advantageous from industrial aspect. The nutritive sources may be those usually employed for the culture of microorganisms. The carbon source may be any utilizable carbon compounds such as glucose, sucrose, lactose, maltose, fructose, molasses, and pyruvic acid. The nitrogen source may be any utilizable nitrogen compounds such as pepton, meat extract, yeast extract, hydrolyzed casein, and alkali extract of soybean. In addition to these, salts such as phosphate, carbonate, sulfate, magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids and specific vitamins may be used as necessary. Culture temperature may be varied as appropriate within the range wherein cells can grow to produce α-glucosidase. In the case of Escherichia coli , the preferable temperature is from 20°C to 42°C. While culture is carried out for somewhat different periods of time depending on culture conditions, it may be terminated at a suitable point when α-glucosidase has been produced in highest yields, which is normally from 12 to 48 hours. The pH of a medium may be varied as appropriate within the range wherein cells can grow to produce α-glucosidase, with preferance given to a pH of 6.0-8.0. While the culture medium containing α-glucosidase-producing cells may be collected from a culture and used as it is, the α-glucosidase-containing solution and cells are, when α-glucosidase is contained in the culture medium, normally separated by conventional methods such as filtration and centrifugation, and then used. When α-glucosidase is contained in cells, the cells are harvested by, for example, filtration or centrifugation of the obtained culture, and then destroyed by a mechanical method or an enzymatic method using lysozyme, after which α-glucosidase is solubilized by adding a chelating agent such as EDTA and/or a surfactant as necessary, whereby α-glucosidase is obtained as an aqueous solution. The thus-obtained solution containing α-glucosidase is subjected to, for example, concentration under reduced pressure, membrane concentration, salting out with ammonium sulfate or sodium sulfate, or fractional precipitation using a hydrophilic organic solvent such as methanol, ethanol or acetone, to allow precipitation. Also, heat treatment and isoelectric treatment are effective means for purification. Purified α-glucosidase can be obtained by gel filtration using an adsorbent or a gel filtration agent, adsorption chromatography, ion exchange chromatography or affinity chromatography. According to the present invention, the nucleotide sequence of α-glucosidase gene, as well as the amino acid sequence of α-glucosidase have been clarified, and α-glucosidase without contamination of amylase can be easily produced in large amounts by genetic engineering. The α-glucosidase obtained by the present invention has higher specific activity and higher purity than those conventionally known. Moreover, the α-glucosidase obtained by the present invention permits precise determination of α-amylase in body fluids, which is attributable to the absence of the contamination of α-amylase. The present invention is more detailedly explained by way of examples in the following. In the examples, α-glucosidase activity was determined as follows. An enzyme was reacted in 33.3 mM phosphate buffer (pH 6.8) containing 2 mM p-nitrophenyl-α-D-glucopyranoside at 60°C for 20 minutes, and absorbance at 400 nm was determined. The amount of enzyme necessary for producing 1 µmol of p-nitrophenol per 1 minute under these conditions was taken as one unit of enzyme activity. Example 1 Determination of N-terminal amino acid sequence of α-glucosidase The α-glucosidase-producing cell Bacillus stearothermophilus ATCC 12016, was cultured in a 10 ℓ-jar fermenter. The medium (pH 7.0) was supplemented with 2% soluble starch, 0.05% meat extract, 0.2% yeast extract, 0.3% K 2 HPO 4 and 0.1% KH 2 PO 4 , and cultured with aeration and agitation at 60°C for 10 hours. The culture medium obtained (5.2 ℓ) was centrifuged, and 80 g of cells was separated. The cells were suspended in 50 mM phosphate buffer. pH 7.0, lysed in Dyno Mill to give 2 ℓ of a cell lysate. The α-glucosidase activity at this time was 5.8 U/ml. After sulfate fractionation, the lysate was desalted on Sephadex® G-25 (Pharmacia LKB). The specific activity at this time was 4.0 U/mg. The desalted solution was applied to a DEAE Sepharose® CL-6B column equilibrated with 50 mM phosphate buffer, pH 7.0, and the adsorbed fraction was eluted by 0-0.3 M NaCl gradient. Thereafter, the obtained fraction was desalted again on Sephadex® G-25. The desalted solution was subjected to HPLC on Q Sepharose® HiLoad 16/10 column for ion exchange chromatography, and then to hydrophobic chromatography using a TSKgel Phenyl-5PW column to give a purified α-glucosidase preparation. The specific activity of the α-glucosidase preparation was 150 U/mg, and activity yield was 13%. The α-glucosidase preparation was subjected to SDS-PAGE. This preparation migrated as a single band and had a molecular weight of 65,000. The purifications so far conducted are summarized in Table 1. α-glucosidase purification Steps total activity (U) specific activity (U/mg) yield (%) α-amylase content(%) cell lysis 10967 0.24 100 19.3 sulfate fractionation 12100 1.88 110 15.4 DEAE Sepharose® 10285 5.50 94 5.2 × 10 -3 Q Sepharose® 5347 87.5 49 1.2 × 10 -3 Phenyl-5PW 1390 150.0 13 5.6 × 10 -5 The purified α-glucosidase preparation was analyzed by an amino acid sequencer, and the N-terminal amino acid sequence of α-glucosidase shown in the Sequence Listing at Sequence No. 1 was determined. Example 2 Separation of chromosome DNA The chromosome DNA of Bacillus stearothermophilus ATCC 12016 was isolated by the following method. This cell line was subjected to a shaking culture in an ordinary bouillon medium (150 ml) at 60°C overnight, and centrifuged (8000 rpm, 10 minutes) to collect cells. The cells were suspended in 5 ml of a solution containing 10% sucrose, 50 mM Tris-HCl buffer (pH 8.0), and 50 mM EDTA, after which 1 ml of a lysozyme solution (10 mg/ml) was added thereto, and the mixture was incubated at 37°C for 15 minutes, followed by addition of 10% SDS (sodium dodecyl sulfate, 1 ml). To the suspension was added an equivalent amount of a chloroform-phenol solution (1:1), and the mixture was stirred. Then, the mixture was centrifuged at 10,000 rpm for 3 minutes to divide same into a water layer and a solvent layer, and the water layer was separated. A two-fold amount of ethanol was gently layered on the water layer, and DNA was isolated by slowly stirring the mixture with a glass rod to allow the DNA to wind around the glass rod, which was then dissolved in a solution of 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (hereinafter abbreviated as TE). After treating the solution with an equivalent amount of chloroform-phenol, the water layer was separated by centrifugation. A two-fold amount of ethanol was added thereto, and the DNA was again isolated in the same manner as above and dissolved in 2 ml of TE. The competent cell of Escherichia coli JM109 was prepared by the method of Hanahan, and used as a host for library construction. Example 3 Preparation of DNA fragment containing a gene encoding α-glucosidase, and recombinant vector containing said DNA fragment The DNA (1 µg) obtained in Example 2 was reacted with 10 units of a restriction enzyme Ava I (manufactured by Toyo Boseki K.K.) at 37°C for 1 hour into complete decomposition. In a similar manner, the DNA was reacted with 0.5 µg of pUC19 cleaved with Ava I , in the presence of 1 unit of T4-DNA ligase (manufactured by Toyo Boseki K.K.) at 16°C for 12 hours to ligate the DNA. The DNA thus ligated was transformed with the competent cell of Escherichia coli JM109. A colony of 1 × 10 6 transformed cells was obtained per 1 µg of the DNA used. The obtained colonies were cultured in an LB medium (1% polypepton, 0.5% yeast extract, 0.5% sodium chloride) supplemented with 50 µg/ml ampicillin and 0.01% 5-bromo-4-chloro-3-indolyl-α-glucopyranoside at 37°C for 18 hours, and the recombinant DNA containing the α-glucosidase gene was screened using blue colony as an index. As a result, one colony displaying a blue color was obtained out of 3000 colonies. The plasmid therein contained an 5.4 kbp DNA fragment insert, and this plasmid was named pSAGH1. Then, the DNA fragment insert was cleaved from pSAGH1 by various restriction enzymes, subcloned into pUC19 to yield pSAGH2-9 containing an 2.2 kbp DNA fragment insert, with which Escherichia coli JM109 was transformed. The restriction enzyme map of the DNA insert of pSAGH2-9 is shown in Fig. 1. Example 4 Determination of nucleotide sequence The 2.2 kbp DNA fragment insert of pSAGH2-9 was cleaved with various restriction enzymes, and subclones were prepared. The various subclones were prepared into a single-stranded DNA by conventional methods, and the single-stranded DNA was analyzed by SEQUENASE® VERSION 2.0 7-deaza dGTP kit (manufactured by Toyo Boseki K.K.) to determine its nucleotide sequence. The nucleotide sequence and the amino acid sequence are shown in the Sequence Listing at sequence No. 2. Example 5 Production of α-glucosidase The aforementioned LB medium (6 ℓ) was dispensed to a 10 ℓ-jar fermenter, autoclaved at 121°C for 15 minutes, allowed to cool, and added with 6 ml of 50 mg/ml ampicillin (manufactured by Nacalai Tesque) and 200 mM IPTG which had been separately sterilized by filtration. Into this medium was inoculated 60 ml of a culture medium of Escherichia coli JM109 (pSAGH2-9) which had been previously subjected to shaking culture at 37°C for 18 hours in a medium having the same composition as above, and the medium was subjected to culture with aeration and agitation at 37°C for 18 hours. The α-glucosidase activity upon termination of the culture was 12.5 U/ml. The culture medium (6 ℓ) was centrifuged to collect cells, and the cells were suspended in 200 ml of 50 mM phosphate buffer (pH 7.0). The cells were lysed by a conventional method using Daino Mill, and centrifuged at 15,000 rpm for 20 minutes to give a crude enzyme solution of α-glucosidase. This crude solution was treated with protamine to remove nucleic acid, and subjected to fractionation using ammonium sulfate. The precipitate was dissolved in 50 ml of 50 mM phosphate buffer (pH 7.0), and heated at 55°C for 16 hours. The resultant solution was desalted on Sephadex® G-25, and subjected to DEAE Sepharose® CL-6B chromatography to give an α-glucosidase active fraction. The α-glucosidase fraction after the DEAE Sepharose® chromatography showed a single band on SDS-PAGE, the specific activity of 187 U/mg and the activity yield of 70.5%. The enzyme properties of the α-glucosidase obtained from a transformant were as follows: molecular weight 65,000, isoelectric point 5.2, optimal pH 6-7, heat resistance not more than 60°C (pH 7.0, 15 minutes treatment), and optimal temperature 60°C. The purifications so far conducted are summarized in Table 2. α-glucosidase purification Steps total activity (U) specific activity (U/mg) yield (%) α-amylase content(%) cell lysis 74850 7.13 100 3.1 × 10 -3 sulfate fractionation 89070 59.0 119 2.4 × 10 -3 heat treatment 65942 92.3 88.1 5.8 × 10 -5 DEAE Sepharose® 52767 187.0 70.5 1.0 × 10 -5 Compared with the results of Table 1, an about 38 times greater amount of α-glucosidase was obtained at higher purity by the same culture scale but by easier purification method.

Claims: A purified protein having an α-glucosidase activity, the protein being characterized by (1) an amino acid sequence as depicted at Sequence No. 3 in the Sequence Listing, (2) a molecular weight of 65,000 (SDS-PAGE), (3) an optimal temperature of 60°C, and (4) being essentially free of contamination by α-amylase. The protein according to claim 1, which is obtainable from a microorganism belonging to the genus Bacillus . The protein according to claim 2, wherein the microorganism belonging to the genus Bacillus is Bacillus stearothermophilus ATCC 12016. A DNA encoding the protein according to any one of claims 1 to 3. A recombinant vector having the DNA of claim 4. A transformant transformed with the recombinant vector of claim 5. A method for producing α-glucosidase as defined in claim 1, which method comprises culturing the transformant of claim 6 in a medium to grow α-glucosidase, and harvesting said α-glucosidase.

Inventors: EMI SHIGENORI, NISHIYA YOSHIAKI, SOGABE ATSUSHI, SOGABE YUKIHIRO, SUZUKI YUZURU, TAKII YUKIO, YAMAMOTO KAZUMI, EMI, SHIGENORI, NISHIYA, YOSHIAKI, SOGABE, ATSUSHI, SOGABE, YUKIHIRO, SUZUKI, YUZURU, TAKII, YUKIO, YAMAMOTO, KAZUMI, EMI, SHIGENORI, C/O TOYO BOSEKI K.K., Nishiya, Yoshiaki, c/o Toyo Boseki K.K., SOGABE, ATSUSHI, C/O TOYO BOSEKI K.K., SOGABE, YUKIHIRO, C/O TOYO BOSEKI K.K., Yamamoto, Kazumi, c/o Toyo Boseki Kabushiki Kaisha
Applicants: TOYO BOSEKI, TOYO BOSEKI KABUSHIKI KAISHA
Application year: 1993
Main classification: C12N15
IPC codes: C12N15, C12N9