WO1999011767A1 - Endoglucanase acc4 - Google Patents

Endoglucanase acc4 Download PDF

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Publication number
WO1999011767A1
WO1999011767A1 PCT/JP1998/003809 JP9803809W WO9911767A1 WO 1999011767 A1 WO1999011767 A1 WO 1999011767A1 JP 9803809 W JP9803809 W JP 9803809W WO 9911767 A1 WO9911767 A1 WO 9911767A1
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Prior art keywords
activity
carboxymethylcellulose
enzyme
saccharification
endoglucanase
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PCT/JP1998/003809
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French (fr)
Japanese (ja)
Inventor
Gentaro Okada
Naoyoshi Matsushita
Shoji Gotoh
Naomi Sumita
Toshiaki Kono
Takashi Yamanobe
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Meiji Seika Kaisha Ltd.
Japan As Represented By Director General Of Agency Of Industrial Science And Technology
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Application filed by Meiji Seika Kaisha Ltd., Japan As Represented By Director General Of Agency Of Industrial Science And Technology filed Critical Meiji Seika Kaisha Ltd.
Priority to AU88860/98A priority Critical patent/AU8886098A/en
Publication of WO1999011767A1 publication Critical patent/WO1999011767A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase

Definitions

  • the present invention relates to endoglucanase, and more particularly to a novel endoglucanase ACC4 derived from the filamentous fungus Acremonium cellus ritus.
  • Cellulose is a major constituent of higher plant cell walls and is widely found in nature.
  • Cellulose is a high-molecular polysaccharide in which glucose is polymerized by 4-glucosidic bonds. Cellulose naturally exists in a crystalline or non-crystalline state. In addition, other components, lignin, and hemicell ports are used. Plants and tissues, and forms complex plant tissues.
  • Cellulase is a general term for a group of enzymes that catalyze the reaction of decomposing cellulose into cellooligosaccharides, cellobiose, and ultimately glucose.
  • endoglucanases, exoglucanases, and ⁇ -glucosidases are known.
  • An object of the present invention is to provide a novel endoglucanase. Disclosure of the invention
  • the present invention is an endoglucanase ACC4 derived from a filamentous fungus Acremonium cellulolyticus and having the following properties.
  • N-terminal amino acid sequence has the sequence shown in SEQ ID NO: 1 in the sequence listing.
  • FIG. 1 is a diagram showing anion exchange chromatography fractionation using MonoQ.
  • FIG. 2 is a diagram showing the optimum pH of the present enzyme.
  • FIG. 3 is a view showing a stable pH range of the present enzyme at 4 and 24 hours, and b shows a stable pH range of the present enzyme at 45 ° C. and 2 hours.
  • FIG. 4 is a diagram showing the optimum temperature of the present enzyme.
  • FIG. 5 is a diagram showing the temperature stability of the present enzyme.
  • FIG. 6 is a diagram showing an SDS-polyacrylamide electrophoresis analysis when the molecular weight of the present enzyme was measured.
  • FIG. 7 is a diagram showing polyacrylamide isoelectric focusing electrophoresis analysis when the isoelectric point of the present enzyme was measured.
  • the endoglucanase ACC4 of the present invention is an active ingredient contained in the cellulase system produced by Acremonium cellus lyticus. less than The present invention will be described in detail.
  • Examples of the microorganisms that produce the cellulase system containing the endoglucanase ACC 4 of the present invention include Acremodium 'cellulolyticus Y-94 strain (FERM B P-5826) and Acremodium' cellulolyticus TN strain (FERM BP-685).
  • the production of cellulase by the present bacterium may be performed, for example, according to the method described in Japanese Patent Publication No. 60-43954 and Japanese Patent Publication No. 63-63197. That is, it can be obtained by culturing the above strain in a medium containing a carbon source and a nitrogen source, and collecting a target cellulase from the culture.
  • the supernatant obtained by removing the culture by centrifugation or the like can be used as a crude enzyme.However, usually, the supernatant is concentrated by ultrafiltration or the like. Add a preservative or the like to make the concentrated enzyme, or use the spray-dry method after concentration to make the powdered enzyme.
  • the endoglucanase ACC4 of the present invention can be obtained by partially or highly purifying these concentrated enzymes or powdered enzymes as necessary.
  • the purification method is a conventional method, that is, a salting-out method such as ammonium sulfate, an organic solvent precipitation method such as alcohol, a membrane separation method, a chromatographic separation method using an ion exchanger, a carrier for hydrophobic chromatography, a carrier for gel filtration, and the like. Alone or as appropriate Can be used.
  • the properties of highly purified endoglucanase AC C4 obtained by the present invention are as follows. This enzyme is a novel enzyme because it has not been reported in the literature up to.
  • N-terminal amino acid sequence has the sequence described in SEQ ID NO: 1 in the sequence listing (based on Protein Sequencer Model 492, manufactured by Pakinkin Elma).
  • the method for measuring the activity of the enzyme and the definition of the amount of enzyme per unit are as follows. It was as below.
  • the enzyme is allowed to act on a 1% Avicel suspension at 40 ° C and the amount of enzyme that produces reducing sugars equivalent to 1 im 01 glucose per minute is defined as 1 unit.
  • the enzyme was allowed to act on a 0.25% carboxymethylcellulose solution at pH 5.6, 30 using a Cannon's Fenske viscometer, and the fluidity (7?) Of the reaction mixture was measured over time.
  • the microorganism was cultured by the following method.
  • the medium was composed of all the following components and used after sterilization by heat in a conventional manner.
  • Acremonium 'cellulolyticus TN strain (FERMBP-685) was inoculated into 500 ml of the culture medium, and cultured at 30 with stirring for 48 hours ( then, the culture solution was used as a seed for 15 L). Scale up to Then, the volume of the culture solution in the 600 L tank was finally adjusted to 300 L, and aeration and stirring culture was performed for 7 days.
  • the resulting culture was filtered through a filter press, concentrated to 15 L by ultrafiltration, lactose (2 kg) was added, and powdered by spray drying.
  • the cellulase preparation obtained by this method was 5.0 kg.
  • Example 1 The bulk powder obtained in Example 1 was dissolved in 20 mM acetate buffer (pH 5.5), and impurities were removed by high-speed cooling centrifugation. The obtained supernatant was purified by the following method as a starting material for enzyme purification.
  • FIG. 2 shows the optimum pH at 30 ° C. of the purified endoglucanase AC C 4 (cellulase activity fraction II) obtained in Example 2.
  • This enzyme showed maximum activity at pH 5.6 in Mcllvaine buffer.
  • the pH stability of this enzyme at 4 ° C is shown in Fig. 3a, and the pH stability at 45 ° C is shown in Fig. 3b.
  • ⁇ and Qin indicate Mcllvain buffer
  • ⁇ and ⁇ ⁇ indicate Britton & Robinson buffer.
  • the optimal temperature of action of the purified endoglucanase AC C4 obtained in Example 2 saccharification activity using carboxymethylcellulose as a substrate was measured. As shown in FIG. 4, the optimal temperature was The temperature was 65 ° C (pH 5.6).
  • Ovalbumin 45 0 0 0
  • polyacrylamide gel isoelectric focusing was performed using a Multi Four II electrophoresis apparatus (Pharmacia Biotech). .
  • a Multi Four II electrophoresis apparatus Pharmacia Biotech.
  • Ampholine PAG Plate pH 3.5 to 5.9
  • IEF IEF
  • the N-terminal amino acid sequence of the purified endoglucanase ACC4 obtained in Example 2 was determined. First, after electrophoretic separation using 8% Gel SDS-PAGE mini (manufactured by Tefco), the protein was applied to a PVDF membrane (manufactured by Millipore) using Multi Four TM (manufactured by Pharmacia Biotech). The mixture was electrically transferred, stained with Kumashi-I 'Brilliant' Blue R250 (manufactured by Nacalai Tesque), washed with water, and air-dried.
  • the enzyme endoglucanase ACC4 of the present invention is a main component ⁇ of a cellulase derived from a microorganism belonging to the genus Acremonium, which is considered to have a strong saccharifying power, and its properties have been clarified for the first time. This enzyme is useful for applications such as feed and silage.

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  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

Endoglucanase ACC4 which originates in a fungus Acremonium cellulolyticus, has an activity at a moderate level of saccharifying avicel and a moderate endo-type ratio of the carboxymethylcellulose-liquefying activity to the carboxymethylcellulose-saccharifying activity, shows an optimum pH value of 5.6 and an optimum action temperature of 65 °C concerning the saccharifying activity with the use of carboxymethyl-cellulose as the substrate, and has a molecular weight of 56,000 when determined by SDS-polyacrylamide gel electrophoresis. This enzyme is useful in feeds, silage, etc.

Description

明 細 書 ェンドグルカナ一ゼ A C C 4 技術分野  Description Endglucanase A C C 4 Technical field
本発明は、 エンドグルカナ一ゼに関し、 詳しくは糸状菌アクレモニゥ ム ·セル口リティカスに由来する新規なェンドグルカナ一ゼ A C C 4に 関する。 背景技術  The present invention relates to endoglucanase, and more particularly to a novel endoglucanase ACC4 derived from the filamentous fungus Acremonium cellus ritus. Background art
セルロースは、 高等植物細胞壁の主要な構成成分であり、 広く天然に 存在する。 セルロースは、 グルコースが 4ーグルコシド結合に より重合した高分子多糖であり、 天然にはセルロースが結晶状、 あるい は非結晶状態で存在しており、 更には他の成分、 リグニン、 へミセル口 —ス類、 ぺクチン類などとも複雑に結合して植物組織を構築している。 セルラ一ゼは、 セルロースをセロオリゴ糖、 セロビオース、 最終的に はグルコースにまで分解する反応を触媒する酵素群の総称であり、 その 作用様式によって、 エンドグルカナ一ゼ、 ェキソグルカナーゼ、 ^ーグ ルコシダ一ゼなどに大別される。 それらの作用様式の詳細まで比較する と、 非常に多数の酵素が存在するので、 従来から様々な作用様式を示す 酵素が互いに補い合って、 相乗効果を発現して植物組織の主成分である セルロースを分解するものと考えられてきた。  Cellulose is a major constituent of higher plant cell walls and is widely found in nature. Cellulose is a high-molecular polysaccharide in which glucose is polymerized by 4-glucosidic bonds. Cellulose naturally exists in a crystalline or non-crystalline state. In addition, other components, lignin, and hemicell ports are used. Plants and tissues, and forms complex plant tissues. Cellulase is a general term for a group of enzymes that catalyze the reaction of decomposing cellulose into cellooligosaccharides, cellobiose, and ultimately glucose.Depending on its mode of action, endoglucanases, exoglucanases, and ^ -glucosidases are known. It is roughly divided into ze etc. Comparing the details of their modes of action, there are a very large number of enzymes, and enzymes that exhibit various modes of action have been complementary to each other, exhibiting a synergistic effect, and converting cellulose, the main component of plant tissue, into cellulose. It has been considered to decompose.
しかし最近、 飼料用酵素分野では、 これらセルラーゼ群の中で特に効 果的なセルラーゼ成分の検討が進み、 ェンドグルカナーゼが主要な役割 を果たしていることが判明しつつある （例えば、 W O 9 5 1 6 3 6 0 公報） 。 糸状菌アクレモニゥム ' セルロリティカス （Acremonium cel lulolyt i cus) F E R M B P— 5 8 2 6の生産するセルラ一ゼは、 糖化力の強 いことが特徴であり、 飼料用途やサイレ一ジ用途での有用性が報告され ている （例えば、 特開平 4 - 1 1 7 2 4 4号公報、 特開平 7 - 2 3 6 4 3 1号公報） 。 また、 含有されているセルラーゼ成分についても報告さ れている （例えば、 Agric. Biol . Chem. , 52, 2493〜2501 (1988)、 同誌 53, 3359-3360 ( 1989) , 同誌 54， 309 〜317(1990) )。 しかし、 そのセ ルラ一ゼ酵素群の中でどの成分が有効成分であるかについては十分に知 られていなかった。 However, recently, in the field of feed enzymes, studies have been made on particularly effective cellulase components among these cellulases, and it has been revealed that endglucanase plays a major role (for example, WO951). 636 publication). Acremonium cellulolyticus (Acremonium cel lulolyt i cus) FERMBP—The cellulase produced by FERMBP is characterized by its strong saccharification, and is useful for feed and silage applications. Have been reported (for example, Japanese Patent Application Laid-Open Nos. HEI 4-1-172464 and H07-2363641). In addition, cellulase components have been reported (eg, Agric. Biol. Chem., 52, 2493-2501 (1988), ibid. 53, 3359-3360 (1989), ibid. 54, 309-317). (1990)). However, it was not sufficiently known which component of the cellulase enzyme group was the active ingredient.
そこで本発明者らは、 アクレモニゥム ' セルロリティカス由来のセル ラーゼ系を構成する種々の酵素を分画、 精製して鋭意検討した。 その結 果、 酵素群の中の有効成分を見出し、 本発明を完成した。 本発明は、 新 規なェンドグルカナ一ゼの提供を目的とするものである。 発明の開示  Therefore, the present inventors fractionated and purified various enzymes constituting the cellulase system derived from Acremonium 'cellulolyticus, and studied diligently. As a result, an active ingredient in the enzyme group was found, and the present invention was completed. An object of the present invention is to provide a novel endoglucanase. Disclosure of the invention
本発明は、 糸状菌アクレモニゥム ·セルロリティカスに由来し、 下記 の性質を有するェン ドグルカナ一ゼ A C C 4である。  The present invention is an endoglucanase ACC4 derived from a filamentous fungus Acremonium cellulolyticus and having the following properties.
(a) 作用 ： アビセル糖化活性が中程度で、 カルボキシメチルセルロース 液化活性とカルボキシメチルセルロース糖化活性の比率が中ェン ド型を 示す。  (a) Action: Avicel saccharification activity is moderate, and the ratio of carboxymethylcellulose liquefaction activity to carboxymethylcellulose saccharification activity shows a medium end type.
(b) 基質特異性：本酵素はカルボキシメチルセルロースによく作用する。 (b) Substrate specificity: This enzyme acts well on carboxymethylcellulose.
(c) 至適 p H及び安定 p H範囲 ： カルボキシメチルセルロースを基質と した糖化活性では、 至適 p Hは 5. 6であり、 p H3. 3〜9. 1 ( 4 °C、 2 4時間） の範囲で安定である。 (c) Optimal pH and stable pH range: For saccharification activity using carboxymethylcellulose as a substrate, the optimal pH is 5.6, and the pH is 3.3 to 9.1 (4 ° C, 24 hours) ) Is stable within the range.
(d) 作用最適温度： カルボキシメチルセルロースを基質とした糖化活性 では、 6 5 °Cである。 (e) 温度安定性： 6 (TC以下で安定である （ρ Η5· 6、 1 0分） 。 (d) Optimum temperature for action: For saccharification activity using carboxymethylcellulose as a substrate, the temperature is 65 ° C. (e) Temperature stability: 6 (stable at TC or less (ρΗ5.6, 10 minutes).
(f) 等電点： p I 4. 0 1  (f) Isoelectric point: p I 4.01
(ポリアクリルアミ ドゲル等電点電気泳動法による） (By polyacrylamide gel isoelectric focusing)
(g) 分子量： 5 6， 0 0 0 (g) Molecular weight: 56, 00
( S D S —ポリアクリルアミ ドゲル電気泳動法による） (SDS—by polyacrylamide gel electrophoresis)
(h) 比活性： 2 9. 4単位/ m g蛋白質 (h) Specific activity: 29.4 units / mg protein
(カルボキシメチルセルロース糖化活性）  (Carboxymethyl cellulose saccharification activity)
： 0. 1 2単位/ m g蛋白質 （アビセル糖化活性） : 0.1 2 units / mg protein (avicel saccharification activity)
(i) N末端ァミノ酸配列：配列表の配列番号 1記載の配列を有する。 図面の簡単な説明 (i) N-terminal amino acid sequence: has the sequence shown in SEQ ID NO: 1 in the sequence listing. BRIEF DESCRIPTION OF THE FIGURES
第 1図は、 MonoQ による陰イオン交換クロマトグラフィー分画図であ 第 2図は、 本酵素の至適 p Hを示す図である。  FIG. 1 is a diagram showing anion exchange chromatography fractionation using MonoQ. FIG. 2 is a diagram showing the optimum pH of the present enzyme.
第 3図は、 aは 4 、 2 4時間での本酵素の安定 p H範囲を、 bは 4 5 °C、 2時間での本酵素の安定 p H範囲を示す図である。  FIG. 3 is a view showing a stable pH range of the present enzyme at 4 and 24 hours, and b shows a stable pH range of the present enzyme at 45 ° C. and 2 hours.
第 4図は、 本酵素の至適温度を示す図である。  FIG. 4 is a diagram showing the optimum temperature of the present enzyme.
第 5図は、 本酵素の温度安定性を示す図である。  FIG. 5 is a diagram showing the temperature stability of the present enzyme.
第 6図は、 本酵素の分子量を測定した際の S D S —ポリアクリルアミ ド電気泳動分析を示す図である。  FIG. 6 is a diagram showing an SDS-polyacrylamide electrophoresis analysis when the molecular weight of the present enzyme was measured.
第 7図は、 本酵素の等電点を測定した際のポリアクリルアミ ド等電点 電気泳動分析を示す図である。 発明を実施するための最良の形態  FIG. 7 is a diagram showing polyacrylamide isoelectric focusing electrophoresis analysis when the isoelectric point of the present enzyme was measured. BEST MODE FOR CARRYING OUT THE INVENTION
本発明のェンドグルカナーゼ A C C 4は、 ァクレモニゥム ·セル口リ ティカスが生産するセルラーゼ系中に含まれる有効成分である。 以下に 本発明を詳細に説明する。 The endoglucanase ACC4 of the present invention is an active ingredient contained in the cellulase system produced by Acremonium cellus lyticus. less than The present invention will be described in detail.
本発明のェンドグルカナ一ゼ A C C 4を含むセルラーゼ系を生産する 微生物としては、 アクレモウム 'セルロリティカス Y— 9 4株 （FERM B P-5826) 、 アクレモウム 'セルロリティカス T N株 （FERM BP- 685 ) な どがあり、 本菌によるセルラーゼの製造については、 例えば特公昭 6 0 - 4 3 9 5 4号公報、 特公昭 6 3 - 6 3 1 9 7号公報に記載された方法 に従って行えばよい。 すなわち、 上記の菌株を炭素源と窒素源を含む培 地に培養し、 培養物から目的とするセルラーゼを採取することにより得 られる。 なお、 アクレモウム 'セルロリティカス Y— 9 4株は、 日本国 茨城県つくば市東 1丁目 1番 3号の通商産業省工業技術院生命工学工業 技術研究所に、 Acremoniura eel lulolyt icus として国際寄託されており Examples of the microorganisms that produce the cellulase system containing the endoglucanase ACC 4 of the present invention include Acremodium 'cellulolyticus Y-94 strain (FERM B P-5826) and Acremodium' cellulolyticus TN strain (FERM BP-685). The production of cellulase by the present bacterium may be performed, for example, according to the method described in Japanese Patent Publication No. 60-43954 and Japanese Patent Publication No. 63-63197. That is, it can be obtained by culturing the above strain in a medium containing a carbon source and a nitrogen source, and collecting a target cellulase from the culture. Acremodium 'Cellulolyticus Y-94' strain was deposited internationally as Acremoniura eel lulolyt icus with the Institute of Biotechnology and Industrial Technology of the Ministry of International Trade and Industry of 1-3-1 Higashi, Tsukuba, Ibaraki, Japan. Cage
(平成 9年 2月 1 9 日に原寄託（FERM P-6867) より移管） 、 その受託番 号は FERM BP-5826である。 また、 アクレモウム ·セルロリティカス T N 株についても同様に、 通商産業省工業技術院生命工学工業技術研究所に、 Acremoniura cel lulolyt icus TNとして国際寄託されており （昭和 5 9年 1 2月 2 0 日に原寄託（FERM P-7894) より移管） 、 その受託番号は FERM(Transferred from the original deposit (FERM P-6867) on February 19, 1997), and its deposit number is FERM BP-5826. Similarly, the Acremodium cellulolyticus TN strain has been deposited internationally as Acremoniura cel lulolyt icus TN with the Ministry of International Trade and Industry at the National Institute of Advanced Industrial Science and Technology (April 20, 1982). Transferred from the original deposit (FERM P-7894) to FERM
BP - 685 である。 BP-685.
上記微生物の培養終了後、 培養物を遠心分離等により除去して得た上 清液を、 粗酵素として用いることもできるが、 通常は、 この上清液を限 外濾過法などにより濃縮し、 防腐剤などを加えて濃縮酵素とするか、 濃 縮後スプレードライ法によって粉末酵素とする。  After the culture of the microorganism, the supernatant obtained by removing the culture by centrifugation or the like can be used as a crude enzyme.However, usually, the supernatant is concentrated by ultrafiltration or the like. Add a preservative or the like to make the concentrated enzyme, or use the spray-dry method after concentration to make the powdered enzyme.
本発明のェンドグルカナ一ゼ A C C 4は、 これら濃縮酵素又は粉末酵 素を必要に応じて部分精製又は高度に精製して得ることができる。 精製 方法としては常法、 即ち硫安などの塩析法、 アルコールなどの有機溶媒 沈澱法、 膜分離法、 イオン交換体、 疎水クロマトグラフ用担体、 ゲル濾 過用担体などを用いるクロマト分離法などを単独又は適宜組み合わせて 用いることができる。 The endoglucanase ACC4 of the present invention can be obtained by partially or highly purifying these concentrated enzymes or powdered enzymes as necessary. The purification method is a conventional method, that is, a salting-out method such as ammonium sulfate, an organic solvent precipitation method such as alcohol, a membrane separation method, a chromatographic separation method using an ion exchanger, a carrier for hydrophobic chromatography, a carrier for gel filtration, and the like. Alone or as appropriate Can be used.
本発明によって得られる高純度に精製されたェンドグルカナーゼ AC C 4の性質は下記の通りである。 この酵素については ·までの文献には 報告されていないので、 新規酵素である。  The properties of highly purified endoglucanase AC C4 obtained by the present invention are as follows. This enzyme is a novel enzyme because it has not been reported in the literature up to.
(a) 作用 ： アビセル糖化活性が中程度で、 カルボキシメチルセル π—ス 液化活性とカルボキシメチルセルロース糖化活性の比率が中エンド型を 示す。  (a) Action: Avicel saccharification activity is moderate, and the ratio of carboxymethylcell π-is liquefaction activity to carboxymethylcellulose saccharification activity is a medium-end type.
(b) 基質特異性： 本酵素はカルボキシメチルセルロースによく作用する。 (b) Substrate specificity: This enzyme acts well on carboxymethylcellulose.
(c) 至適 pH及び安定 pH範囲 ： カルボキシメチルセルロースを基質と した糖化活性では、 至適 p Hは 5. 6であり （第 2図） 、 pH3. 3〜9. 1 ( 4 °C、 2 4時間） の範囲で安定であり （第 3図、 a ) 、 4 5 °C、 2時 間では pH4. 6〜7. 8の範囲で安定である （第 3図、 b) 。 (c) Optimum pH and stable pH range: For saccharification activity using carboxymethylcellulose as a substrate, the optimum pH is 5.6 (Fig. 2), and the pH is 3.3 to 9.1 (4 ° C, 2 ° C). 4 hours) (Fig. 3, a), and stable at 45 ° C and 2 hours, pH 4.6-7.8 (Fig. 3, b).
(d) 作用最適温度： カルボキシメチルセルロースを基質とした糖化活性 では、 6 5 °Cである （第 4図） 。  (d) Optimal temperature for action: The saccharification activity using carboxymethylcellulose as a substrate is 65 ° C (Fig. 4).
(e) 温度安定性： 6 0°C以下で安定である （pH5. 6、 1 0分） （第 5 図） 。  (e) Temperature stability: Stable below 60 ° C (pH 5.6, 10 minutes) (Fig. 5).
(0 分子量： 56， 0 0 0 ( S D S—ポリアクリルアミ ドゲル電気泳動法 による） （第 6図）  (0 molecular weight: 56, 000 (by SDS-polyacrylamide gel electrophoresis) (Fig. 6)
(g) 等電点： P I 4. 0 1 (ポリアクリルアミ ドゲル等電点電気泳動法 による） （第 7図）  (g) Isoelectric point: PI 4.01 (by polyacrylamide gel isoelectric focusing) (Fig. 7)
(h) 比活性： 29. 4単位 Zmg蛋白質  (h) Specific activity: 29.4 units Zmg protein
(カルボキンメチルセルロース糖化活性） ： 0. 1 2単位/ mg蛋白質 （アビセル糖化活性） (Carboquin methylcellulose saccharification activity): 0.1 2 units / mg protein (Avicel saccharification activity)
(i) N末端アミノ酸配列 ：配列表の配列番号 1記載の配列を有する （パ 一キンエルマ一社製、 プロテインシーケンサー Model 492による） 。 ここで、 当該酵素の活性測定法と 1単位の酵素量の定義については以 下の通りとした。 (i) N-terminal amino acid sequence: has the sequence described in SEQ ID NO: 1 in the sequence listing (based on Protein Sequencer Model 492, manufactured by Pakinkin Elma). Here, the method for measuring the activity of the enzyme and the definition of the amount of enzyme per unit are as follows. It was as below.
•アビセル糖化活性  Avicel saccharification activity
p H5. 6, 4 0 °Cで 1 %アビセル懸濁液に酵素を作用させ、 1分間に 1 im 0 1のグルコースに相当する還元糖を生成する酵素量を 1単位と 疋義し Γこ  pH 5. The enzyme is allowed to act on a 1% Avicel suspension at 40 ° C and the amount of enzyme that produces reducing sugars equivalent to 1 im 01 glucose per minute is defined as 1 unit.
• カルボキシメチルセルロース糖化活性  • Carboxymethylcellulose saccharification activity
ρ H5. 6 , 3 0°Cで 0. 2 5 %カルボキシメチルセルロース溶液に酵素 を作用させ、 1分間に 1 //mo 1のグルコースに相当する還元糖を生成 する酵素量を 1単位と定義した。  ρH5.6 At 30 ° C, the enzyme was allowed to act on a 0.25% carboxymethylcellulose solution, and the amount of enzyme that produced reducing sugars equivalent to 1 // mo 1 glucose per minute was defined as 1 unit. .
• カルボキシメチルセルロース液化活性  • Carboxymethylcellulose liquefaction activity
キャノ ン ' フェンスケ粘度計を用いて、 p H5. 6, 3 0 で0. 2 5 %カ ルポキシメチルセルロース溶液に酵素を作用させ、 経時的に反応混液の 流動度 （7? ) を測定し、 比流動度 s p. = 1 / τ? ) をカルボキシメ チルセルロース液化活性とした。  The enzyme was allowed to act on a 0.25% carboxymethylcellulose solution at pH 5.6, 30 using a Cannon's Fenske viscometer, and the fluidity (7?) Of the reaction mixture was measured over time. The specific flow rate sp. = 1 / τ?) Was defined as the liquefaction activity of carboxymethyl cellulose.
次に、 本発明を実施例により詳しく説明する。  Next, the present invention will be described in more detail with reference to examples.
実施例 1 (酵素原末の調製）  Example 1 (Preparation of bulk enzyme powder)
ァクレモ二ゥム属微生物由来のセルラーゼ製剤を得るため、 下記の手 法により微生物の培養を行った。 培地は、 すべて以下の組成から構成さ れ、 常法により加熱殺菌したものを用いた。  In order to obtain a cellulase preparation derived from the microorganism of the genus Acremonium, the microorganism was cultured by the following method. The medium was composed of all the following components and used after sterilization by heat in a conventional manner.
セルロース 4 %、 リ ン酸水素二力リウム 1. 2 %、 ノくク トペプトン 1 %、 硝酸力リゥム 0. 6 %、 尿素 0. 2 %、 塩化力リゥ厶 0, 1 6 %、 硫酸マグネ シゥム ·七水塩 0. 1 2 %、 硫酸亜鉛 ·七水塩 0. 0 0 1 %, 硫酸マンガン •七水塩 0. 0 0 1 %、 硫酸銅 ·五水塩 0. 0 0 1 %。  Cellulose 4%, dihydrogen hydrogen phosphate 1.2%, noctopeptone 1%, nitric acid rim 0.6%, urea 0.2%, chlorinated lime 0,16%, magnesium sulfate · Seven hydrate 0.12%, Zinc sulfate · Seven hydrate 0.01%, Manganese sulphate • Seven hydrate 0.001%, Copper sulphate · Pentahydrate 0.001%.
培地 5 0 0 m 1 にァクレモニゥム 'セルロリティカス TN株 （F E R M B P— 6 8 5 ) を接種し、 3 0でで 4 8時間攪拌しながら培養した ( 次に、 その培養液をシードとして 1 5 Lにスケールアップし、 更にスケ ールアップを続けて最終的に 6 0 0 Lタンク中の培養液量を 3 0 0 Lと し、 通気攪拌培養を 7日間行った。 Acremonium 'cellulolyticus TN strain (FERMBP-685) was inoculated into 500 ml of the culture medium, and cultured at 30 with stirring for 48 hours ( then, the culture solution was used as a seed for 15 L). Scale up to Then, the volume of the culture solution in the 600 L tank was finally adjusted to 300 L, and aeration and stirring culture was performed for 7 days.
得られた培養液をフィルタ一プレスで濾過した後、 限外濾過により 1 5 Lまで濃縮し、 乳糖 2 k gを添加してスプレードライにより粉末化し た。 この方法で得られたセルラーゼ製剤は 5. 0 k gであった。  The resulting culture was filtered through a filter press, concentrated to 15 L by ultrafiltration, lactose (2 kg) was added, and powdered by spray drying. The cellulase preparation obtained by this method was 5.0 kg.
実施例 2 (エンドグルカナ一ゼ AC C 4の精製） Example 2 (Purification of endoglucanase AC C4)
実施例 1で得られた原末を、 2 0 mM 酢酸緩衝液 （pH5. 5 ) に溶 解し、 不純物を高速冷却遠心分離により除去した。 得られた上清を酵素 精製の出発材料として以下に示した方法で精製した。  The bulk powder obtained in Example 1 was dissolved in 20 mM acetate buffer (pH 5.5), and impurities were removed by high-speed cooling centrifugation. The obtained supernatant was purified by the following method as a starting material for enzyme purification.
①強塩基性陰イオン交換クロマトグラフィ一： QAE— トヨパール 550C (東ツー （株） ） に上清を吸着させ、 酢酸緩衝液 （ 2 OmM、 Η5. 5) 中に N a C 1各 0、 0. 0 4、 0. 1 5、 0. 5 Mを含有せしめ、 ステップヮ ィズ溶出を行い、 0. 1 5M N a C 1で溶出されるセルラ一ゼ活性画分 を分取した。  (1) Strongly basic anion exchange chromatography: QAE—Adsorb the supernatant to Toyopearl 550C (Higashi-Two Co., Ltd.), and add NaC 1 in acetate buffer (2 OmM, Η5.5) to 0,0. 0.4, 0.15, and 0.5 M were contained, and a stepwise elution was performed, and a cellulase-active fraction eluted with 0.15 M NaC1 was collected.
②疎水クロマトグラフィ一 ： ブチルトヨパール （東ソ一 （株） ） に前 記①の分取画分を吸着させ、 酢酸緩衝液 （ 2 0mM、 H4. 0) 中に （ NH4)2 S〇4 の濃度が 0. 7， 0. 5, 0. 4， 0Mとなるように溶解せし めた各緩衝液及び脱ィォン水を用いて、 順次ステップワイズ溶出を行つ た。 脱イオン水により溶出されたセルラーゼ活性画分を分取した。 (2) Hydrophobic chromatography: Adsorb the preparative fraction described in (1) above to Butyl Toyopearl (Tosoichi Co., Ltd.), and add (NH 4 ) 2 S〇 4 in acetate buffer (20 mM, H4.0). Stepwise elution was carried out sequentially using each of the buffer solutions and deionized water dissolved at a concentration of 0.7, 0.5, 0.4, and 0M. The cellulase activity fraction eluted with deionized water was collected.
③ MonoQ を用いた陰イオン交換クロマトグラフィー： MonoQ カラムに、 前記②で得たセルラーゼ活性画分を吸着させ、 F P L Cを用いて溶出を 行った。 酢酸緩衝液 （ 2 0mM、 pH4. 5) 中の N a C 1濃度が 0から 0. 1 Mに上昇するよう、 直線濃度勾配をかけ室温で溶出した。 その結果、 セルラーゼ活性を有する I及び Πの 2個のタンパク質画分が溶出された。 これらの画分のタンパク質量とカルボキシメチルセルロース （CMC) 糖化活性の測定値を第 1図に示した。 これらのうち、 セルラーゼ活性画分 IIは Native- 及び SDS—ポリア クリルァミ ドゲル電気泳動で単一のタンパク質染色バンドを示す、 高純 度セルラーゼ標品であった。 (3) Anion exchange chromatography using MonoQ: The cellulase activity fraction obtained in (1) above was adsorbed to a MonoQ column, and elution was performed using FPLC. Elution was performed at room temperature by applying a linear concentration gradient so that the NaC1 concentration in acetate buffer (20 mM, pH 4.5) increased from 0 to 0.1 M. As a result, two protein fractions I and を having cellulase activity were eluted. The protein content of these fractions and the measured values of carboxymethylcellulose (CMC) saccharification activity are shown in FIG. Among these, the cellulase activity fraction II was a high-purity cellulase sample showing a single protein staining band on Native- and SDS-polyacrylamide gel electrophoresis.
実施例 3 Example 3
実施例 2で得た精製ェンドグルカナーゼ AC C 4 (セルラ一ゼ活性画 分 II) の 3 0°Cにおける至適 pHを第 2図に示す。 本酵素は、 Mcllvaine 緩衝液で pH5. 6のときに最大活性を示した。 また、 本酵素の 4°Cにお ける pH安定性を第 3図 aに、 4 5 °Cにおける p H安定性を第 3図 bに それぞれ示す。 図中の騸と秦は Mcllvain緩衝液を示し、 ロと〇は Britton & Robinson緩衝液を示す。  FIG. 2 shows the optimum pH at 30 ° C. of the purified endoglucanase AC C 4 (cellulase activity fraction II) obtained in Example 2. This enzyme showed maximum activity at pH 5.6 in Mcllvaine buffer. The pH stability of this enzyme at 4 ° C is shown in Fig. 3a, and the pH stability at 45 ° C is shown in Fig. 3b. In the figure, 騸 and Qin indicate Mcllvain buffer, and ロ and 示 す indicate Britton & Robinson buffer.
実施例 4 Example 4
実施例 2で得た精製ェンドグルカナーゼ AC C 4の作用最適温度を調 ベるために、 カルボキシメチルセルロースを基質とした糖化活性を測定 したところ、 第 4図に示したように、 至適温度は 6 5 °C (p H5. 6 ) で あつた。  In order to determine the optimal temperature of action of the purified endoglucanase AC C4 obtained in Example 2, saccharification activity using carboxymethylcellulose as a substrate was measured. As shown in FIG. 4, the optimal temperature was The temperature was 65 ° C (pH 5.6).
次に、 本酵素の温度安定性を pH5. 6、 1 0分間の条件下で測定した ところ、 第 5図に示したように、 6 0°C以下で安定であった。  Next, when the temperature stability of this enzyme was measured under the conditions of pH 5.6 and 10 minutes, it was stable at 60 ° C or lower as shown in FIG.
実施例 5  Example 5
実施例 2で得た精製ェンドグルカナーゼ AC C 4の分子量を決定する ため、 SDS—ポリアクリルアミ ドゲル電気泳動を行った。 すなわち、 12. 5 %ゲルを用いて 2 0 m Aの定電流で室温にて 5 0分間通電した。 その結果、 本酵素の分子量は約 56, 0 0 0と算出された （第 6図） 。 なお、 この測定に用いた標準タンパク質及びその分子量は以下の通りで あ o  In order to determine the molecular weight of the purified endoglucanase AC C4 obtained in Example 2, SDS-polyacrylamide gel electrophoresis was performed. That is, a current was passed for 50 minutes at room temperature using a 12.5% gel at a constant current of 20 mA. As a result, the molecular weight of this enzyme was calculated to be about 56,000 (FIG. 6). The standard proteins and their molecular weights used in this measurement are as follows:
標準タンパク質 分子量  Standard protein molecular weight
Phosphorylase b 97, 0 0 0 Serum albumin 66, 0 0 0 Phosphorylase b 97, 0 0 0 Serum albumin 66, 0 0 0
Ovalbumin 45, 0 0 0  Ovalbumin 45, 0 0 0
Carbonic anhydrase 31, 0 0 0  Carbonic anhydrase 31, 0 0 0
Trypsin inhibitor 1, 5 0 0  Trypsin inhibitor 1, 5 0 0
Lysozyme 14, 4 0 0  Lysozyme 14, 4 0 0
実施例 6 Example 6
実施例 2で得た精製ェンドグルカナーゼ AC C 4の等電点を決定する ため、 マルチフォー II電気泳動装置 （フアルマシアバイオテク社製） を 用いてポリアク リルアミ ドゲル等電点電気泳動を行った。 泳動用ゲルに は、 Ampholine PAG Plate ( p H3. 5〜5. 9) for IEF (フアルマシア バイオテク社製） を用いて 1 0 °C、 1 5 0 0 V、 5 OmA. 3 0Wの泳 動条件で 9 0分間通電した。  In order to determine the isoelectric point of the purified endoglucanase AC C4 obtained in Example 2, polyacrylamide gel isoelectric focusing was performed using a Multi Four II electrophoresis apparatus (Pharmacia Biotech). . For the gel for electrophoresis, use Ampholine PAG Plate (pH 3.5 to 5.9) for IEF (Pharmacia Biotech) at 10 ° C, 150 V, 5 OmA. For 90 minutes.
その結果、 本酵素の等電点 （p i ) は 4. 0 1 と算出された （第 7図） ， なお、 この測定に用いた標準タンパク質の p Iは以下の通りである。 標準タンパク質 I  As a result, the isoelectric point (p i) of this enzyme was calculated to be 4.01 (Fig. 7). The pI of the standard protein used for this measurement is as follows. Standard protein I
Amyloglucosidase 3. 5 0  Amyloglucosidase 3.5 0
Soybean trypsin inhibitor 4. 5 5  Soybean trypsin inhibitor 4.5 5 5
/3 -し actoglobul in A 5. 2 0  / 3 -shi actoglobul in A 5.2 0
Bovin carbonic anhydrase B 5. 8 0  Bovin carbonic anhydrase B 5.80
Human carbonic anhydrase B 6. 5 5  Human carbonic anhydrase B 6.5 5
Horse myoglobin - acidic band 6. 8 5  Horse myoglobin-acidic band 6. 8 5
Horse myoglobin - basic band 7. 3 5  Horse myoglobin-basic band 7.3.5
Lentil lectin - acidic band 8. 1 5  Lentil lectin-acidic band 8. 1 5
Lentil lectin - middle band 8.4 5  Lentil lectin-middle band 8.4 5
し entil lectin- basic band 8. 6 5  Shi entil lectin- basic band 8. 6 5
Trypsinogen 9. 3 0 実施例 7 Trypsinogen 9.30 Example 7
実施例 2で得た精製ェンドグルカナ一ゼ A C C 4の N末端アミノ酸配 列を決定した。 まず、 8 % Gel SDS-PAGE mini (テフコ社製） を用いて 電気泳動分離を行った後、 マルチフォー Π (フアルマシアバイオテク社 製） にて、 P V D F膜 （ミ リポア社製） に、 タンパク質を電気的にうつ しとり、 クマシ一 'ブリ リアント ' ブルー R 2 5 0 (ナカライテスク社 製） で染色した後、 水で洗浄し、 風乾した。 ここから分子量 5 6, 0 0 0 のタンパク質がプロッ 卜されている部分を切り出し、 プロテインシ一ケ ンサ一 Model 492 (パーキンエルマ一社製） に供し、 N末端アミノ酸配 列の決定を試みたが、 ェドマン分解により切り出されるァミノ酸が得ら れず、 N末端側ァミ ノ酸が修飾保護されていることが判明した。  The N-terminal amino acid sequence of the purified endoglucanase ACC4 obtained in Example 2 was determined. First, after electrophoretic separation using 8% Gel SDS-PAGE mini (manufactured by Tefco), the protein was applied to a PVDF membrane (manufactured by Millipore) using Multi Four ™ (manufactured by Pharmacia Biotech). The mixture was electrically transferred, stained with Kumashi-I 'Brilliant' Blue R250 (manufactured by Nacalai Tesque), washed with water, and air-dried. From this, a portion where a protein having a molecular weight of 56,000 was plotted was cut out and applied to a protein sequencer Model 492 (manufactured by PerkinElmer) to determine the N-terminal amino acid sequence. However, no amino acid cleaved out by Edman degradation was obtained, indicating that the N-terminal amino acid was modified and protected.
そこで、 先の染色し、 洗浄したメンブレンから分子量 5 6, 0 0 0の夕 ンパク質を切り出し、 ポリ ビニルピロリ ドン一 4 0 (シグマ社製） にて 膜上のタンパク質未結合部分をブロックし、 Podel l . D. N. 等， Biochem. Bi ophys. Res. Co醒 un. , 81 , 176 (1978)の方法に従って、 牛肝臓ピロ グル夕メートべプチダ一ゼ （ベ一リ ンガー社製） を用いて処理すること により、 修飾 Ν末端残基を除去した。 しかる後、 再度前記したプロティ ンシーケンサ一により、 Ν末端側ァミノ酸配列を 2 1残基決定した。 得 られた配列は配列表の配列番号 1に示される通りであった。 産業上の利用可能性  Therefore, protein having a molecular weight of 6,000 was cut out from the previously stained and washed membrane, and the non-protein-bound portion on the membrane was blocked with polyvinylpyrrolidone-140 (manufactured by Sigma). l. DN, et al., Biochem. Biophys. Res. Co., Ai., 81, 176 (1978), using beef liver pyroglucose mate beptidase (manufactured by Behringer). As a result, the modified Ν-terminal residue was removed. Thereafter, 21 residues of the Ν-terminal amino acid sequence were determined again by the above-mentioned protein sequencer. The obtained sequence was as shown in SEQ ID NO: 1 in the sequence listing. Industrial applicability
本発明の酵素ェンドグルカナ一ゼ A C C 4は、 糖化力が強いとされる ァクレモ二ゥム属微生物由来のセルラ一ゼの主成分の ^であり、 その 性質が初めて明らかになった。 本酵素は、 飼料、 サイレージなどの用途 に有用である。  The enzyme endoglucanase ACC4 of the present invention is a main component ^ of a cellulase derived from a microorganism belonging to the genus Acremonium, which is considered to have a strong saccharifying power, and its properties have been clarified for the first time. This enzyme is useful for applications such as feed and silage.

Claims

請 求 の 範 囲 The scope of the claims
1 . 糸状菌アクレ乇ニゥム 'セルロリティカスに由来し、 下記の性質を 有するェンドグルカナーゼ A C C 4。 1. Endoglucanase ACC4, which is derived from the filamentous fungus Acredinium cellulolyticus and has the following properties:
(a) 作用 ： アビセル糖化活性が中程度で、 カルボキシメチルセルロース 液化活性とカルボキシメチルセルロース糖化活性の比率が中ェンド型を 示す。  (a) Action: Avicel saccharification activity is moderate, and the ratio of carboxymethylcellulose liquefaction activity to carboxymethylcellulose saccharification activity is a medium-end type.
(b) 基質特異性：本酵素はカルボキシメチルセルロースによく作用する。 (b) Substrate specificity: This enzyme acts well on carboxymethylcellulose.
(c) 至適 p H及び安定 pH範囲 ： カルボキシメチルセルロースを基質とし た糖化活性では、 至適 p Hは 5. 6であり、 p H 3. 3〜9. 1 ( 4 °C、 2 4 時間） の範囲で安定である。 (c) Optimal pH and stable pH range: For saccharification activity using carboxymethylcellulose as a substrate, the optimal pH is 5.6, and the pH is 3.3 to 9.1 (4 ° C, 24 hours) ) Is stable within the range.
(d) 作用最適温度： カルボキシメチルセルロースを基質とした糖化活性 では、 6 5でである。  (d) Optimal temperature for action: 65 for saccharification activity using carboxymethylcellulose as a substrate.
(e) 温度安定性： 6 0 °C以下で安定である （ p H5. 6、 1 0分） 。  (e) Temperature stability: Stable below 60 ° C (pH 5.6, 10 min).
(f) 等電点： p i 4. 0 1 (ポリアクリルアミ ドゲル等電点電気泳動法 による）  (f) Isoelectric point: p i 4.01 (by polyacrylamide gel isoelectric focusing)
(g) 分子量： 5 6, 0 0 0  (g) Molecular weight: 5, 6, 0 0 0
( S D S —ボリアクリルアミ ドゲル電気泳動法による） (SDS—by polyacrylamide gel electrophoresis)
(h) 比活性： 2 9. 4単位/ m g蛋白質 (h) Specific activity: 29.4 units / mg protein
(カルボキシメチルセルロース糖化活性） ： 0. 1 2単位/ m g蛋白質 （アビセル糖化活性） (Carboxymethylcellulose saccharification activity): 0.12 units / mg protein (Avicel saccharification activity)
(i) N末端ァミノ酸配列 ：配列表の配列番号 1記載の配列を有する。 (i) N-terminal amino acid sequence: has the sequence shown in SEQ ID NO: 1 in the sequence listing.
PCT/JP1998/003809 1997-08-28 1998-08-27 Endoglucanase acc4 WO1999011767A1 (en)

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
WO2008139641A1 (en) * 2007-05-07 2008-11-20 National Institute Of Advanced Industrial Science And Technology Method for producing cellulase and hemicellulase having high hydrolytic activity
WO2011021616A1 (en) 2009-08-20 2011-02-24 明治製菓株式会社 Novel protein having β-glucosidase activity, and use thereof
WO2011121768A1 (en) 2010-03-31 2011-10-06 明治製菓株式会社 Novel cellulase gene
JP2016039821A (en) * 2015-11-20 2016-03-24 Ｍｅｉｊｉ Ｓｅｉｋａファルマ株式会社 Novel cellulase gene

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008139641A1 (en) * 2007-05-07 2008-11-20 National Institute Of Advanced Industrial Science And Technology Method for producing cellulase and hemicellulase having high hydrolytic activity
US8034596B2 (en) 2007-05-07 2011-10-11 National Institute Of Advanced Industrial Science And Technology Method for producing cellulase and hemicellulase having high hydrolytic activity
WO2011021616A1 (en) 2009-08-20 2011-02-24 明治製菓株式会社 Novel protein having β-glucosidase activity, and use thereof
US8975057B2 (en) 2009-08-20 2015-03-10 Meiji Seika Pharma Co., Ltd. Protein having β-glucosidase activity and uses thereof
US10125355B2 (en) 2009-08-20 2018-11-13 Meiji Seika Pharma Co., Ltd. Protein having B-glucosidase activity and uses thereof
WO2011121768A1 (en) 2010-03-31 2011-10-06 明治製菓株式会社 Novel cellulase gene
US10053679B2 (en) 2010-03-31 2018-08-21 Meiji Seika Pharma Co., Ltd. Cellulase gene
US10774318B2 (en) 2010-03-31 2020-09-15 Meiji Seika Pharma Co., Ltd. Cellulase gene
JP2016039821A (en) * 2015-11-20 2016-03-24 Ｍｅｉｊｉ Ｓｅｉｋａファルマ株式会社 Novel cellulase gene

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