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
Authority
WO
WIPO (PCT)
Prior art keywords
activity
carboxymethylcellulose
enzyme
saccharification
endoglucanase
Prior art date
Application number
PCT/JP1998/003809
Other languages
English (en)
Japanese (ja)
Inventor
Gentaro Okada
Naoyoshi Matsushita
Shoji Gotoh
Naomi Sumita
Toshiaki Kono
Takashi Yamanobe
Original Assignee
Meiji Seika Kaisha Ltd.
Japan As Represented By Director General Of Agency Of Industrial Science And Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
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/fr

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Classifications

    • 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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  • Chemical & Material Sciences (AREA)
  • 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

L'invention concerne l'endoglucanase ACC4, prenant naissance dans un champignon du type Acremonium cellulolyticus, dont l'activité s'établit à un niveau modéré d'avicel de saccharification et à un rapport modéré de type endo entre l'activité de liquéfaction-carboxyméthylcellulose et l'activité de saccharification-carboxyméthylcellulose, qui présente en outre une valeur de pH optimale égale à 5,6 ainsi qu'une température d'action optimale égale à 65° C en ce qui concerne l'activité de saccharification dans laquelle la carboxyméthylcellulose tient lieu de substrat, et qui présente enfin un poids moléculaire de 56 000, ce poids étant déterminé par électrophorèse sur gel de SDS. L'enzyme considérée est utile dans les produits de nourrissage, les produits d'ensilage, etc.
PCT/JP1998/003809 1997-08-28 1998-08-27 Endoglucanase acc4 WO1999011767A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU88860/98A AU8886098A (en) 1997-08-28 1998-08-27 Endoglucanase acc4

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP9/245977 1997-08-28
JP24597797 1997-08-28

Publications (1)

Publication Number Publication Date
WO1999011767A1 true WO1999011767A1 (fr) 1999-03-11

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP1998/003809 WO1999011767A1 (fr) 1997-08-28 1998-08-27 Endoglucanase acc4

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AU (1) AU8886098A (fr)
WO (1) WO1999011767A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008139641A1 (fr) * 2007-05-07 2008-11-20 National Institute Of Advanced Industrial Science And Technology Procédé de fabrication de cellulase et d'hémicellulase ayant une activité hydrolytique élevée
WO2011021616A1 (fr) 2009-08-20 2011-02-24 明治製菓株式会社 NOUVELLE PROTÉINE POSSÉDANT UNE ACTIVITÉ β-GLUCOSIDASE ET UTILISATION ASSOCIÉE
WO2011121768A1 (fr) 2010-03-31 2011-10-06 明治製菓株式会社 Nouveau gène de cellulase
JP2016039821A (ja) * 2015-11-20 2016-03-24 Ｍｅｉｊｉ Ｓｅｉｋａファルマ株式会社 新規セルラーゼ遺伝子

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS59166083A (ja) * 1983-03-09 1984-09-19 Agency Of Ind Science & Technol セルラ−ゼの生産方法
JPS61162167A (ja) * 1985-01-07 1986-07-22 Agency Of Ind Science & Technol 新規なアクレモニウム・セルロリテイカスｔｎ株
JPH02119774A (ja) * 1988-01-13 1990-05-07 Mokuzai Seibun Sogo Riyou Gijutsu Kenkyu Kumiai セルラーゼの製造法
JPH04117244A (ja) * 1990-09-05 1992-04-17 Agency Of Ind Science & Technol サイレージ用酵素剤
WO1995016060A1 (fr) * 1993-12-06 1995-06-15 White Eagle International Technologies, L.P. Procede de preparation de materiaux et revetements ceramiques composites a haute temperature
JPH07236432A (ja) * 1994-03-01 1995-09-12 Asahi Denka Kogyo Kk 固形成形物の製造方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS59166083A (ja) * 1983-03-09 1984-09-19 Agency Of Ind Science & Technol セルラ−ゼの生産方法
JPS61162167A (ja) * 1985-01-07 1986-07-22 Agency Of Ind Science & Technol 新規なアクレモニウム・セルロリテイカスｔｎ株
JPH02119774A (ja) * 1988-01-13 1990-05-07 Mokuzai Seibun Sogo Riyou Gijutsu Kenkyu Kumiai セルラーゼの製造法
JPH04117244A (ja) * 1990-09-05 1992-04-17 Agency Of Ind Science & Technol サイレージ用酵素剤
WO1995016060A1 (fr) * 1993-12-06 1995-06-15 White Eagle International Technologies, L.P. Procede de preparation de materiaux et revetements ceramiques composites a haute temperature
JPH07236432A (ja) * 1994-03-01 1995-09-12 Asahi Denka Kogyo Kk 固形成形物の製造方法

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008139641A1 (fr) * 2007-05-07 2008-11-20 National Institute Of Advanced Industrial Science And Technology Procédé de fabrication de cellulase et d'hémicellulase ayant une activité hydrolytique élevée
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 (fr) 2009-08-20 2011-02-24 明治製菓株式会社 NOUVELLE PROTÉINE POSSÉDANT UNE ACTIVITÉ β-GLUCOSIDASE ET UTILISATION ASSOCIÉE
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 (fr) 2010-03-31 2011-10-06 明治製菓株式会社 Nouveau gène de cellulase
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 (ja) * 2015-11-20 2016-03-24 Ｍｅｉｊｉ Ｓｅｉｋａファルマ株式会社 新規セルラーゼ遺伝子

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Publication number Publication date
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