WO2014025203A1 - Composition de mannanase hydrosoluble, et son procédé de préparation - Google Patents

Composition de mannanase hydrosoluble, et son procédé de préparation Download PDF

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Publication number
WO2014025203A1
WO2014025203A1 PCT/KR2013/007122 KR2013007122W WO2014025203A1 WO 2014025203 A1 WO2014025203 A1 WO 2014025203A1 KR 2013007122 W KR2013007122 W KR 2013007122W WO 2014025203 A1 WO2014025203 A1 WO 2014025203A1
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WO
WIPO (PCT)
Prior art keywords
composition
mannanase
kcl
water
soluble
Prior art date
Application number
PCT/KR2013/007122
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English (en)
Korean (ko)
Inventor
최대건
박양순
Original Assignee
주식회사 씨티씨바이오
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Application filed by 주식회사 씨티씨바이오 filed Critical 주식회사 씨티씨바이오
Publication of WO2014025203A1 publication Critical patent/WO2014025203A1/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
    • C12N9/98—Preparation of granular or free-flowing enzyme compositions
    • 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)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/2488—Mannanases
    • C12N9/2494—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
    • 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)

Definitions

  • the present invention relates to a water-soluble mannanase composition having a high water solubility and excellent enzyme stability and a method for producing the same.
  • Hemicellulose is a constituent of the plant cell wall and is a combination of cellulose and lignin. Hemicellulose is the second most abundant polysaccharide in nature after cellulose. Mannanase is an enzyme that decomposes a mannan-containing substance, which is a major component of hemicellulose, and its use is increasing. Along with xylanase and glucanase, the most important of the availability of the hemicellulase met nanase is the glycation of hemicellulose, a plant resource, into a carbon source available to organisms.
  • Hemicellulase is currently in practical use, such as coffee, chocolate, cocoa, tea, cereals in addition to processed food products such as industrial applications such as pulp production.
  • a metase is used as a feed additive to increase the utilization of the feed containing hemicellulose has a great advantage that can reduce the cost of raising livestock.
  • met kinase has been sold in the form of a direct or purified culture of the met kinase production strain, or commercially available in lyophilized powder form.
  • the culture solution is not easy to handle, there is a problem that long-term storage stability is not good because it is a liquid.
  • the water solubility is practically impossible to use mixed with the drinking water for livestock, which caused various inconveniences in handling or use.
  • the problem to be solved by the present invention is to provide a high water-soluble mannase-containing dry composition, in particular a composition of the powder formulation and a method for producing such a composition.
  • a composition can be used by spraying a feed having a high water solubility in animal drinking water, and is easy to handle, convenient to transport, and can be distributed in a dry state to increase enzyme stability.
  • the present invention is prepared by lyophilizing a liquid mixture comprising (1) mannase and (2) KCl, NaCl or a mixture thereof (preferably KCl), mannase and KCl, NaCl Or a mixture thereof (preferably KCl).
  • the present invention can stably maintain the titer of the metase during the lyophilization process by adding KCl (potassium chloride) or NaCl (sodium chloride) prior to lyophilization of the mannase liquid composition (suspension, solution, concentrate, etc., preferably concentrate). It is based on the surprising finding that the water solubility of the lyophilisate formed is dramatically improved.
  • the present inventors have tested various materials for improving water solubility, preserving the titer during the manufacturing process, improving stability, improving the ease of handling of the formed lyophilisate, securing the benefits as a feed additive, and ensuring the ease of the manufacturing process. Only NaCl was able to achieve the desired effect, with KCl being particularly preferred for various purposes of the present invention.
  • the content of KCl, NaCl or a mixture thereof in the lyophilized composition is 0.2 to 0.5 parts by weight based on the total weight of the met kinase after lyophilization, more preferably 0.2 to 0.4 parts by weight relative to the total weight of the met kinase after lyophilization And, it is even more preferred that it is 0.2 to 0.3 parts by weight relative to the total weight of the met kinase.
  • the present invention provides a method for preparing a water-soluble mannase composition, which is lyophilized by dissolving KCl, NaCl or a mixture thereof in a mannase suspension, a solution or a concentrate, preferably a concentrate.
  • KCl, NaCl, or a mixture thereof is included in an amount of 0.01 to 0.2 parts by weight based on the total weight of the metnanase concentrate, and more preferably 0.03 to 0.15 parts by weight, based on the total weight of the metnanase concentrate, Even more preferably, 0.1 parts by weight is included.
  • the meetnase concentrate is removed from the culture medium incubated with a Bacillus strain producing a metase by the method disclosed in US Patent No. 6,984,406 for about 24-30 hours until the maximum production of metnase with 0.45 ⁇ m membrane, It may mean a concentrate concentrated about 20 times through an ultrafilter, but the present invention is not limited to such a concentrate.
  • metnanase concentrate was prepared using the metnanase production strain and culture method disclosed in US Pat. No. 6,984,406 for Bacillus sp. WL-1 strain deposited with accession number KCTC 0800BP. Can be prepared.
  • the invention also provides a water soluble mannanase dry composition prepared by the production process according to the invention.
  • the mannase is endo- ⁇ -mannanase, and in particular, the mannase is a gene bank of the Korea Institute of Science and Technology Currently renamed to the Korea Institute of Bioscience and Biotechnology, Biotechnology Center, 125, Gwahak-ro, Yuseong-gu, Daejeon, Korea.
  • the strain was Bacillus sp. WL-1, which was deposited with the deposit number KCTC 0800BP dated June 12, 2000. It is more preferred that it is a metnanase produced.
  • the present invention also provides a lyophilized composition containing the mannase and KCl, NaCl or a mixture thereof according to the present invention and a nanase containing composition comprising a pharmaceutically or veterinary acceptable excipient.
  • Such compositions may be formulations such as powders, granules, etc., but the invention is not limited to such formulations.
  • the pharmaceutically or veterinary acceptable excipients may be glucose, sorbitol, fructooligosaccharide, trehalose, mannitol, maltose, sugar, maltotextrin or lactose alone or in combination, and the final composition containing mannanase
  • glucose, maltodextrin, sorbitol, fructooligosaccharide, trehalose and the like are more preferable, and glucose is more preferable.
  • the present invention provides a mannanase composition containing 3-4% by weight of the mannose according to the present invention and lyophilisate containing KCl, NaCl or a mixture thereof and 96-97% by weight of anhydrous crystalline glucose.
  • Lyophilized compositions comprising the metnase according to the present invention and KCl, NaCl or mixtures thereof (preferably KCl) and metannase containing powders comprising such lyophilized compositions and pharmaceutically or veterinary acceptable excipients
  • the powder or granule composition may be used in a manner that is mixed (preferably dissolved) in the drinking water for the livestock and sprayed onto the feed, or the livestock may consume the drinking water itself to which the composition of the present invention is added, and It can be used by spraying the pellets after preparation of the pellet feed, or by blending the powder or granule composition itself into the feed.
  • the present invention is not limited to this method of using the composition of the present invention.
  • the present invention provides a water-soluble mannanase composition and a method for preparing the same.
  • the titer of the met kinase can be kept high, and the water solubility is very high and has various usefulness.
  • the met kinase concentrate was prepared in advance as follows. Cells were removed from the Bacillus strain (KCTC 0800BP) culture medium secreting the metase through a vibrating membrane filter equipped with a 0.45 ⁇ m membrane. Thereafter, the filtrate was concentrated 20 times through an ultrafilter to prepare a concentrated mannanase. It was then stored at 4 ° C. to minimize the titer loss of the metase.
  • KCTC 0800BP Bacillus strain
  • FD 80 (CUDDON, New Zealand) was used as a lyophilization device, and the freeze-drying conditions were dried for about 32 hours while raising the temperature sequentially to 30 ° C after freezing at about 9 hours at minus 25 ° C.
  • Example 2 96% by weight of metnanase + 4% by weight of KCl
  • Example 3 Mannanase Concentrate 95% by weight + Maltodextrin 5% by weight Comparative Example 1 Mannanase Concentrate 100% by weight
  • Example 1-3 Residual titer recovery and solubility before and after lyophilization of Example 1-3 and Comparative Example 1 were evaluated by the following method.
  • the titer was 3,5-dinitrosalicylic acid (DNS) method.
  • LBG locust bean gum
  • NDS 3,5-dinitrosalicylic acid
  • LBG locust bean gum
  • the reaction was stopped by the addition of DNS reagent, allowed to stand for 5 minutes in the water to be developed, and the absorbance was measured at 540 nm.
  • Mannose was used as the standard sample to determine the amount of free reducing sugars by comparing the absorbances of color developed under the same conditions.
  • One unit of enzyme titer was defined as the amount of enzyme that produced 1 ⁇ mol of the corresponding reducing sugar from LBG for 1 minute under the above conditions.
  • the solubility was evaluated by placing 5 g of the sample in 100 ml of distilled water, shaking it well, checking the melting time, and visually confirming the presence or absence of precipitation.
  • Example 1 100 ⁇ (bad)
  • Example 2 100 ⁇ (excellent)
  • Example 3 89 ⁇ (excellent) Comparative
  • Example 1 83 ⁇ (normal)
  • the melt time is between 11 and 15 seconds
  • the melt time is between 16 and 20 seconds
  • Example 2 While storing Example 2 and Comparative Example 1 at room temperature and low temperature (4 ° C), the change in titer was evaluated in the same manner as in Experimental Example 1.

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

Abstract

La présente invention concerne une composition de mannanase hydrosoluble lyophilisée possédant une solubilité élevée dans l'eau et une stabilité supérieure, et une composition comportant la composition lyophilisée et un excipient acceptable en pharmacie ou en médecine vétérinaire. La présente invention concerne également un procédé pour la préparation d'un matériau hydrosoluble lyophilisé possédant une solubilité élevée dans l'eau, qui empêche une diminution du titre de celui-ci, et qui permet une préparation facile.
PCT/KR2013/007122 2012-08-08 2013-08-07 Composition de mannanase hydrosoluble, et son procédé de préparation WO2014025203A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2012-0086780 2012-08-08
KR20120086780 2012-08-08

Publications (1)

Publication Number Publication Date
WO2014025203A1 true WO2014025203A1 (fr) 2014-02-13

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PCT/KR2013/007122 WO2014025203A1 (fr) 2012-08-08 2013-08-07 Composition de mannanase hydrosoluble, et son procédé de préparation

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KR (1) KR101507818B1 (fr)
WO (1) WO2014025203A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20010052736A (ko) * 1998-06-10 2001-06-25 피아 스타르 신규한 만난나제
KR20020000013A (ko) * 2000-06-20 2002-01-04 김성린, 조호연 만난아제를 생산하는 신규한 바실러스 속 ｗｌ-1 균주
JP2005505277A (ja) * 2001-09-25 2005-02-24 ビーエーエスエフ アクチェンゲゼルシャフト 水溶性酵素の油状懸濁液の製造方法
KR20070101152A (ko) * 2006-04-10 2007-10-16 크래프트 후우즈 홀딩즈 인코포레이티드 안정화된 효소 조성물
US20080064064A1 (en) * 2006-07-18 2008-03-13 Direvo Biotech Ag Mannanases
JP4801872B2 (ja) * 2000-09-29 2011-10-26 Ｍｅｉｊｉ Ｓｅｉｋａファルマ株式会社 β−グルコシダーゼ活性を有する新規酵素とその用途

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5117154A (en) * 1990-12-31 1992-05-26 Welch Allyn, Inc. Metal halide discharge lamp with improved shank loading factor

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20010052736A (ko) * 1998-06-10 2001-06-25 피아 스타르 신규한 만난나제
KR20020000013A (ko) * 2000-06-20 2002-01-04 김성린, 조호연 만난아제를 생산하는 신규한 바실러스 속 ｗｌ-1 균주
JP4801872B2 (ja) * 2000-09-29 2011-10-26 Ｍｅｉｊｉ Ｓｅｉｋａファルマ株式会社 β−グルコシダーゼ活性を有する新規酵素とその用途
JP2005505277A (ja) * 2001-09-25 2005-02-24 ビーエーエスエフ アクチェンゲゼルシャフト 水溶性酵素の油状懸濁液の製造方法
KR20070101152A (ko) * 2006-04-10 2007-10-16 크래프트 후우즈 홀딩즈 인코포레이티드 안정화된 효소 조성물
US20080064064A1 (en) * 2006-07-18 2008-03-13 Direvo Biotech Ag Mannanases

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KR101507818B1 (ko) 2015-04-07
KR20140020204A (ko) 2014-02-18

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