• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    A method for producing cholesterol oxidase, involving the cultivation of a producing microorganism, including the genus Streptomycetaceae, on a nutrient medium containing yeast extract, N "Heralized salts, MgSO 71120, FeSO47HjO, and water, followed by release of the enzyme from the culture fluid and from the human organism, from the culture fluid and from the human body, and from the human body, and from the human body. in order to reduce the cost of the process, Streptcmq ces griseofuscus DSM 40191 or StreptOTayces higroscopicus DSM 40771 and HI and Streptoroyces acidonyceti cus SMM 40798 aprof a method, using a patch, or a Streptocyscenecetaceae, is used to make the process cheaper. 1, that is, with the fact that the cultivation is carried out on a medium that also contains starch, peptone, and mineral salts of CaCO, SHOZ, NaCl, which are additionally introduced with the following ratio of components, wt.%; Soluble i CO starch10-30 Peptone.2-10 Yeast extract 2-10, 5-3 ClNRO 0.2-2 t SO47HAO0.5-3 Had0.2-2 FeS047H, iO0.01-0.05 Bona Remaining ro o: yes SL OS
    公开号:SU1026656A3
    申请号:SU802930501
    申请日:1980-06-04
    公开日:1983-06-30
    发明作者:Гауль Хельмгард；Шаволь Георг；Зайдель Ханс；Бокамп Клаус
    申请人:Берингер Маннхайм Гмбх (Фирма)；
    IPC主号:
    专利说明:

    The invention relates to microbial industry and relates to a method for producing cholesterol-Dase from microorganisms. Various types of microorganisms are known - producers of cholesterol oxidase and methods for producing this enzyme. The closest in technical essence and the achieved result to the proposed is a method for cholesterol oxidase, providing for the cultivation of the producing microorganism, including the genus Streptomycetaceae, on a nutrient medium containing yeast extract, mineral salts, MgSO47H2O, FeSO4 VHGO And water , with the subsequent release of the enzyme from the culture fluid and / or from the cells of Š. The disadvantage of this method is the need to add inducers to increase the activity of the enzyme, which is small and reaches a small amount The order of 100 and / l. The purpose of the invention is to reduce the cost of the process. This goal is achieved in that according to the method for producing cholesterol oxidase, which involves cultivating a producing microorganism, including the genus Streptomycetaceae, on a nutrient medium containing yeast extract, mineral salts K2HPO4, FeSO THAO, and VOLU, / or from cells, Strep myces grlseofuscus DSM 40191 or Streptomyces higroscopicus DSM 407 or Streptomyces acidomyceticus DSM 40798 and / or Arthrobacter parafneus DSM 312 are used as the producing microorganism of the genus Streptoraycetaceae. In addition, the cultivators lead on the medium, which additionally contains soluble starch, peptone and mineral salts CaCOj, CLOSE, NaCl in the following ratio of components, wt.%: Soluble starch Peptone Yeast extract 1-3 1-3-3-3. 2 KjHPOi 0.5-3 - - 0.2-2 Fes647H O 0.01-0.05 Remain Water The essence of the invention is to use cholesterol oxidase producing strains capable of producing an enzyme without an inducer on a nutrient medium balanced by growth factors. . Below are the characteristics of the microorganism strains used according to the invention. Streptomyces griseofuscus BMTi 1616, DSM 40191, ATCC 23916 General characteristic: gram-positive, aerobic organism that forms aerial mycelium. Coloring: colonies of airborne mycelium with growth on yeast and malt agar, oatmeal agar, starch agar with mineral salts and glycerol-aspartic agar can be a series of red or gray tones (cinnamon-brown-gray.) no specific pigments are visible to the back of the colonies. Soluble pigments: no formation of melanoid pigments in peptone-yeast agar with iron, tyrosinovsim agar or tryptone-yeast nutrient solution, as well as the formation of pigments in yeast-malt agar, oat flakes, starch agar with mineral salts or glycerol-aspara-agar, asparagus, oatmeal agar, glycerol-asparate agar or glycerol-asparagus agar, oatmeal agar, glycerol-agar with oleaginous agar or tryptone-yeast nutrient solution. . Spore chain morphologists: spiral sections. Mature, narrowly spiral chains of spores contain 10-50 spores, the surface of the spores is smooth. Using C-sources. The use of D-glucose, L-arabinose, D-xylose, D-mannitol and D-fructoe. No growth or scant growth on sucrose, inositol, rhamnose, raffinose and lactose. Samples of aliphatic fatty acids: representatives of the Streptomycetaceae family are characterized by the predominant presence of iso / anteiso-CI-15 and iso / anteoiso-C-17. missing. Streptomyces nigroscopicus BMTU2093, DSM 40771, ATCC 10976 General characteristic: gram-positive, aerobic organism that forms aerial mycelium. Coloring: colonies of aerial mycelium with growth on yeast-malt agar, oatmeal agar and starchy agar with mineral salts may be a series of red and gray tones (cinnamon-brown-gray. On the back side of the colonies
    no specific pigments are visible.
    Soluble pigments: there is no formation of melanoid pigments in peptone-yeast agar, as well as the formation of pigment in yeast-malt agar, starch agar with mineral salts and oatmeal agar.
    Spore chain morphology: spiral plots. Chains of spores in narrow, x closed x spirals, often with legs, consist of 10-50 spores, the surface of the spores is smooth.
    Using C-sources.
    Use of D-glucose, P-xylose, B fructose and D-mannitol. No growth or very poor growth on L-arabinose, rhamnose, sucrose, raffinose, inositol and lactose.
    Samples of aliphatic acids: representatives of the Streptmycetaceae family are characterized by the predominant presence of iso / anteiso-C-15 and iso / anteiso-C-17-aliphatic acids.
    The cell wall contains LL-diaminopimelic acid typical of Streptomyces. DL-diaminopimelic or hydroxy-O-diaminopimelic acid is absent.
    Streptomyce, s acidomyceticus
    VMTi 1.873, DSM 40798,
    ATCC 11611
    General characteristics: gram-positive, aerobic organism, which forms aerial mycelium.
    Coloring: colonies of aerial mycelium with growth on yeast-malt agar, oatmeal agar and starch agar with mineral salts may be a series of red (gray) tones (pink, brown korytsevy). On the back side of the colonies, growth on yeast-malt agar shows a yellow-brown pigment, on the oatmeal agar — red-brown color and starch agar with mineral salts — black.
    Soluble pigments: the formation of melanoid pigments in pepton-yeast agar with iron. Other pigments of the type mentioned are not formed.
    Spore chain morphology: Retinaculiaperti patches with many strands of spore chain growing on yeast-malt agar. On agar from oat flakes and starch agar with mineral salts, the formation of hooks, loops and short irregular spirals. The surface of the dispute is smooth.
     Using C-sources.
    Using D-glucose., L-apabinose, D-fructose and sucrose. No growth on rhamnose, lactose, raffinose, D-mannitol and inositol.
    For the members of the family of Streptomycetaceae, iso / antyoiso-C-15 and iso / anteiso-C-17 samples of aliphatic acids are characteristic.
    The cell wall contains LL-diaminopimelic acid typical of Streptomyces. DL-diaminopimelic acid or oxy-DTJ-diaminopimelic acid are absent.
    Arthrobacter paraffineus. BMP 501, DSM 312.
    Morphology: from short to medium length, cinnamic bars
    with typical snapping off. There is no clearly pronounced cycle of development, the cells in all phases of development are columnar. Cells are gram-positive and immobile. No
    education dispute. The colonies on the HD-arara are white-cream colored and shiny} there is no pigment formation.
    Physiological properties.
    Reference to oxygen - a pronounced aerobic formation of glucose and lactose acids does not occur; catalyzate is formed; oxidase not detected} nitrate reduction - positive} splitting of starch, gelatins and casein negative; tributurin splitting is weakly positive; urease is formed; the formation of indole and HjS is negative; using
    citrate - positive; salt tolerance - calculation in case of 5% NaCl, no growth at 10% NaCl.
    Peptidoglyc "cell wall contains a meso-diamine-pymeline kislot.
    Example. Streptomyces grlseofuscus DSM 40191 (equivalent to ATCC 23916 and iFO 12870) is cultivated in the medium of the following composition,
    / Soluble
    starch.
    Yeast Extract
    (Difco) 4
    Peptone (m clear) 5
    Calcium carbonate 3
    Potassium Nitrate 1
    C2HRO Diphosphate 0.5
    MgSO47H, zO1.03
    Sodium chloride, 5
    FeSO47H2O.0,02
    The strain is cultured for 2 days in 10 ml of medium in a 100 ml Erleimeier flask, then transferred to 40 ml of the same
    the medium in an Erlenmeyer flask (3 days), a sample is taken and the activity of the cholesterol dose is checked in the separated culture solution and in the ex-raw path. Raw extract is obtained by centrifuging 5 ml of a culture solution, washing the biomass with 0.05 M phosphate buffer pH 7, using a toric suspension in 5 ml of the same buffer and distributing through the addition of O, 2 ml of a 10% aqueous solution of Triton-X-100. For 10 minutes at room temperature, the mixture is centrifuged and the separated culture solution is obtained in the form of an extract of a salt.
    To determine activity, cholestenone formation is measured by increasing extinction (extrinsing) at 240 mm, at pH 7, in 0.5 mol / l phosphate buffer. Each time the blank value is determined by checking the value without adding cholesten.
    The following U / L values were obtained:
    General - culture solution 5741
    Raw extract,
    derived from
    this solution 1118
    Separated culture solution 4623
    EXAMPLE 2 The method of Example 1 is repeated using Streptomyces higroscopicus DSM 4077 (corresponding to ATCC 10976).
    The determinations were carried out after 3 and 4 days IB during the culture.
    The activity values are given in the table ..
    In the case of cultivation in the presence of 2% cholesterol as
    inductor activity gain is less than half.
    EXAMPLE 3 The method of example 1 is repeated using Streptomyces acidomyceticus DSM 40789 ((corresponds to ATCC 11611 and iFO 3125.
    Results with cultivation times of 3 and 4 days are shown in the table.
    When culture is carried out in the presence of 2% cholesterol as an inducer, the increase in activity increases by 5-0%.
    EXAMPLE 4. The method of example 1 is repeated using Arthrobacten paraffineus DS 312 (it corresponds to ATCC 15591), but provided that 3% cholesterol is added to the medium as a suspension in a yeast extract (total amount 10 g / l). After four days of cultivation, a U / L value of 4470 was found in the raw extract. A significant activity was also found in the culture solution after separation.
    The use of the invention makes it possible to significantly reduce the cost of production method due to the achievement of higher cholesterol oxidase activity values. In this case, a particular advantage lies in the possibility of eliminating the inductor cholesterol oxidase, which leads not only to an additional cost reduction of the method, but also to a significant increase in the reproducibility of the method.
    权利要求:
    Claims (2)
    [1]
    METHOD FOR PRODUCING cholesterol oxidase, comprising culturing the producing microorganism, including the genus Streptomycetaceae, in a nutrient medium containing yeast extract, νμηθralnye salt Q 2 HPO 4, MgS0 4 '7H 2 O, FeSO 4 * 7H 2 O and water, followed by isolation enzyme from the culture fluid and / or cells, characterized in that, in order to reduce the cost of the process, Streptontyces griseofuscus DSM 40191 or Streptontyces higroscopicus DSM 40771 or Streptontyces acidomyceticus DSM. 40798 and / or Arthrobusactus are used as a producing microorganism from the genus Streptomycetaceae DSM 312.
    [2]
    2. The method no. 1, about t l and h a torn and th with the fact that the cultivation is carried out on an environment in which soluble starch, mite and mineral salts of CaCO 4 , ZhO 3 , NaCl are additionally introduced in the following ratio of components , weight.%:
    Peptone Soluble Starch Yeast extract CaCO,
    SKC
    K 2 YRO 4
    IDZOD-TH ^ O NaCl
    FeSO 4 · 7H 2 O
    Water
    10-302-10 § 2-10 1-30.5-30.2-2 R 0.5-30.2-2 § 0.01-0.05 Rest about.
    u <&>
    SL>
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    同族专利:
    公开号 | 公开日
    AT976T|1982-05-15|
    US4334023A|1982-06-08|
    JPS565094A|1981-01-20|
    CA1145278A|1983-04-26|
    DE3060364D1|1982-06-24|
    EP0021311A1|1981-01-07|
    DE2924875A1|1981-01-29|
    JPS6012029B2|1985-03-29|
    EP0021311B1|1982-05-05|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    FR2047147A5|1969-05-06|1971-03-12|Kyowa Hakko Kogyo Kk|
    US4186251A|1973-03-01|1980-01-29|Miles Laboratories, Inc.|Composition and method for determination of cholesterol|
    JPS5721981B2|1974-12-10|1982-05-11|
    US4035237A|1975-11-07|1977-07-12|Eastman Kodak Company|Method for the preparation of cholesterol oxidase|
    US4052263A|1975-12-11|1977-10-04|Eastman Kodak Company|Production of cholesterol esterase using Nocardia cholesterolicum|
    JPS6120268B2|1977-12-02|1986-05-21|Banyu Pharma Co Ltd|DE3046241A1|1980-12-08|1982-07-15|Boehringer Mannheim Gmbh, 6800 Mannheim|METHOD AND REAGENT FOR DETERMINING CHOLESTERIN|
    IT1140326B|1981-12-11|1986-09-24|Anic Spa|METHOD FOR THE PRODUCTION OF CELLS CONTAINING CHOLESTEROL OXIDASE|
    JPH0542270B2|1984-07-23|1993-06-28|Asahi Chemical Ind|
    US5238816A|1989-07-24|1993-08-24|Asahi Kasei Kogyo Kabushiki Kaisha|Omega carboxyalcohol oxidase enzyme|
    JP2786679B2|1989-07-24|1998-08-13|旭化成工業株式会社|Novel ω-carboxy alcohol oxidase|
    US5206148A|1989-07-24|1993-04-27|Asahi Kasei Kogyo Kabushiki Kaisha|Method for assaying aliphatic alcohol, aliphatic aldehyde or ω-carboxylic acid derivatives thereof|
    KR100464674B1|2002-08-27|2005-01-03|학교법인 계명기독학원|A method for producing cholesterol oxidase from Streptomyces sp.|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    DE19792924875|DE2924875A1|1979-06-20|1979-06-20|METHOD FOR OBTAINING CHOLESTERIN OXIDASE|
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