• 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:
    The invention relates to a process for the production of the alpha-galactoxidase enzyme by culturing yeasts of the Saccharomyces cerevisiae genus in a temperature range from 20 DEG C. to 40 DEG C. The invention also relates to a process for enzymic hydrolysis of raffinose by alpha-galactoxidase from Saccharomyces cerevisiae. Such hydrolysis may take place with the yeast cells being present or also in the presence of enzymic extracts, both as such and enriched. An important advantage of the invention is the high alpha-galactoxidasic activity of the selected microorganisms, together with the absence of any invertasic activity.
    公开号:SU1090262A3
    申请号:SU813288200
    申请日:1981-01-30
    公开日:1984-04-30
    发明作者:Витобелло Винченца；Брандуцци Паоло；Чимини Надя
    申请人:Э.Н.И.Энте Национале Идрокарбури (Фирма)；
    IPC主号:
    专利说明:

    FIELD OF THE INVENTION The invention relates to the microbiological industry, namely the method of curing about. -galacto CeZa, used to hydrolyze raffinose. A method of obtaining ot-gal tosidase is known, according to which c-hapak tosidase is obtained by cultivating the mold of Penicilfium duponti (Tataronryces thermophiXus) in an aqueous medium containing 1 sugar with -1 d-D-ralactopyranosyl link and subsequent separation of the mycelium. Cultivation is carried out in an aqueous suspension obtained by boiling immature bean pods l. It is also known to use the preparation of C-hapaktosvdaza obtained from the mycelium of Mortieretfa vinacca var raffinoseutifizer (ATCC 20034), which does not contain invertase, which provides a decrease in raffinosis | 1 and an increase in sucrose in beet syrup 2j. The closest to the present invention is a method for producing α-galacto zidase, which involves cultivating its producers on a nutrient medium containing sources of carbon, nitrogen and mineral salts in the presence of one of 12 Sugars in 0.5- 1.0% of the mass of the medium at the optimum temperature and pJ process with the subsequent determination of the biomass of cells The et-galactosidase activity on a dry basis is 1,49710 units / g Cz. . . However, the known methods are characterized by an insufficiently high rate of enzyme biosynthesis and its activity. The purpose of the invention is to increase the activity and speed up the process. The supplied chain is achieved tbm according to the method of producing β-lactosidase, which involves cultivating its producers on a nutrient medium containing sources of carbon, nitrogen and mineral salts in the presence of one of 12 Sugars in an amount of 0.5-1.0% by weight of the medium at an optimum The temperature and pH of the process followed by the separation of the cell biomass as producers use Sacchoromyces cerevisial var oleaceus NRKLY 12056 or Sacchoromyces cerevisial var oleaginosus NRRL Y 12057 microorganism strains. The microorganisms used are separated from waste Lok, and processing of olives for squeezing press them respectively assigned numbers: NRRL Y 12056 and NRRL Y 12057. The following are characteristics of culturing these microorganisms and their ziologicheskie formologicheskie properties. A. Characteristics of growing. 1. Solid Wednesday (3 days). Maltese agar. The colonies are oily, cream-colored and shiny. 2.Izdka Wednesday (3 days). Maltese extract. A precipitate forms (in S cerevisial, var ojeaginosus a weak ring is observed) .: B. Morphological properties. 1. Characteristics of vegetative cells. Cells are ellipsoidal, cylindrical and sometimes elongated. 2 Formation of pseudomorphs or true mycelium. The rudimentary pseudomycelium is present under anaerobic conditions. C. Sex characteristics. Vegetative cells are transformed directly into which they contain from one to four spheroidal spores. D. Ziological properties. 1. Use of carbon sources. S.cerevi- S.cereviFerment sial var. s, ial var. Glucose Galactose Maltose Trealose Me11ibioza Rafinoza Assimily Qi Glucose Galactose Maltose Trealose Melibiosis Rafinoza D-Mannitol D-Glucytol Ethanol Glycerol and lactic acid Other carbon compounds are not assimilated. 2. Assimilate cyanogenous compounds. Potassium nitrate is negative, 3. Growth at 37 ° C is positive. For classification, they are guided by the scheme described by the author: I.P. Van der Walt in Book, Jhe Jeast 2 nd ed. T. Lodder. The cultures of the proposed strains can be prepared under aerobic conditions by any known method, for example, by surface dilution or by immersion of the grown crops using a 1-shoe fermenter. The culture medium for the dilution, which can be either solid or liquid, contains a source of assimilated carbon, nitrogen source and mineral salts; glucose, mepibiosis, raffinose and other sugars, glycerin and sodium acetate can be used as carbon sources. Inorganic and organic nitrogenous compounds, such as meat extract, yeast extract, peptone, tryptone, casein hydrolysis products in amino acid media, soybean flour, and ammonium salts can be used as nitrogen sources. In addition, when these microorganisms are diluted on glucose, a enzyme is formed (this enzyme constitutes a constitutive). . The nutrient medium.dil dilution, for example, has the following composition, g / l: Yeast extract, 5-20 Glucose10 Traces H2P04, MgS04, (NH4) jS041 The pH of the nutrient medium for dilution is from ADO 7 and preferably from 5.0 to 5.5, the temperature of the nutrient medium is from 20 to, preferably from 25 to 28 s. Enzyme formation can be accelerated by adding small amounts of melibiose, which is a disaccharide obtained by partial hydrolysis of raffinose. Meliobiose can be injected directly into the growth medium before inoculum, or when the logarithmic growth stage is completed. The induction period can range from 16 to 72 hours and range from 40 to 48 hours. 1 Cells that accumulate during the fermentation process or after completion of the fermentation can be used as such or as a dried powder. In addition, raw or purified extracts of such cells can be used. The cells are separated by any known method, using a crude or purified extract containing the enzyme. . Further as technical, so. and the economic advantage can be achieved by fixing the enzyme by mixing it with macromolecular compounds by forming chemical bonds with the main body or by ion bonding, and also by physically fixing the enzyme or cells. The cells thus obtained are introduced as such or immobile into the reaction medium containing molasses, having a pH ranging from 4 to 7, the temperature of which is from 30 to 60 C. The raffinose contained in the molasses is hydrolyzed to of galactose and sucrose, the yield of the latter being higher. . . Example 1. Nutritional broth is prepared for growth having the following composition of water, g / l: (NH4) jS04.5 MgS047HjO5 NajHPO UH O - 4.6 KHjP043: NaCl0.1, 05 Yeast extract 10 Melibiose - 10 These compounds are dissolved in deionized water and acidified with hydrochloric acid to a pH of 3.5. The nutrient solution thus prepared is distributed into 500 ml wide-necked Erlennayer flasks (100 ml nutrient solution per flask, sterilizing the flasks at 116 ° C for 30 minutes). The contents of the flasks are inoculated with a 1 MP culture of the Saccharomyces cerevisiat, var oteaceus strain in 250 ml coibas containing 50 ml of the indicated nutrient solution, the microorganisms are grown for 16 hours with stirring with 1 stirrer rotating at 180 rpm. The fermentation flasks are placed in incubation conditions where stirring takes place (at a rotation speed of 180 rpm) at 25 ° C. After 40 hours from the moment of inoculation, culture cells of the nutrient solution (broth culture) are harvested by centrifugation and washed with phosphate buffer (0). , 1 M, pH 5.6). From 100 ml of the feed solution (broth), 0.55 g of dried cells is obtained. Wet cells, each of 100 ml of the nutritional solution, are re-suspended in 100 mp of phosphate buffer solution (0.1 mph, pH 5.6) and the enzymatic activity is determined. One gram of dried cells contains approximately 110 enzyme units. Enzyme activity is measured as follows. To 1 ml of the cell suspension containing buffer, add 4 mp of phosphate buffer solution (O, 1 M; pH 5.6) and a few drops of toluene to break the cell septa. After 15 minutes of incubation with stirring at 40 ° C, 10 ml of a 1% aqueous solution (w / v) of the bios stream in a phosphate buffer solution (0.1 M, pH 5.6) are added to the suspension. The reaction is incubated for 2 hours in an agitated water bath and is stopped by boiling for 15 minutes of samples taken from the reaction mixture. The glucose concentration obtained by hydrolysis of the melibiose during this reaction is determined by a calorimetric method using a I-Iannheim GrnB COD-Period calorimeter. The optical density of the colored samples is measured at room temperature in the Coleman 55 spectrophotometer, the boat optical path 0 , 1 dm with a long wavelength of 436 microns. If the amount of enzyme that forms 1 µg of glucose for 2 hours under the above assay conditions is taken per unit of quantity, then the amounts of enzyme per 1 hour of dried cells can be calculated using the following liu-llZluhHSi JAlO dried klestart. current, g 2 is the optical density of the sample taken after 2 hours; optical density of the sample, taken as zero (O h); optical density STANDYRG mill. Dart glucose solution, which contains 18.2 μg of glucose per ml; C is the number of grams of dried cells in 100 ml of nutrient solution. Example 2. A nutrient solution for dilution is prepared having the following composition of water, g / l: (NH4) 2S04 MgS04 KH PO4 Nad Yeast extract Glucose The compounds listed above are dissolved in deionized water, and then the pH is adjusted to 5.3 with hydrochloric acid. The cultures in the indicated nutrient solution of the Saccharomyces cerevisiat var oteaceus strain, prepared as described in Example 1, are incubated by kneading (rotation speed 180 rpm) and temperature. After 39 hours from the moment of inoculation, the nutrient broth yupetas are collected by centrifugation and washed with a phosphate solution (0.1 moles pH 5.6). From 100 ml of nutrient solution for scattering, 0.546 g of dried cells is obtained. Cells extracted from 100 ml of nutrient broth are re-suspended in 100 ml of phosphate buffer solution (O, 1 M} pH 5.6) and their enzymatic activity is determined: 1 g of dried cells contains 9.410 enzyme units. Example 3 A nutrient medium is prepared having the following composition: 0 g / l; (No.4) 2804 MgSO-yHaO NajHPQ 12H20 KHjPO Yeast extract Glucose10 Melibios1 The compounds listed above are dissolved in deionized water and the pH is adjusted to 5.3 with hydrochloric acid. Kuytury Saccharomyces cerevisiat strain, var. oleaceus, prepared as described in Example 1, is incubated with circular rotation (180 rpm) under stirring. After 43 hours from the moment of inoculum, the broth cultures are harvested by centrifugation and washed with phosphate-bm buffer solution (0.1 M). pH 5.6). 0.594 g of dried cells are obtained from 100 MP of the shale culture. Cells isolated from 100 ml of nutrient solution (bulba) were suspended in 100 ml of phosphate buffer solution (O, 1 M; pH 5.6), and the enzyme activity was determined: 1 g of dried cells contained 1.6 -10 enzyme units. PRI IMER 4. Prepare a nutrient medium having the following composition, g / l: (NH4) jS04 NaHPO KHjP04 Yeast extract Glucose Melibiosis The compounds listed above are dissolved with deionized water, and the pH is adjusted to 5.3 by adding saline acid. Saccharomyces cerevisiati ,, var oleaceus cultures in such a nutrient solution for dilution, prepared as described in Example 1, are incubated by crossing with circular rotation (at 180 rpm) at 29 s. After 48 hours from the moment of inoculation of the nutrient solution, the cells are harvested by centrifugation and washed with phosphate buffer solution (0.1 M; pH value 5.6). 0.365 g of dried cells are obtained per 100 ml of nutrient solution for dilution. 6 MP of nutrient solution (broth) are introduced in 50 ml of Brix molasses (at 35 C) i containing 1.6% raffinose (based on the total amount of solid), and the pH of the solution is adjusted to 5.2 with sulfuric acid . The treatment is carried out at 40 ° C for 16 hours while stirring. The concentration of galactose, the product of raffinose hydrolysis during this reaction, is determined by the ultraviolet analysis method Lactose / Galactose, developed by Boehringer MannheimGmbH. With the above mentioned; conditions, 153 micromole of galactose is obtained, which is equivalent to the hydrolysis of 30% of all the raffinose present.
    权利要求:
    Claims (1)
    [1]
    (54I57) METHOD FOR OB-GALACTOSIDASE PRODUCTION, which provides for the cultivation of its producers on a nutrient medium containing carbon, nitrogen and mineral salts in the presence of one of 12 sugars in an amount of 0.5-1.0% by weight of the medium at the optimum temperature and pH of the process followed by separation of biomass kpe, current, characterized in that, in order to increase activity and accelerate the process, microorganisms Sacchoromyces cerevisiat var oieaceus NRRLY 12056 or Sacchoromyces p cerevisiab var__oteaginosus NRRLY 12057 SU, .., 1090262 * are used as producers.
    >
    - 1 1090262
    The invention relates to the microbiological industry, and in particular to a method for producing cC -galactoei Dase used for hydrolysis of raffinose.
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    同族专利:
    公开号 | 公开日
    DE3049308A1|1981-09-24|
    US4450238A|1984-05-22|
    IE802742L|1981-08-01|
    YU5881A|1983-10-31|
    YU42232B|1988-06-30|
    NO162347B|1989-09-04|
    GB2068972A|1981-08-19|
    FR2481315A1|1981-10-30|
    NO810306L|1981-08-03|
    CA1163939A|1984-03-20|
    JPS56121485A|1981-09-24|
    BE887293A|1981-07-29|
    PL127026B1|1983-09-30|
    SE8100741L|1981-08-02|
    IT8019618D0|1980-02-01|
    DD157564A5|1982-11-17|
    FI70596B|1986-06-06|
    SE453836B|1988-03-07|
    BG42525A3|1987-12-15|
    FI804090L|1981-08-02|
    HU183303B|1984-04-28|
    PL229117A1|1981-09-04|
    FI70596C|1986-09-24|
    RO80915A|1983-02-01|
    DK557080A|1981-08-02|
    NL8100476A|1981-09-01|
    IT1130242B|1986-06-11|
    IE51009B1|1986-09-03|
    US4376167A|1983-03-08|
    FR2481315B1|1984-10-12|
    CS248014B2|1987-01-15|
    LU83094A1|1981-09-10|
    JPH0253029B2|1990-11-15|
    DK153410B|1988-07-11|
    TR21577A|1984-10-16|
    DE3049308C2|1982-11-11|
    GB2068972B|1983-04-13|
    DK153410C|1989-06-05|
    NO162347C|1989-12-13|
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    US4643901A|1983-06-10|1987-02-17|Universal Foods Corporation|Yeast strains, method of production and use in baking|
    JPH0154036B2|1985-04-09|1989-11-16|Orientaru Kobo Kogyo Kk|
    US5055401A|1987-04-10|1991-10-08|Alko Ltd.|Construction of new α-galactosidase producing yeast strains and the industrial application of these strains|
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    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    IT19618/80A|IT1130242B|1980-02-01|1980-02-01|PROCEDURE FOR THE PRODUCTION OF THE ALPHA-GALACTOSIDASE ENZYME AND FOR THE HYDROLYSIS OF THE REFINOSE BY USING THE ENZYME ITSELF|
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