• 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 bacterial cell aggregate having increased particle hardness is produced by contacting a mass of bacterial cells with a cross-linking reaction product of (1) glutaraldehyde, cyanuric halide or combinations thereof and (2) a specific cationic polymer obtained by polymerization of an epihalohydrin and an alkylene polyamine, recovering the resulting aggregate and drying the aggregate.
    公开号:SU929014A3
    申请号:SU792745296
    申请日:1979-03-26
    公开日:1982-05-15
    发明作者:Бальтзер Борглум Джеральд
    申请人:Майлз Лабораториз Инк (Фирма)；
    IPC主号:
    专利说明:

    39
    Streptom ces olivaceus thermal cells, which implies that the mass of these bacterial cells is kept in contact with a crosslinking agent in an alkaline medium for 0 hours, and the product of the crosslinking agent is 12–65% by weight of a compound selected from groups including glutaraldehyde, cyanuric acid trichloride, and their combination, and; 88-3.9 weight D of a water-soluble cationic polymer — a product of the polymerization of epichlorohydrin with an alkylene polyamine of the formula (2NRH, 2;
    where R is lower Cp-Ck is alkylene, and R and RQ are lower, are alkyls,
    the specified crosslinker is used in an amount of 6.5 to 55 wt.% of the dry weight of the cells, and the process is carried out at pH 8-9 and temperature, followed by isolation of the target product.
    After isolation, the aggregate is dried.
    As a crosslinking agent, use was made of a product of the weight% glutaraldehyde and + 2.9 weight%. water-soluble cationic polymer in the amount of 17.5 weight. in terms of the dry weight of the cells.
    As a crosslinking agent, the interaction product is also used, 9 wt. glutaraldehyde and 3.6 wt. cyanuric acid trichloride, taken as glutaraldehyde, and 1.5% by weight of a water-soluble cationic polymer in an amount of 18.2% by weight, calculated on the dry weight
    cells.
    I
    Bacterial cells containing active glucose isomerase, which can be used in the implementation of the proposed method, can be obtained in accordance with known technology. Enzyme-containing. cells are obtained by growing in the submerged state in aerobic fields of Streptomyces olivaceus culture (usually strain 3583) or their mutants in medium containing the appropriate nutrients in a known manner. The resulting bacterial cells are separated from the fermentation medium by filtration and centrifugation -. Components that are used in the implementation of such a method are readily available. A special epihalohydrin-polyamine polymer, which is used in the implementation of the proposed method, is a technically available product produced under the Betz Laboratories, Inc., BETC 1180 trademark, Trek, Pencil pieces. The molecular weight of the BETC 1180 product is less than 1,000,000, its molecules contain approximately 0.288 mmol, amino groups per gram of solution (according to the ninhydrin test), and the product itself goes on sale in the form of a solution containing 30 wt. dry matter in terms of the total weight of the solution. This product is known and characterized as a water-soluble cationic polymer obtained by the polymerization of chlorohydrin with alkylene polyamine, corresponding to the general formula Rj RqNRNHn, where R is lower alkylene containing from 2 to 6 carbon atoms, and each of the R and R symbols denotes a lower alkyl containing from 1 to 6 carbon atoms, the molecular ratio between chlorohydrin and polyamine being in the range from 0.60: 1 to 2.7: 1) during this polymerization, the reaction of an alkylene polyamine from 50-90 epi is provided of the amount to be polymerized, the continuation of this reaction until the viscosity of the reaction medium is almost homogeneous and the reaction of the remaining part of epichlorohydrin in gradually increasing amounts, resulting in a cationic polymer, the polymerization temperature ranges from 60 to 120 WITH. In a further part of this description, this material is called a polyamine polymer.
    As a cross-linking reaction product, necessary for the practical implementation of the proposed invention, in the process of producing an aggregate of bacterial cells, one of three possible compositions can be used. You can carry out the reaction of the polyamine polymer with glutaraldehyde or cyanuric anhydride, or with glutaraldehyde or with cyanuric acid trichloride at the same time. Glutaraldehyde trichloride cyanuric acid, and their combination, collectively referred to as glutaraldehyde, is used to react with a polyamine polymer. With a pH in the range from 6 to 10 and a temperature from O to ZO-C for from 0.5 to 2.5 hours. In general, the cross-linking reaction product contains from 12 to b5.1 weight .. glutaraldehyde and from about 3 9 to 88 weight. polyamine polymer based on the total weight of the active components. Preferably, the crosslinking agent resulting from the reaction between glutaraldehyde and the polyamine polymer is obtained at pH values from 8 to 9, temperatures of 18 and for about 0.3 hours. Glutaraldehyde should be present in a molar ratio of at least 1: 1 amino groups in polyamine polymer molecules, which prevents undesired cross-linking of the polyamine polymer with glutaraldehyde. The reaction between cyanuric acid trichloride alone and a polyamine polymer is preferably carried out at pH values ranging from 8 to 9 and temperatures ranging from 0 to 2 to 2 hours. Cyanuric acid trichloride should be present in a molar ratio of less than measure 1: 1 amino groups in polyamine polymer molecules, which helps prevent undesirable transverse crosslinking of polyamine polymer molecules by cyanuric acid halide. In the cyanuric acid trichloride molecule, they have three halogen reactive sites. One of these sections reacts at a temperature of 0 ° C or higher. After completion of the reaction in this first area at a temperature of from 30 to 50 ° C, the second section reacts, and the third section reacts at a temperature of 90 to. It is desirable to initially use for the reactions with the polyamine polymer only the first sites of the molecules of cyanuric acid trichloride. The formed cross-linking reaction product is used in reaction with bacterial cells, after which it is heated to a high temperature during the drying process, the remaining active sites of cyanuric acid react with the polyamine polymer molecules to provide additional cross-linking of the bacterial cell aggregate. The reaction between the polyamine polymer and the combination of glutaraldehyde with cyanuric acid trichloride is carried out in several stages. First, cyanuric acid trichloride is reacted with a polyamine polymer at a pH of 8 to 9 and a temperature of 0 to IDC for 1 to 2 hours. In a preferred embodiment, the starting reagents for carrying out this step should be used in a molecular ratio of 1 mole of cyanuric trichloride. acids per 2 mol of amino groups in polyamine polymer molecules. An excess amount of glutaraldehyde is then added and the reaction is carried out under the same pH and temperature conditions for 0.5 hours. The crosslinking reaction product used is not a cationic polyelectrolyte, since the amino groups in the polyamine polymer molecules that originally reported this cationic polyelectrolyte characteristics, by this time already reacted with glutaric aldehyde and / or cyanuric acid trichloride, resulting in the absence of free groups. Bacterial cell aggregates are obtained by contacting the mass of bacterial cells with a cross-linking reactive agent obtained in accordance with the proposed one, with a pH value of from 8 to 9 and a temperature of from 5 TO 30-C for 0.5 to 1.5 hours. Crosslinking the reaction agent is used in such amounts and concentrations that the bacterial cells come into contact with approximately 6.555 wt. active components of the crosslinking reaction product, calculated on the dry weight of the cells.
    After the completion of this reaction, the bacterial cell aggregate of the preferred embodiment is usually extruded or molded to give the product the desired shape and dried at a temperature of approximately several hours. The resulting dry aggregate can be stored until needed for the enzymatic process. In this case, the dried aggregate is rehydrated and prepared for use.
    The obtained samples are subjected to a hardness test to evaluate their suitability for use in reactors. This hardness is expressed in resistance to compression of particles of the bacterial cell aggregate. The tests were carried out using an Instron tensile testing machine in combination with a block of compressive load NO.CCI in accordance with the previously described method. This device is manufactured by Instron Corporation Canton, Massachusetts.
    The following is the test procedure for determining the hardness after rehydration.
    The rehydration solution is prepared by mixing 9.68 g of cobalt hydrochloride hexahydrate, 28.0 g of magnesium hydroxide and 5b, 0 g of anhydrous citric acid in 600 ml of distilled water at ° C. This mixture is further stirred and heated to 60 ° C with the dissolution of all salts. The mixture is then cooled to 25 ° C and adjusted to pH 8.5 by the addition of sodium hydroxide. Next, the solution is filtered and its volume is adjusted to 1.0 L by adding distilled water. A 2.5 m dose of the specified solution is mixed with 130 ml of water, 70.3 g of dextrose, 2.228 g of tris (oxymethyl) aminomethane and the pH is adjusted to 8.55 by adding sodium hydroxide at 25 ° C. The volume is then made up to 200 ml by the addition of distilled water.
    5 particles of the dried bacterial cell aggregate were converted using 2.5 ml of this solution for rehydration in a Petri dish and kept at 60 ° C in a water bath for 1 hour, and then left to stand at room temperature until cooled290U8
    deni Thereafter, the particles are removed from the solution, the excess surface fluid is also removed from it, and tests are carried out using
    5 Instron bursting machine. Before using this instrument for measurement, it is heated for at least 30 minutes together with the mounted device.
    10 block compressive load. The slider's movement speed is set at 5.1 mm / min and the chart movement speed is 51 mm / min. Translation t handle Return to half
    15 stroke, bringing it to the level of 0.9 by 5 mm for the surface of the load block. Set the handle to the length of the test portion of the sample so as to clean the edge of the sample cup. Ask on the self-recording device the range of the entire scale. This is typically 5 kg. Standardize the recorder to read the zero number with the sample cup on the load block and 1 pound (0.5 kg), with the cup and 1 pound (0.45 kg) of the standard weight on the load block. Place a single particle after reuse.
    30 hydration per dish for a sample that is centered with a slider. Manually lower the slider onto the upper part of the particle and press the Move button. On the recorder, read the force in pounds (kilograms) at a distance of 0.03 inch 0.72 mm from the point at which the slider touches the particle. Thus, hardness is expressed in effort (in pounds or kilograms.),
    4Q required to compress the particle by 0.03 inch (0.762 mm). This experiment was repeated on several particles, and the results were averaged.
    D5 Example 1. A cross-linking reaction product is obtained by adding 2.25 g of BETZ 1180 solution containing 0.675 g of active material and 0 mmol of amino groups in 100 ml of 1.25%
    (weight per volume) of a solution of glutaraldehyde containing 13.2A mmol of active material, with a pH value of 9. This mixture is stirred for 30 minutes, resulting in a solution of deep yellow color. The final product is obtained from a reaction mixture that contains 64.9% by weight of glutaraldehyde and 35-1% by weight of polyamine polymer, calculated on the total weight of glutaraldehyde and polyamine polymer.
    The mutant culture of Streptomyces olivaceus NRRh 3583 is grown in a fermentor with mixing and aeration, which contained the appropriate nutrient medium. The pH of the fermentor nutrient medium, containing the mass of bacterial cells, is adjusted to 8.2 by adding appropriate buffer materials. A portion of the prepared solution is added to a portion of the nutrient medium of the fermenter in an amount that is necessary to achieve an equivalent of k wt.% Glutaraldehyde (21.6 wt.% Of the total amount of the reaction product), calculated on the dry weight of the bacterial cells. After a 30-minute reaction time at 25 ° C and a pH of 8.2, the treated medium is filtered. Then the filter cake is extruded through a syringe with a hole diameter of 2.2 mm. The extruded filaments thus molded are cut into 30 mm lengths and dried overnight at 65 ° C in an oven with forced circulation of hot air flow. A similar portion of the fermentor nutrient medium is treated with only one glutaraldehyde at a concentration of 14 wt.%
    290Н10
    in terms of the dry weight of the bacterial cell mass. Then the treated cells are filtered, extruded and dried in the same 5 way to obtain control particles. Both the control product and the product obtained according to the invention are subjected to hardness tests. The hardness of the control sample of 0.36 kg
    10 whereas the product treated in accordance with the invention, has a high hardness of 1.27 kg.
    Example 2. Portions of the nutrient medium of the fermenter with the microorganism Streptamyces olivaceus, similar to that mentioned in Example 1, are treated with only one 20 glutaraldehyde (control experiment) and various combinations of cross-linking agents at a pH of 9. and temperature. Different crosslinker agents are prepared in accordance with the one described in Example 1 above using different amounts of glutaraldehyde and a polyamine polymer. Then, the treated bacterial cells are filtered, extruded, dried and subjected to a hardness test.
    Table 1 shows the reaction mixture for the preparation of the composition,
    35 of which receive cross-linking reaction product.
    Table 1
    The use of a glutaraldehyde-based crosslinking agent and a polyamine polymer makes it possible to substantially increase the hardness compared with that obtained using only glutaraldehyde in accordance with the known method. Example 3- A cross-linking reaction agent is prepared by dissolving 0.188 g of cyanuric acid trichloride (0, mmol) in 10 ml of acetone, and then this solution is added to 70 ml with ice-cooled water with stirring to give a finely dispersed precipitate. A ratio of 2.25 A g of BETZ 1180 solution (containing 0.675 g of active material and 0.6A8 mmol of amino groups) is diluted by adding 20 ml of water and the mixture is added to a suspension of 1: cyanuric acid trichloride. The final mixture is stirred and kept at a pH of 9 and a temperature of 0-5 ° C for 1-2 hours, then diluted to a volume of 100 ml. Cyanuric acid trichloride is dissolved, indicating reaction with a polyamine. This reaction product is obtained from the reaction mixture, which contains 21.8 wt. cyanuric acid trichloride as a polyamine colimer. A part of the nutrient medium, similarly indicated in example 1, from a ferptor with the microorganism Streptomyces olivaceus is mixed with a part of the indicated reaction product with a concentration of 32.0 wt. o, 2 (weight per volume) of an aqueous solution of sodium bicarbonate is added to maintain the pH at 9- After 1, 5 hours has elapsed at pH 9 and the temperature is 25 ° C, the treated nutrient medium Do filter, extrude and dry. Another part of the nutrient medium of the fermenter is treated according to the method for 0.5 h. In the initial stage, sodium bicarbonate is not added, but the filtered cells are washed with sodium bicarbonate solution with a concentration of 1 wt. when the value is not pH 9 before extrusion and you are dried. To obtain the control product, glutaraldehyde is added to a separate portion of the nutrient medium from the fermenter at a concentration of 1A weight. in terms of the dry weight of the bacterial cell mass. The resulting mass is treated with glutaraldehyde for 30 min 12 at a pH of 8.2 and temperature, followed by filtration, extrusion and drying. The hardness of the control product is 1 kg, whereas as a result of treatment with the specified crosslinking product for 0.5 h, a product with a hardness of 1.73 kg is obtained, and as a result of treatment with the same 1.5 h, the hardness reaches 1.82 kg. Example 4. A cross-linking reaction product is obtained by adding 5 g of BETZ 1180 solution (containing 1.35 g of active material and 1.29 mmol of amino groups) in 0.118 g (0, mmol) of finely divided cyanuric acid trichloride in ice-cooled water. The pH is adjusted to 9 and maintained at this level in an ice bath (at 0 ° C) for 2 hours. Then 1.25 g (13, mmol) of glutaraldehyde is added at pH 9 and a low temperature is maintained for Approximately 0.5 h. The mixture becomes dark yellow. The reaction product is obtained from the reaction mixture which contains 3 wt. cyanuric acid trichloride, A6.0% by weight glutaraldehyde (50.3% by weight glutaraldehyde in total) and 49.7 weight% polyamine polymer in the form of glutaraldehyde. A portion of the nutrient medium, similar to the nutrient medium of example 1, from the fermentor with a culture of the microorganism Streptomyces olivaceus is mixed with a portion of the specified cross-linking product to achieve a concentration of 30.4 wt.% The reaction product, calculated on the dry weight of the bacterial cell mass. After the 0.5 hour reaction period has elapsed at a pH of 9 and a temperature of 25 ° C, the treated nutrient medium is filtered, washed with an aqueous solution of sodium bicarbonate with a concentration of 5 wt. at pH 9, extruded and dried. A control sample is obtained in the manner described in Example 3. The hardness of the control product is 0.36 kg, while the hardness achieved using the specified crosslinker product reaches 1.73. kg Example 5 - Portions of a nutrient medium from a fermentor with a culture of the microorganism Streptomyces otivaceus, similar to that mentioned in Example 1, are treated with only one glutaraldehyde (control experiment) and various combinations of crosslinking products at pH 9 and temperatures of 25 and 5 ° C. In all cases, the cross-linking reaction products that are used include 1 mol of cyanuric acid trichloride per 2 mol of amino groups in poly25 ° C. 100 Control As can be seen from the above data, the bacterial cells treated nnye crosslinking reaction products according to the present invention have a markedly increased hardness as compared with bacterial cells which have been treated in accordance with the known method only one glutaraldehyde.
    When using a crosslinking reaction product obtained from glutaraldehyde and a polyamine polymer, the preferred composition is prepared from a mixture of wt.% Glutaraldehyde and 2.9 wt.% Polyamine polymer 9290 5 10
    in terms of the total weight of active ingredients. In a preferred embodiment, this composition should also be used in an amount of 17.5% by weight, calculated on the dry weight of the bacterial cells. In the case when using cross-linking reaction product obtained from glutaraldehyde, cyanuric acid trichloride and polyamine polymer, in accordance with the preliminary version, the composition should be obtained from a mixture of 5.9 wt.% Glutaraldehyde and 3.6 weight. cyanuric acid trichloride and 41.5 wt.% of the polyamine polymer, calculated on the total U amine polymer. Then combinations are prepared as indicated in Table 2. Then, the treated bacterial cells are filtered, extruded, dried and subjected to hardness testing. Table 2 shows the composition of the reaction mixture to obtain a cross-linking reaction product. Table 2 15 weight active ingredients. In a preferred embodiment, this composition should also be applied in an amount of 18.2 wt.D, calculated on the dry weight of the bacterial cells. All bacterial aggregates prepared as described above show the ability to convert glucose to fructose. The practical implementation of the proposed method does not impair glucose isomerase activity. The main advantage and technical and economic efficiency of the present invention is to achieve the hardness of the bacterial cell aggregate after rehydration in comparison with the bacterial cell aggregates obtained by previously known methods. Form T1A of the invention 1. A method of a half-body of an aggregate of Streptomyces oliva ceus bacterial cells, providing for keeping the mass of bacterial cells in contact with a crosslinking agent in an alkaline medium for 0, 5 hours, characterized in that, in order to increase the hardness of the obtained product, 12-65.1% by weight of a compound selected from the group consisting of glutaraldehyde, trichloric anhydride and cyanuric acid and their combination, and 88-3.9% by weight of a water-soluble cationic field a measure of the product of polymerization of epichlorohydrin with an alkylene polyamine of the formula, where K is lower alkylene, and Rq is lower - alkyls, and the specified crosslinking agent is used in the amount of 6.5-55 wt.% of the dry weight of the cells, and the process is carried out at pH 8-9 and temperature 5-30 ° C, followed by separation of the target product. 2. Method according to Claim 1, about the fact that after separation the aggregate is dried. 3. The method according to claim 1, about tl and h ayusch and the fact that as the cross-linking agent using the product of the interaction 57.1 wt D glutaraldehyde and A2.9 weight. water-soluble cationic polymer in the amount of 17.5 weight. in terms of the dry weight of the cells. . The method according to claim 1, characterized in that the interaction product is used as a crosslinking agent, 9 wt. glutaraldehyde and 3.6 wt. cyanuric acid trichloride, taken as glutaraldehyde and 41.5% by weight of a water-soluble cationic polymer in an amount of 18.2% by weight. in terms of the dry weight of the cells.
    权利要求:
    Claims (2)
    [1]
    Claim
    1. A method of obtaining an aggregate of bacterial cells Streptomyces olivaceus, comprising maintaining the mass of bacterial cells in contact with a crosslinking agent in an alkaline medium for 0.5-1.5 hours, characterized in that, in order to increase the hardness of the obtained product, as a crosslinking agent use the product of the interaction of 12-65.1 wt.% compounds selected glutaric acid from the group of, including aldehyde, trichlor35
    Claim 1, characterized in that the product of the interaction is 57.1 wt.% glutaraldehyde and 42.9 wt.% water-soluble cationic polymer in an amount of 17.5 wt.% calculated on the dry weight of the cells.
    excellent quality
    4. The method according to claim 1, and with the fact that the crosslinking agent use the product of the interaction
    54.9 wt.% Glutaraldehyde and
    [2]
    3.6 wt.% Cyanuric acid trichloride, taken as glutaraldehyde and 41.5 wt.% Water-soluble cationic polymer in the amount of 18.2 wt.%, Calculated on the dry weight of the cells.
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    同族专利:
    公开号 | 公开日
    DE2911557C3|1981-01-22|
    DE2911557A1|1979-10-04|
    RO78777A|1982-06-25|
    HU182535B|1984-02-28|
    JPS5715879B2|1982-04-01|
    SE7902150L|1979-09-28|
    DK151637B|1987-12-21|
    IT1114710B|1986-01-27|
    AU512660B2|1980-10-23|
    ES478940A1|1980-01-01|
    GB2033396B|1982-07-28|
    YU73279A|1984-04-30|
    IL56732A|1981-11-30|
    NO790984L|1979-09-28|
    NL7902083A|1979-10-01|
    IL56732D0|1979-05-31|
    NL188359B|1992-01-02|
    DK151637C|1988-06-20|
    US4212943A|1980-07-15|
    AR222482A1|1981-05-29|
    PL214277A1|1979-12-17|
    IT7948478D0|1979-03-26|
    BE875032A|1979-07-16|
    DK122379A|1979-09-28|
    YU41859B|1988-02-29|
    CA1110185A|1981-10-06|
    GB2033396A|1980-05-21|
    JPS54129183A|1979-10-06|
    AU4537479A|1979-10-04|
    DE2911557B2|1980-04-03|
    ATA223579A|1982-02-15|
    NL188359C|1992-06-01|
    FR2421213A1|1979-10-26|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US3625828A|1969-04-16|1971-12-07|Miles Lab|Process for production of glucose isomerase|
    US3779869A|1971-05-13|1973-12-18|Miles Lab|Enzyme stabilization|
    BE785810A|1971-07-09|1973-01-04|Reynolds Tobacco Co R|ENZYMATIC TRANSFORMATION PROCESS|
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    US3935069A|1974-12-23|1976-01-27|R. J. Reynolds Tobacco Company|Enzymatic process using immobilized microbial cells|US4337313A|1980-12-08|1982-06-29|Miles Laboratories, Inc.|Immobilization of biocatalysts|
    US4355105A|1981-03-30|1982-10-19|Miles Laboratories, Inc.|Glutaraldehyde/polyethylenimine immobilization of whole microbial cells|
    US4390627A|1981-10-26|1983-06-28|Miles Laboratories, Inc.|Immobilization of the sucrose mutase in whole cells of protaminobacter rubrum|
    US4543332A|1982-03-29|1985-09-24|Miles Laboratories, Inc.|Method for the preparation of spherical microorganism cell aggregates|
    KR101100770B1|2009-04-14|2011-12-29|전북대학교산학협력단|Recovery method of valuableness metals|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    US05/890,500|US4212943A|1978-03-27|1978-03-27|Production of bacterial cell aggregate|
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