SU979354A1 - Process for producing sorbent - Google Patents

Description

The invention relates to the field of enzymology and is intended for the preparation of a sorbent for the isolation and purification of cofactor-dependent enzymes - oxidation, kinases, etc.

For isolation and purification of cofactor-dependent enzymes, along with classical methods, biospecific chromatography using group sorbents based on immobilized i-factors has been widely used ..

However, sorbents of this type, despite their high specificity, have significant drawbacks: high cost, which determines the high cost of carrier and cofactors used for the synthesis of sorbents, the complexity of methods for activating and modifying cofactors, low chemical stability of the resulting sorbents, which eliminates the possibility of using sorbents for preparative isolation. and purification of cofactor-dependent enzymes.

More promising for preparative isolation and purification of cofactor-dependent enzymes are

sorbents containing dyes as a ligand, structural analogs of coenzymes, and first of all the active dye cybacron blue. Thus, there is a known method for obtaining a sorbent based on an agarose matrix with an attached cybron blue dye l.

However, the possibility of preparative use of sorbents with associated dyes despite the chemical stability of the latter is limited by the disadvantages of the agarose matrix used for the synthesis of sorbents (high cost, low chemical, microbiological and mechanical stability).

The closest in technical essence is the method of obtaining

20 sorbent based on the dye cybacron blue F-jGA and microspherical porous silica gel SG 120, including activation of the carrier. f-aminopropyltriethoxysilane, separate from the carrier, activation of water-soluble dextran with BrCN in the medium of sodium bicarbonate at a pH of 9.0 in a ratio of 50 mg of BrCN per 200 mg of dextran and modification

30 media of BrCN-activated dextran at pH 9.0 in a column of 200 mg of dextran per 1.0 g of carrier by recycling the activated dextran through the column for 24 hours, washing the modified carrier, attaching dye cybacron blue and washing the resulting sorbent water. Typically, washing of the modified carrier is carried out with an IM solution of NaCP and WATER 2. However, this method of preparing a sorbent has a number of significant drawbacks. First, the process of modifying an activated inorganic carrier is non-technological and is complicated by the use of dextran T40 to attach to the carrier BrCN, which is a costly, toxic and dangerous substance when used for preparative purposes, secondly when used for modifying the inorganic carrier dextr activated by BrCN, forming a bond between the carrier and the dextral wound is chemically unstable 3, which leads to the loss of the dye being added during long-term using a sorbent, and hence reducing sorbent capacity; Thirdly, the existing β-modifying method of an inorganic carrier allows 5–10 µM dye per gram of sorbent to be introduced into the sorbent and thereby limits its use to isolate and purify those cofactor-dependent enzymes that have a low dye agent. On the other hand The relatively low concentration of the injected white cell affects the degree of sorption capacity of the sorbent as a whole in relation to the proteins secreted with it. The purpose of the invention is to simplify the process of obtaining a sorbent (by reducing the number of stages, the total labor input of the process, and also to eliminate the use of contaminated substances) and to increase the sorption capacity of the sorbent. The goal is achieved by the method of obtaining sorbent, which includes processing of inorganic silica containing f-aminopropyltriethoxysilane material and modification of the obtained derivative followed by washing and addition of the active dye cybacron blue, modifying the zvodnogo carried out in 1-2 hours Techa of 25-50-fold excess of glu NH taraldegida against -groups processed product. The cybacron blue F GA active dye is used in the amount of 20100 μM / g. processed product. As an inorganic silica-containing material, porous glass or silochrome with an average radius of 300-800 A is used. Washing the modified product before attaching the dye is carried out with water, 10-20 volumes of 0.09-0., 15 M NaHCO solution containing 0.2 -0.3% sodium borohydride for 2-3 hours. The method allows 10–40 µM dye per gram of sorbent to be introduced into the sorbent, i.e. allows to increase its sorption capacity. The modification of the carrier containing amino groups is carried out with glutaraldehyde followed by a reducing agent, which contributes to the formation of a stable bond between it and the carrier and to the formation of a stable bond between the attached dye and the modified carrier. The method of modifying glutaraldehyde is available, simple in technological design, since it does not require the use of hard-to-reach and poisonous substances, and allows the selection of the ratio of reactants to regulate the concentration of dye introduced into the sorbent and, accordingly, the sorption capacity of the sorbent in a wide range of values. The method is more technological and simpler than the known one. The inorganic matrix can be used both in the form of silochromes and porous glasses, followed by amination with -TG-aminopropyltriethoxysilane, or in the form of ready-made NH-containing derivatives, produced by foreign companies and NPO Viochim-reagent. The optimal content of NH-groups is 100-448 μM / g. Example. Getting sorbent on the basis of silochrome C-80. 10 g of silochrome C-80 are poured with 10 g of absolute acetone, 10 g of α-aminopropyltriethoxysilia are added and the reaction mixture is stirred for 24 hours at the boiling point of the mixture. The reaction product is filtered off, washed 4-5 times with acetone (50 ml) and dried. 9.4 g of the amino derivative silochrome C-80 are obtained. 2.3 g of silanized silochrome C-80 (concentration of NH4. -Rpynn 120 μM / 1 g) are filled with 5.8 ml of 25% glutaral dialdehyde and 2.6 ml of water, stirred for 1 hour at room temperature, washed on the filter with water, then in small portions (15-20 ml) with 0.1 M NaHCO (200 ml) containing 0.2-0.3% YaABK (for 2 hours and again washed with water. To the moist glutaraldehyde modified C-80 silochrome, add 3 ml of water are added with a stirring, while a solution of 252.5 mg (234-8 microns of cybron blue in 14.0 ml of water) is added dropwise. Stirring is continued for 0.5 h. They are heated for 1 hour, then the temperature is raised to 80 ° C, 200 mg of Na is added, and stirred for 2 hours. The resulting sorbent is washed with cold and hot water, BO-EO C, until the washings are colorless. The absorbent is determined by the difference between the amount taken from it and that found in the wash water using the theoretical extinction coefficient E = 13,600 M. (X di 610 nm). The concentration of cybacron blue F GA is 37.0 µM / g dry sorbent. Example 2: Preparation of group sorbent based on porous glass CPG 550. 6.7 g of 25% glutaraldehyde are poured onto 6.7 g of silanized glass CPG 550 (concentration of NH, j, -rpynn 100 µM / g) 23.3 ml of water are added and stirred for 1 hour at room temperature, washed on the filter with water, then 400 ml of 0.1 M H 2 O 3 (portions of 30-50 ml) containing 0.2-0.3% NaBH, water is added to the wet modified glass for 10 hours. 10 ml of water are added and a solution of 660, 1 mg (613.9 µM) of cybacron blue EZ GA is added dropwise with stirring at 60 ° C. Further, the reaction is carried out as in Example 1, but the amounts of NaCE added and, respectively, 5.5 g and 493 mg. The concentration of cybacron blue is 25 µM / g dry sorbent. To determine sorbents suitability for cleaning cofactor-dependent enzymes are used a base formulation of yeast hexokinase obtained after autolysis of baker's yeast, acid fractionation, two-fold fractionation with ammonium sulfate, dialysis and liofil hydrochloric drying, according to the initial steps of enzyme purification schemes following the procedure f43 and base formulation yeast alcohol dehydrogenase obtained after autolysis of the yeast, heat treatment, fractionation with ammonium rulphate, dialysis, and lyophilization according to the beginning lnym step by the method 5. The specific activity of enzymes in an average of 10-25 U / mg for hexokinase and 5-10 U / mg protein for alcohol dehydrogenase. Froze Purification of yeast hexokinase. A chromatographic column (1, .0 cm) was charged with 1.0 ml of the sorbate obtained in Example 1 using a 25-fold excess of glutaraldehyde with a dye concentration of 15 μM / g of sorbent. The sorbent was equilibrated with 10 mM Tris-HCf buffer pH 6.5, containing 5 mM MdSrB.O. and 0.5 mM EDTA, and 1.5 ml of the enzyme solution in the original buffer was applied. The protein concentration is 6.0 mg / ml, the specific activity is 19.8 units / mg of protein. When eluted from 50 ml of the original buffer, the ballast proteins are washed out. Elution of hexokinae was carried out with a gradient of sodium chloride from 0-1.0 M in the initial buffer. The specific activity of hexokinase after desorption is on average 72 units / mg. The yield on the activity of 98-100%. Sorbent capacity 140 u / ml. Example Over. A chromatographic column (as in Example 3) was charged with 1.0 ml of sorbent obtained in Example 1 using a 25-fold excess of glutaraldehyde with a dye concentration of 40.6 μM / g of sorbent. The sorbent was equilibrated with buffer (as in Example 3) and 2.0 ml of the enzyme solution in the initial buffer was applied. The protein concentration is 8.8 mg / ml, the specific activity is 16.5 units / mg protein. Upon elution with 100 ml of the original buffer solution, the ballast proteins are washed out; The active hexokinase is desorbed with a linear gradient of sodium chloride from 01.0 M in the initial buffer. The specific activity of hexokinase after desorption is 82.0 units / mg of protein, the yield for activity is 100%. Sorbent capacity is 225 units / ml. PRI me R Zb. A chromatographic column (as in Example 3) was loaded with 1.0 ml of the sorbent obtained in Example 1 using a 50-fold excess of glutaraldehyde with a dye concentration of 15 µM / g of the sorbent, equilibrated with the initial buffer, and 1 ml of a 8.8 protein concentration was applied to hexokinase. mg / ml and specific activity of 16.4 units / mg protein. When elution from 80 ml of initial buffer, ballast proteins are washed out. The active hexokinase is desorbed with linear hail; NaCf envelope from 0-1.0 M in the initial buffer. The specific activity of hexokinase after desorption is on average 80 units / mg of protein, the yield for activity is 100%. Sorbent capacity is 142 units / ml. PRI me R Sv. A chromatographic column (as in Example 3) was loaded with 1 ml of sorbent obtained in Example 1 using a 50-fold excess of glutaraldehyde with a dye concentration of 37} O μM / g of sorbent, was equilibrated with initial buffer and 1.5 ml of solution was applied

hexokinase with a protein concentration of 8.5 mg / ml and a specific activity of 21.1 units / mg protein. When chromatography was carried out under conditions identical to example 3, the specific enzyme activity was 69 units / mg of protein with a yield of 72%. The capacity of the sorbent is 224.5 units / ml.

PRI me R 3g. A chromatographic column (as in example 3) was charged with 2.8 ml of sorbent based on porous glass (as in example 2) using a 50-fold excess of glutaraldehyde and a dye concentration of 15 μM / 1 g of sorbent. After equilibration of the sorbent with the initial buffer, 3 ml of hexokinase solution with a protein concentration of 9.6 mg / ml and a specific activity of 33 units / mg of protein are applied .. When chromatography is carried out under the conditions of example 3, the specific activity of the desorbed enzyme reaches 150 units / mg of protein with a yield of activity 80%. Sorbent capacity 333 u / ml.

PRI me R 4. Purification of yeast alcohol dehydrogenase.

A chromatographic column (1.5-3.0 cm) filled with 2.0 ml of sorbent synthesized (as in Example 2) using a 25-fold excess of glutaraldehyde with a dye concentration of 15 μM / g of sorbent, and equilibrated with 10 mM trsNSSS buffer pH 6.5, containing. 5 mM MgCe, j; 6H5 0 ,. 0., 5 mM EDTA, and 0.05 m of mercaptoethanol, is applied with 3.0 ml of the enzyme solution of elcool dehydrogenase. A beam concentration of 14.7 mg / m has a specific activity of 7.9 units / mg of protein. When the column was washed with stock buffer (140 ml), ballast proteins broke. Elution of the enzyme was carried out with a specifically linear gradient of NAD from 0-10 mM (50 NN) in the original buffer. Active alcohol dehydrogenase is desorbed at 3 mM NAD with a specific activity of 120 units / mg protein and a yield of 47%. The sorption capacity of the sorbent is 140 U / ml sorbent.

Note E. 4c. In a chromatographic column (as in example 4), filled with 1 ml of sorbent obtained on the basis of silochrome C-80 (as in example 1) using a 25-fold excess of glutaraldehyde with a dye concentration of 15 μM / g, balanced with the initial buffer, put 2 ml of alcohol dehydrogenase solution with a specific activity of 7.6 units / mg protein and a protein concentration of 10.6 mg / ml. By washing with 60 ml of the original buffer, the ballast proteins are washed out. The elution of alcohol dehydrogenase is carried out with a linear gradient of NAD from 0-10 mM in the initial buffer. The active enzyme was desorbed at 5 mM NAD

With a specific activity of 55 U / mg Bel and an activity yield of 48%. The capacity of the sorbent is 120. U / ml of sorbent.

Note .. A chromatographic column (as in Example 4) filled with 1 ml of sorbent based on C-80 silochrome (as in Example 1) using a 25-fold excess of glutaraldehyde with a condensation of 40.6 μM / g dye, balanced the original buffer, put, 2.0 ml of an alcohol dehydrogenase solution with a protein concentration of 11.6 mg / ml and a specific activity of 8.3 units / mg of protein. After washing with 60 ml of the original buffer, the ballast proteins are washed out. The elution of the alcohol dehydrogenase is carried out with a linear gradient. OVER from 0-10 mM in the initial buffer. Alcohol dehydrogenase active is desorbed at 5 mM NAD with a specific activity of 140 units / mg protein. The sorption capacity of the sorbent is 130 units / ml of sorbent.

Example 4-. In a chromatographic column (as in example 4), filled with 1 ml of sorbent, obtained on the basis of silochrome C-80 (as in example 1) using a 50-fold excess of glutaraldehyde with a dye concentration of 15 μM / g, equilibrated with the initial buffer, put 2 ml of alcohol dehydrogenase solution with specific activity of 7.6 units / mg of protein and protein concentration of 10.6 mg / ml. After washing the ballast proteins from 60 ml of the original buffer, the active alcohol dehydrogenase is desorbed with a linear gradient of NAD from 1-10 mM in the original buffer. The specific activity of the enzyme desorbed with 4 mM NAD 110 u / mg protein with a yield of 43%. I eat | Costi sorbent 90 units / ml sorbent.

Example 4. In a chromatographic column (as in example 4), filled with 1.0 ml of sorbent based on C-80 silochrome (as in example 1) using a 50-fold excess of glutaraldehyde with a dye concentration of 37 μM / g , apply 2 ml of an alcohol dehydrogenase solution with a specific activity of 7.6 units / mg of protein and a protein concentration of 10.6 mg / ml. When performing chromatography as in Example 4-4, alcohol dehydrogenase is desorbed with 5 mM NAD With a specific activity of 115 units / mg of protein and an activity yield of 41%. The capacity of the sorbent 112 u / ml.

Claims (5)

The proposed method for the preparation of a group sorbent based on a modified inorganic carrier and dye tsibacron blue Fj GA provides an advantage over the known methods for obtaining sorbents. High efficiency of the sorbent in relation to cofactor-dependent enzymes, i.e. the possibility of increasing the activity of the purified enzymes in a single stage of sorption — desorption, on average, 4–15 times, while maintaining the activity of the enzyme by 40–100%. The sorbent obtained by the proposed method has a high volumetric sorption capacity of enzymes, amounting to 90-330 yards. activity / ml of sorbent. The availability of the starting materials necessary for the preparation of the sorbent, the simplicity and accessibility of the method for the preparation of the sorbent, make it possible to introduce synthesis technology into production for the production of enlarged batches of the sorbent. . The combination of the listed advantages determines the efficiency of using the proposed method for producing a sorbent for its industrial production and use in the technology of obtaining highly purified cofactor-dependent enzymes. Formula 1. A method for producing a sorbent that includes treating inorganic silica-containing material with T-amnoprptiltriethoxysilane, is a subject. washing and joining the active dye cybacro blue, distinguishing with the fact that , in order to simplify the process and decrease the sorption capacity of the sorbent, the modification is carried out within 1-2 hours with glutaraldehyde, taken in 25-50-fold excess relative to the content of NHj-groups of the processed product. 2. A method according to Claim 1, characterized in that the cybron blue active dye is used in an amount of 20-100 µM / g of the product to be processed. 3. Method according to Claims 1 and 2, characterized in that porous glass or silochrome with an average pore radius of 300-800 A is used as an inorganic silica-containing material. 4. Method according to Claims 1-3, characterized in that the washing of the modified product before the addition of the dye is carried out with water, 10-20 volumes of 0.09-0.15 M solution of NaHCQv containing 0.20, 3% sodium borohydride for 2-3 hours. Sources of information taken into account in the examination 1.Burgett M.W., GreenEey L.V. Amer. Lab., 1977, 9.78. 2.Anderson P.A., Jervis L. Affinity purification of maCate dehydrogenase and Eactate dehydrogenase from fish muscCe on microsphericaC porous ceramic adsorbents. - Biochem.Soc.Trans., 1977, 5 ,. 728. 3.Turkova Y. Leakage of coupfed affinant, - J.Chromatography, 1978, 12, 189. 4..Biochem.Biophys., 1973, 158, 451-457. 5.Kochetov G.A. A practical guide to enzymology. M., Higher School, 1978, 128.