CS256821B1 - Immunosorbents for iso-amylase isolation and method for their preparation - Google Patents

Abstract

The subject of the solution is an immunosorbent for the isolation of α-amylase, where a specific antibody against α-amylase is bound to a starting carrier consisting of regenerated cellulose in the form of spherical particles provided with diethylamine or primary amino groups through a linker formed by reacted glutaraldehyde. Method of preparing immunosorbents for? The isolation of β-amylase consists in activating the sorbent, pearl cellulose with diethylamine or primary amino groups, in a buffered solution at pH=7 for 1 to 2 hours at laboratory temperature with a 2.5 to 5% solution of glutaraldehyde in water, the activated carrier is washed with water, left to react with a solution of antibodies against β-amylase in a buffer of pH-8.0 to 8.5 for 1 to 2 hours, and the immunosorbent is finally washed with a solution of sodium bicarbonate, sodium chloride or a buffer of pH=4 to 5 at concentrations of 0.1 to 1 mol/l.

Description

The invention relates to immunosorbents for the isolation and purification of oC-amylase and to a process for their preparation, and can be used, for example, in the food industry.

Purification of the enzymatic products of Bacilus subtilis gives a product (γ-amylase enzyme) of higher activity and selectivity. The presently described state includes purification of the native enzyme by precipitation, gel chromatography, ion exchange chromatography, hydrophobic chromatography, and some attempts have been made to use affinity chromatography.

In the 1970s, precipitation of the enzyme solution with calcium acetate and an equal portion of acetone at 5 ° C was used (M.H. Maseley, L.Keay: Biotechnol. And Bioeng. 12, 251 (1970)). Better results were obtained using cellulose ion exchangers or dextran gels. In the first step, the native enzyme solution was purified on a CM-cellulose column, followed by gel filtration on Sephadex G75 and ion exchange chromatography on DEAE Sephadex A50 (yield in the first step was 70%). The disadvantage is the multi-stage process, which is further complicated by the fact that the elution of the enzyme from ion exchange columns always required the use of an ionic strength (NaCl) gradient, which must be removed by dialysis before the next stage (H. Matsuzaki,

K. Yamane, B.Mario: Biochemica and Biophysica Acta 365, 235 (1974)). A similar procedure was to precipitate the protein fraction of bacterial culture with ammonium sulfate solution, purify on a CM-cellulose column and concentrate the product by ultrafiltration (K. Yamane, B. Maro. J. Bacteriol. 120, 792 (1974)).

Hydrophobic chromatography was used to purify C-amylase on agarose containing 1-alkoxy-2-hydroxypropyl groups with different alkyl sizes (C 1, C 6 and C 8) and a concentration of 25-50 µmol) cm -1 (E.Iaga, S. Katon, T. Inone, M. Shiozawa: Biotechnol and Bioeng., 27, 514 (1985)).

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Affinity chromatography has been described for the purification of α-amylase from glucoamylase. For this purpose, cross-linked starch has been used as a carrier, but which has very poor mechanical properties and is therefore practically unusable for column work (H. D. Schell, M. Matescu: Anal. Letters 14, 1501 (1981)). Iβ-amylase was purified by biospecific type affinity chromatography using their hydroxyl groups, starch was again used as the sorbent (P. Revert: FEBS Letters 47, 86 (1974)).

Summary of the state of the art has shown the historical development of enzyme purification methods based on classical salt precipitation methods, through ion exchange chromatography using non-specific ionic interactions between carrier and enzyme to affinity chromatography, where specific interaction of the desired enzyme with carrier ligands is applied. The most specific interaction in biochemistry is the reaction of the antigen with the antibody. Based on the simplistic concept of the lock and key, it is theoretically possible to find an antibody produced by the immune systems of living organisms against any extraneous substance sent into the body. This antibody, when separated from the body, is able to react with only a single antigen in the surrounding medium to form a complex. The mechanism of action of the immunosorbent of the invention also rests on this principle. In order to separate the antibody-antigen complex, the antibody is bound to a solid support. After mixing with the antigen solution, it reacts with the immobilized antibody and the resulting complex is firmly attached to the carrier. Mixing is carried out batchwise, in a stirred vessel, or by pouring the antigen solution through a bed of immunosorbent placed in the column. After saturation of the antibodies with antigens, all undesirable impurities are removed by simple filtration and washing of the buffer solution. Finally, the complex is cleaved with a salt solution of higher ionic strength and the released pure antigen is collected in solution, while the bound antibodies are capable of further use in the next purification cycle. In the case of the present invention, the antibody is contained in a globulin fraction of rabbit serum suitably immunized with the antigen-Q (amylase isolated from Bacilus · substilis), of course, the use of the above antibody is only one of many possibilities. so-called monoclonal antibodies prepared very specifically in vitro.

256 821

The process for preparing immunosorbents for isolating the amylase of the present invention simplifies the ligand immobilization process and separation process, and reduces the cost of separation even at higher process yields (up to 82%). This is achieved by using pearl cellulose with diethylamine or amine groups as the sorbent and ligand binding by glutaraldehyde. The bead cellulose consists of solid, porous spherical particles having a diameter of 0.55 to 1.0 mm, which are practically incompressible in the column, making it possible to utilize immunosorbents in column type technology without increasing the layer resistance at flow.

Immunosorbent for the isolation and purification of ^ (- amylases of the invention are folded, characterized in that the sorbent is used role bead cellulose diethylaminovými ^C or activated amine groups with glutaraldehyde prior to immobilization of the sorbent.

The process for preparing immunosorbents for isolation of γ-amylase is carried out as follows. The bead cellulose with diethylamine or amine groups is activated with a solution of glutaraldehyde in phosphate buffer at room temperature. The excess glutaraldehyde is washed with water and the sorbent is incubated with the serum solution containing the antibody (ligand) at room temperature. After washing, the immunosorbent thus prepared is used for purification of .beta.-amylase. Unreacted groups do not interfere with the purification process and do not need to be chemically saturated, generally by purifying the solution containing the protein mixture including .beta.-amylase and other impurities. the mixtures selectively react immobilized antibodies with β-amylase to form a solid complex. After saturation, the column is washed with a buffer with a salt solution of increasing ionic strength and β-amylase binding is released.

The present preparation method makes it possible to simplify the process by reducing the number of individual cleaning steps. A benefit is also the use of a sorbent of readily available raw materials that is sufficiently strong to allow the use of column type process equipment for separation.

The examples are intended to characterize the invention without limiting its scope in any way.

Example 1

256,821 parts of wet bead cellulose with diethylamine groups (0.91 mmol / g, water retention 5.2 g water per g dry sorbent, particle size 100 µm) were suspended in 10 parts 5% glutaraldehyde solution in 0.5 mol / ml 1 phosphate buffer pH 7.0 and tempered to 25 ° C for 1 hour. Excess glutaraldehyde was then removed by washing with water on a frit (300 parts) and the sorbent sealed with 4 parts of a globulin fraction of rabbit serum immunized with (.alpha.-amylase from bacilus subtilis (Hartree protein content of 35 mg / ml) in 16 parts 0.1 mol / ml. The mixture was incubated at 25 ° C for two hours on a shaker, the sorbent was decanted and washed with 150 parts of a 0.1 mol / L sodium bicarbonate solution, 100 parts of 1 mol. / L sodium chloride (NaCl) solution and 100 parts of acetate buffer pH 4.1 The protein content of Hartree was determined in the eluate and wash water and the sorbent-bound protein content was calculated to be 13.5 mg / g dry. sorbent.

The immobilized ligand sorbent was loaded into a 20 cm long and 1 cm diameter column and equilibrated with 0.17 mol / L phosphate buffer pH 6.34. 100 parts of the amylosubtiline preparation (with an activity of 3000 units / g preparation) were dissolved in 100 parts of 0.17 mol / l phosphate buffer pH 6.34 and 20 parts of this solution were applied to the equilibrated column at a rate of 2.3 ml / hour. Elution (β-amylase) was conducted in a sodium chloride concentration gradient in starting buffer from 0 to 0.5 mol / L NaCl. The enzyme yield was 80%. The relative β-amylase activity increased 2.5-fold. Polyacrylamide gel electrophoresis showed the presence of a single substance corresponding to? -amylase.

Example 2 parts of wet pearl cellulose with diethylamine groups were activated with glutaraldehyde as in example 1. Then the sorbent was poured over 20 parts of a 0.1 mol / l sodium bicarbonate solution (NaHCO-j) and pH 8.08 containing 10 mg of immunoglubulirium-G, isolated from rabbit serum immunized with Bacilus γ-amylase

256 821 subtilis by sodium sulfate precipitation (Na ₂ SO ₃ followed by DEAE-cellulose chromatography.) The mixture was incubated on a shaker for 2 hours at 25 and decanted and the sorbent poured over 20 parts of 1 mol / L glycine solution. at room temperature and the sorbent washed as in Example 1. The washed conjugate contained 8 mg protein per gram dry sorbent.

The immobilized ligand sorbent was loaded into the column and used to purify the β-amylase as in Example 1. The yield from the-amylase by activity was 82%. The relative β-amylase activity increased 2.5-fold.

Claims (2)

SUBJECT OF THE INVENTION Immunosorbent for the isolation of cC-amylase ₍ characterized in that a specific antibody against cC-amylase is coupled to the starting carrier consisting of regenerated cellulose in the form of spherical particles provided with diethylamine or primary amino groups by means of a glutaraldehyde-reacted linker. 2. A process for the preparation of immunosorbents for the isolation of oC-amylase according to claim 1, characterized in that the sorbent, bead cellulose with diethylamine or primary amino groups is activated in a buffered solution at pH = 7 for 1-2 hours at room temperature with a 2.5-5% glutaraldehyde solution. water, the activated support is washed with water, in a buffer of pH = 8.0 to 8.5 1 to 2 hours to react with the anti-N-amylase antibody solution, and the immunosorbent is finally washed with sodium bicarbonate, sodium chloride or pH 0.1 to 1 mol / L buffer.