CS264992B1 - A method for preparing a cross-linked protein - Google Patents

Abstract

The solution relates to the preparation of a spliced protein for the purposes of preparing standard chromolytic substrates. The essence of the solution is that the protein is cross-linked using a homobifunctional agent derived from the L-amino acid components of the primary structure of the protein, such as L-ethyl ester of 2,6-diisothiocyanatohexane acid in an amount of 0.5 to 10% by weight. based on the weight of the dry protein, at a pH of 7 to 12 adjusted using solutions of dilute alkalis or acids or buffer solutions of 1 to 50 mmol.l-^, preferably phosphate or borate, at a temperature of 10 to 50 °C for a period of 10 to 120 min.

Description

The present invention relates to a process for the preparation of fused proteins for the purpose of preparing standard chromolytic substrates, affinity proteolytic enzymes of general type, protein type hydrogels, or carriers for immobilizing enzymes and cells.

Previously known protein crosslinking methods use predominantly homobifunctional glutaraldehyde-type substances (S. Avrameas, Immunochemistry, 6/1969/43), or heterobifunctional 2-chloromethyloxirane type substances (AO 194 592; AO 218 709). While the preparation of glutaraldehyde crosslinking results in undefined oligomeric and polymeric structures containing alpha-unsaturated aldehydes, which also participate in the crosslinking reaction (F. M. Richards, J. R. Knowles,

J. Mol. Biol. (231) The Schiff bases that arise during crosslinking do not guarantee the stability of the resulting product. In the case of 2-chloromethyl-oxirane cross-linking, a relatively high alkalinity (pH 14) is required to convert the intermediate chlorohydrin to the oxirane group, which is accompanied by degradation reactions in the primary protein structure. The above-mentioned crosslinking reaction is accompanied by high salt formation, which negatively affects the hydrodynamic properties of the prepared protein gel. When preparing larger amounts of cross-linked protein using 2-chloromethyloxirane, technological problems arise with homogeneous liquor distribution across the cross-section of the reaction mixture, resulting in uneven gel cross-linking on the one hand and local degradation of the protein to water-soluble fragments due to high alkalinity.

The above-mentioned drawbacks are eliminated by the process according to the invention, wherein in the first step the protein is crosslinked using a homobifunctional agent derived from the L-amino acid components of the primary structure of the protein, such as 2,6-diisothiocyanato-hexanoic acid L-ethyl ester, in an amount of 0.5. up to 10 wt. based on the weight of the dry protein, in a solution medium having a pH of 7 to 12, preferably borate or phosphate buffer, at a temperature of 10 to 50 ° C for 10 to 120 minutes. In a second step, the cross-linked protein is washed from buffer components and adjusted for saler use. The fused protein hydrogel can be used as a selection platform to monitor microbial producers of proteolytic type enzymes or in the dry state after defibrating in a high speed mixer and then drying and sieving as an affinity to isolate proteolytic enzymes from the enzyme mixture or as insoluble protein to prepare to determine the activities of proteolytic enzymes in chemical treatment with compounds containing chromophoric groups.

The advantage of this process is that there is no byproduct formation during the reaction, the crosslinking reaction is controlled in one direction without the formation of concomitant salts, allows the preparation of new protein gels over a wide range of physical and chemical properties for the specific needs of their use. The dried fused protein is well stored. Depending on the reaction conditions, the crosslinking reaction may be conducted so that only the thiol groups of the protein, or both the thiol groups and the primary amino groups of the protein, react preferentially.

Example 1

Lyophilized bovine serum albumin (5 g) was dissolved with stirring in an electromagnetic mixer in 100 ml borate buffer (10 mmol.l; pH 12) with the addition of 0.01 ml n-octanol.

0.5 g of lysine 2,6-diisocyanate ethyl ester was added to the soluble protein with rapid stirring and the reaction mixture was poured into a mold (40x40 cm) of 0.5 cm height. The crosslinking reaction is terminated after 10 minutes at 50 ° C. The resulting hydrogel of the fused protein is washed in distilled water from the borate buffer residues. The product obtained after washing and swelling in water has a dry weight of 1.1% and a modulus of elasticity of 26 mm.

Example 2

The procedure of Example 1 except that ovalbumin is used as the protein. The product obtained has a dry weight of 1.8 t and a modulus of elasticity of 24 mm penetrometrically.

Example 3

The procedure of Example 1 except that casein is used as the protein. The crosslinking reaction was carried out in phosphate buffer (40 mmol.I ^-1 ; pH 7) and 25 mg of L-lysine 2,6-diisothiocyanate ethyl ester was added. The crosslinking reaction was terminated after 10 minutes at 10 ° C. The product obtained is wet-defibrated in a high speed mixer, washed with distilled water and ethanol, and dried at room temperature (20 ° C). The obtained dry gel is screened to a particle size of 0.06-0.3 mm with a swelling volume of 33 ml.g ³ and used as an affinity of proteolytic enzymes (pepsin, trypsin, chymotrypsin) of the general type.

The invention has broad application in particular in enzymatic engineering, food processing and in general applied microbiology.

Claims (1)

OBJECT OF THE INVENTION A process for the preparation of a cross-linked protein, characterized in that the protein is cross-linked using a homobifunctional reagent derived from the L-amino acid components of the primary structure of the protein, such as 2,6-diisothiocyanato-hexanoic acid L-ethyl ester in an amount of 0.5-10% by weight. based on dry protein, at pH 7-12 adjusted with dilute solutions of acids or acids, or with buffer solutions of 1-50 mmol.l ³ , preferably phosphate or borate, at 10 to 50 ° C for 10 to 120 minutes .