CS239826B1 - A method of preparing a salivary alpha amylase isoenzyme - Google Patents

Abstract

The invention relates to a method for preparing salivary alpha amylase isoenzyme from biological material. The essence of the method for preparing salivary alpha amylase is that an acetone solution of saliva after separation of mechanical impurities and rapidly sedimenting fractions containing the enzyme glycoprotein of salivary alpha amylase is reacted with covalently immobilized Tectin isolated from potatoes or wheat, specific for N-acetyl-D-glucose or sialic acid. The enzyme alpha amylase is released from the above complex by the effect of a buffered solution of N-acetyl-D-glucosamine or a solution of glycogen with a pH of 7 to 7.2. The invention is used in clinical biochemistry, in the preparation of pure biochemicals and in the isolation of enzyme glycoproteins of the amylase type.

Description

239826

The invention relates to the preparation of a saline isoenzyme of alpha amylase.

In addition to the chromolytic methods on gel-locel from molecular sieves and ion exchangers, the techniques of separation and isolation of enzymes include the affinity chromatography method. E. Starkenstein and O. Holmbergh used n-starch starch to isolate alpha-mylase from biological material. Stadkenstein, O. Holmbergh: Biochem. Issue 24, 21-0 (1910); O. Holmbergh: Biochem. Z. 25-8,134 (1933)]. Native starch starch of the general type was further used to isolate alpha amylase from Clostridium ace-to-butilicum [D. J. D. Hockenhull, D. Herbert: Biochem. J. 39, 102 (1945)] and alpha a-mylases from ja-n. Schwimmer, A. K.Balls: J. Biol. Chem. 179, 1063 (1949)]. The affinity ligand used was a) a glyco gene, covalently linked to the surface of the modified dextran [R. Tkachuk: FEBS Lett. 52, © 6 (1975)]. Native starch substrate with respect to particle homogeneity and unsuitable hydrodynamic properties does not possess the properties or affinity ligand of α-myolytic enzymes [R, M. Sandsted, S.Ueda: Japan Soc. Starch. Sci. This procedure, or the use of other affinity bases of modified alpha-glucan, did not provide protection of the amylase glycoprotein of salivary isoamylase pre-d effect of ombacid terralas located in the oral cavity [Μ]. P. Silvanovich, R. D. Hill: Anal. Biochem. 73, 430 (1976)]. The optimal procedure does not prevent the use of a protein alpha-amylase inhibitor isolated from the fraction of wheat grain albumin bound to the modified dextran by covalent bonding. In fact, the high-specific carrier sorption of the matrix is applied to the prion-free protein and the low efficacy of the affinity ligand bound to the n-α surface without the pacifier, as indicated by the results of the alpha pancreatic amylase, fu-shade and alpha-amylase of the Tenebriomolor [V. Buonocore, E. Poerico, F. Gramenzi. V. Sielano :. J. Chromatogr. 114, 109 (1975)].

The present invention solves these shortcomings, which is that fresh secretions of human saliva or biological fluids containing salivary alpha amylase isoenzyme are diluted by one tenth of the volume of acetone and the resulting solution centrifuged at 2,000 to 3,000 gpo 10-20 min, the supernatant is adjusted to pH 7 and the resulting concentration of acetone in solution at 40-50 ° C is added dropwise with acetone cooled to -10 to -20 ° C. The volume of the obtained suspension is centrifuged at 2000 to 30-00 g for 10 to 20 minutes, and then the recovered alpha amylation-containing supernatant is purified by affinity using N-acetyl-D-glucose-α-mine and acid-affinity to the surface of the bead cellulose, and subsequently the enzyme alpha amylase is liberated from the complex with lectin effect of 0.02 to 0.1 mol. 11 µl of N-acetyl-D-glucose-mine solution, or 2 to 5% by weight of glycogen in 0.0 µl to 0.05 mol. I ' 1 phosphate solution and pH up to 7.2 and a volume of 5-10 times higher than the swelling of the used affinity. The advantage of the proposed coupling is the high affinity of the co-bound lectin, specific to N-acetyl-D-glucosamine and alkyl-silane to the enzyme glycoprotein of the strong alpha amylase isoenzyme, and the high efficacy of the inactivity, low In addition, the cost and the fact that alpha amylase is sorbed on the surface of the support: with a covalently bonded lectin of the type described herein is stabilized against the inactivating effects of the hydrolases of the microorganisms present in the oral cavity. EXAMPLE 1 Fresh secretion of human saliva (200 ml) is diluted with 20 ml of acetone and the resulting solution is centrifuged at 2000 g for 20 minutes and the recovered supernatant is adjusted to pH 7 with 22-0 ml of 0.02 mol. The phosphate buffer was added dropwise and 330 ml of acetone, cooled to -10 ° C, was added dropwise (30 ml). The resulting suspension is centrifuged at 2000 g for 20 minutes, and the supernatant containing -alpha-amylase-o-activity is added to 40 ml of swollen cellulose gel activated with 2-chloromethyloxirane-cellulose oxirane ( dry matter 1-3%; degree of substitution of the oxi-frame cellulose 0.42 mmol.g "1) with a co-bound N-acetyl-D-glulcosamine specific acid-specific lectin (prepared from 40 mg / ml cellulose Triticum vulgarisj Immobilized lectin alpha-mylase enzyme for 1 hour with agitation at 20 ° C, pearl cellulose with co-bound lectin-α-tartrate the azan alfa amylase is separated by dacantation and washed with 500 ml of 0.2 mol / l phosphate buffer solution of opH7 and the purified enzyme glycoprotein-alpha-alpha amylase is released with 400 ml of 0.02 mol / l N-acetyl-D-glucosamine in 0.01 µmol / l phosphate buffer solution pH 7. Total activity a 1α amylase 1 cattle at a specific activity of 0.25 pkat, mg -1, and staple purification of 14.1. Example 2

The procedure is as in Example 1, except that the enzyme solution diluted with 10% acetone is centrifuged at 3000 g for 10 minutes, adjusted to 0.02 moles. The pH of 7-phosphate buffer solution I-1 is adjusted dropwise with 226 acetone buffered to -20 ° C, and the resulting suspension is centrifuged at 3000 g for 10 minutes. For affinity pre-purification, a similar type of imin

Claims (1)

However, it was isolated from potatoes (Solarium). Displacement of the alpha amylase enzyme from the complex with immobilized lectin transferred 400 ml of 0.05 mol. I-1 with a phosphate buffer solution of pH 7.2 containing 0.1 mol. to N-acetyl-D-glucosamine. The yield of alpha amylase and the degree of purification of the alcohols is given in Example 1. EXAMPLE 3 The alcohol in Example 1 was followed except that 200 was used to displace the alpha amylase enzyme from the lectin complex. ml of solution 2% by weight of glycogen (from the rabbit. The alpha amylase yield was 1.04 m with a specific activity of 0.24 jukat.mg-1 and the purification step of Example 1. Example 2 is followed by in that 200 ml of 5 θ / o weight glycogen (from Saccharomyces cerevisiae yeast are used to displace the alpha amylase enzyme), giving alpha amylase * with a total activity of 1.08 and a specific activity of 0.25, Purpose of purification was found to be widespread in medical and clinical biochemistry for the preparation of high purity glyphosylated alpha amylase standards for the purpose of checking the accuracy and accuracy of the methods used in the present invention. h in routine analysis of body fluids, especially in differential biochemical diagnosis of pancreatic and paro-thyroid disorders. Saline isoenzyme -alpha amylase can continue to be used in basic biochemical and enzymatic research and for maltose-type recombinant oligosaccharides. r RED Μ ET A method of preparing a shaded alpha-amylase isoenzyme, characterized in that fresh secreted saliva or biological fluid-bearing shaded alpha amylase isozyme is diluted with one-tenth of the volume with acetone and the resulting solution is centrifuged at 2,000 to 3,000grams for 10 to 20 minutes, the supernatants were adjusted to pH 7 and the resulting acetone concentration in the solution was adjusted dropwise to 40-50% by volume with acetone cooled to -10 to -20 ° C and the suspension obtained was centrifuged at 2,000 to 3,000 g for 10-20. minutes, the obtained alphaamylase-containing supernatant is purified by affinity method using N -cetyl-D-glucosamine-specific lectin and acidic sialic bound binding to the surface of pearl cellulose and subsequently released by the enzyme a complex of lectin complexes having an effect of 0.02 to 0.1 mol. I-1 with a solution of N-acetyl-D-glucosamine, or 2 to 5% by weight of glycogen in 0.01 to 0.05 mol. The I-1 phosphate buffer solution and the pH of 7 to 7.2 and the volume of 5 to 10 co-higher alcoal is the n-appendant volume of the used.