CS265540B1 - A method of isolating alpha-amylase from biological materials - Google Patents

Abstract

The solution concerns a new method of isolation and purification of alpha-amylase in industrial production from biological materials. It is based on the use of the existence of an isoelectric point of this enzyme, where it is separated by autofocusing under the action of a direct current at a voltage of 200 to 1,000 V, from a mixture of enzymes and proteins in a solution with a conductivity of 100 to 800 pS.cnT1 at a pH of 5.82 to 6.12. This fraction is subjected to a separation technique using different molecular weights, preferably gel chromatography. By repeating or interchanging both techniques, it is possible to achieve a superpure enzyme needed mainly in industrial production.

Description

265540 2

The invention relates to a process for the isolation and purification of the alpha-amylase enzyme (EC 3.2.1.1.) In industrial production, which utilizes the isoelectric point of this enzyme as a purity criterion.

According to the prior art, alpha-amylase has been produced by several processes including precipitation with ammonium sulfate, ethanol, acetone, other gel and ion exchange systems, or electrophoresis in a variety of configurations. Their common disadvantage was the difficulty in the technical equipment, while the higher purity was hampered by the fact that at the final stage of isolation the protein mixture was approximately the same molecular weight, where a substantial part was the enzyme but the impurity content was + 5 to 10%. These ballast proteins reduce specific activity and cause other side effects, such as gradual degradation of the isolate. In our inventions CS-A-211 856 and 234 801, in principle, a new method of self-acting isoelectric focusing is protected by the action of unidirectional laundry in a uniform solution of the diluent, which is mechanically stabilized, as well as a simple apparatus for carrying out the process.

The above described drawbacks of isolation and purification of the enzyme are substantially eliminated by the invention, wherein dialysis or ultrafiltration adjusts the conductivity of the alpha-amylase-containing biological material to 100 to 800 pS.cm 1 and adjusts the protein content to 1 to 30 g of solution is autofocused with unidirectional electrical underwear at a tension of 200 to 1000 volts, a solution forming a fraction with the highest enzymatic activity, preferably at a pH of 5.82 to 6.18, is isolated and subjected to a different molecular technique the weight of the divided substances, preferably gel chromatography, depending on the purity required, one or both separation techniques are repeated at least once.

The invention is suitable for obtaining super-pure alpha-amylase in the process of manufacturing this enzyme for the needs of multiple industries. Přikladl

The bacterial culture of Bacillus subtilis strain is centrifuged and the supernatant obtained is concentrated by vacuum evaporation. The conductivity of the thickened supernatant is adjusted to 100 to 800 p.S.cm by dialysis or ultrafiltration with the addition of Mcllvari buffer 0.01 mol pH 6.0 containing citrate ions which stabilize the alpha-amylase activity. The protein content is adjusted to 10-30 g / l by dilution with buffer. The treated solution is filled with autofocusing apparatus with a number of chambers 20 and unidirectional electrical-controlled laundry is applied after closing to a temperature of 200 to 1000 V regulated so that the laundry linen and the tension do not exceed 3 W. Autofocusing takes place under a cold to +4 ° C. The autofocusing process is over when 1000 V drops to zero. The split mixture self-generates an upward pH gradient over which individual protein fractions are formed into peaks according to their isoelectric points. The alpha-amylase is found in the fractions with the highest enzymatic activity, mainly in the chambers with a pH between 5.82 and 6.18, while the enzymatic activity is determined on the basis of the "oxidative activity of the cleaved starch enzyme. Proteins from this range are unified and applied to a column of 80x50 cm filled with Spheron P-40 molecular sieve, equilibrated with Clclvain buffer, and chromatographed at 4 ° C at a flow rate of 120 ml / h and 30 ml fractions were collected, and the protein content of Lawry and alpha-amylase activity was determined in all fractions after all gel filtration. Goal filtration by autofocusing of the isolated protein is divided into four vertically pure alpha-amylases located in the third peak from the beginning of fraction collection. The individual steps can be repeated according to the degree of purity required, which is defined by the single electrophoresis of the only one. but it is also not a specific activity to be achieved by at least 3 U.mg according to international evaluation. Thus, up to 99.99% purity of the enzyme is obtained.

Claims (1)

Method for isolating alpha-amylase from biological materials, characterized in that by conducting dialysis or ultrafiltration, the conductivity of the biological material solution is adjusted to 100 to 800 pS.cm ¹ and the protein content is adjusted to 1 to 30 g and the solution is autofocused by direct current at 200 to 1000 volts until the current drops to zero and the solution forming the fraction with the highest enzymatic activity, preferably at pH 5.82 to 6.18, is isolated and subjected to a separation technique using a different molecular weight of the separated substances, preferably by gel chromatography wherein one or both of the separation techniques are repeated at least once depending on the desired purity.