CS277433B6 - A method of isolating lysozyme enzyme from biological materials - Google Patents

Abstract

The purpose of the solution is to improve the isolation of the lysozyme enzyme from biological materials in industrial production. The stated purpose is achieved by using the existence of the isoelectric point of the lysozyme enzyme, where it is separated from a mixture of proteins and enzymes in a solution with a conductivity of 100 to 800 lS.cnf1 at a pH of 10.00 to 10.60 by autofocusing under the action of a direct electric current. This fraction is subjected to a separation technique using different molecular weights, preferably gel chromatography. By repeating or interchanging both techniques, a pure enzyme can be obtained. The method for isolating the lysozyme enzyme from biological materials has applications in the chemical and food industries.

Description

The invention relates to a method for isolating the lysozyme enzyme (EC 3.2.1.17) from biological materials.

According to the prior art, lysozyme has been obtained by several processes, including barbecue precipitation systems and separations, as well as chromatographic processes in various configurations. Their common disadvantage was labor, equipment complexity, and the purity of the enzyme was at the level of the protein mixture approximately the same molecular weight, where a substantial part was lysozyme, but their primes were from 5 to 10%. These ballast proteins reduce specific activity and also cause other side effects, such as the gradual degradation of the isolate. In our inventions / CS author's certificate No. 211 856, 234 801 / a fundamentally new method of separation and purification of substances is protected on the basis of their isoelectric point by automatic isoelectric focusing by direct current in a diverse solution of the separated substance, which is mechanically stabilized as well as several types of devices for carrying out this method.

The above-described shortcomings of the current purification and isolation procedures for obtaining lysozyme are substantially eliminated by the invention, which consists in adjusting the conductivity of biological material containing lysozyme activity to 100 to 800 μS.cm ^{1 by} dialysis or ultrafiltration, and the protein content adjusts to values from 1 to 30 gl ” ¹ . The solution is then autofocused by direct current at a voltage of 200 to 1000 V and a power input of at most 3 Wl ^-1, and after autofocusing, the fractions with the highest activities, preferably at pH 10.00 to 10.60, are isolated and subjected to a separation technique using different molecular weights of the separated substances, preferably gel permeation chromatography, one or both of which are repeated at least once, depending on the desired purity.

The present invention eliminates the need to use salts for salting out and precipitating proteins, which indicate considerable environmental pollution and difficulty in neutralizing them. The substance is purified according to two criteria, in contrast to the previous methods, where it was purified only on the basis of its molecular weight.

Example 1 Egg white from 1,200 eggs giving about 30 liters of protein is diluted 1: 2.5 with tap water to give about 100 liters of a protein solution at a concentration of up to 3 g / ^l . The solution thus treated was dialyzed against tap water for 12 hours at 10 ° C. The solution is then applied to a 100 L autofocus apparatus, which is connected to a 3 W / ¹ DC power source. Autofocusing takes place at a temperature of 10 ° C until the current drops at a limit voltage of 1000 V to at least one tenth of the flood value as at the beginning of autofocusing. In the autofocus apparatus, a rising pH interface is formed in each working chamber from the anode to the cathode. Lysozyme is found in fractions with a pH between 10.00 and 10.60. The contents of the chambers are unified at the indicated pH and subjected to refocusing in an autofocuser of a volume of 10 l. near zero, the lysozyme is present in chambers with a pH between 10.20 and 10.40 in a purer state. 180 g of lysozyme enzyme are obtained.

Example 2

Proceed as. in Example 1 with the difference that after autofocusing, the lysozyme fractions are subjected to gel chromatography on a modular suture column (Spheron P-40). The majority fraction contains lysozyme, whose activity is 20,000 U / mg protein (95% shows lysozyme activity). The 1 U activity represents a decrease in the absorbance of the Micrococcus luteus suspension (strain 331) at 450 nm, pH 6.6 and 30 ° C in 1 min by 0.001.

The invention may find: use in the production of the enzyme lysozyme on a technological scale for its production for the needs of the food industry, the chemical industry, scientific research and other industries.

Claims (1)

PATENT CLAIMS Methods of isolating the enzyme lysozyme from biological materials, characterized in that the protein content in the solution of the biological material exhibiting lysozyme activity in water was adjusted by dilution to 1 to 30 gl ^-1 and the conductivity was adjusted by ultrafiltration or dialysis to 100-800 μΒ.απΓ ¹ and the solution with enzymatic activity is then autofocused by direct current at a voltage of 200 to 1000 V and 3 Wl ^-1 until the current value falls below one tenth of the original value and the solution with the highest enzymatic activity, preferably at pH 10.00 to 10.60 is isolated and subjected to a separation technique using different molecular weights of the separated substances, preferably gel permeation chromatography, one or both of which, depending on the desired purity, are separated. techniques are repeated at least once.