SU510479A1 - Method of immobilization of proteolytic enzymes - Google Patents

Description

The invention relates to the production of enzymes and can be used in the food, medical and microbiological industries.

Methods of immobilization of proteolytic enzymes on carriers of organic and inorganic origin, such as cellulose, polyacrylamide, dextra, and other derivatives, as well as bentonite, wollastonite, silica gel, aluminum oxide and nickel, hydroxysate, and porous glass are currently used. This means that inorganic carriers are more resistant to the action of microorganisms, alkalis, acids, temperatures, do not swell and do not change their volume depending on the pH of the medium and the ionic strength of the solutions.

There is a method of immobilizing proteolytic enzymes by shifting at an acidic pH value the enzyme, activator and silica-containing carrier pretreated with an aminoalkylsilane derivative, followed by separating and washing the immobilized enzyme.

In order to improve the quality of enzymes in the proposed method, silochrome is used as a silica-containing carrier, and the shift is carried out at a pH of 4.5-5.5 and batch addition of an activator.

The method is carried out as follows.

The proteolytic enzyme, for example, pepsin, is brought into contact with a water-soluble activator of the carboxyl groups of the enzyme, carbodiimide and a silica-containing carrier, silochrome, previously treated with an aminoalkylsilane derivative.

Mixing lead in distilled water at a pH of 4.5-5.5 and a portion of the addition of the activator. After completion of the reaction, the aqueous solution is removed by decantation, and the immobilized enzyme is washed successively with solutions of hydrochloric acid, sodium chloride, and acetate buffer at 5 ppm to be removed from the adsorbed media.

due to the complete exchange of the enzyme.

Example. 50 mg of pepsin, 22 mg of water-soluble p-toluenesulfonate-1-cyclohexyl-3- (2-morphominoethyl) -carbodiimide and 0.5 g pre-treated with aminoalkylsilane silochrome are mixed in distilled water for one hour at 20 ° C, maintaining the pH a mixture of 4.5-5.5.

Then, 22 mg is added to the mixture again.

carbodiimide and continue stirring for two hours at 20 ° C and rp 4.5-5.5.

Next, the aqueous solution is removed by decanting, and the immobilized pepsin is washed successively with 0.01 M HC1 solution.

1.0 M NaCl and 0.1 M acetate buffer at

3

pH 5.0 to remove the enzyme adsorbed from the carrier metabolism from the carrier.

The amount of enzyme covalently attached to the carrier is determined by hydrolysis of the immobilized enzyme in the presence of 5.7 M HC1 at 105 ° C for 24 hours in a vacuum, and it has been established that 40-50 mg of the enzyme is added covalently to 1 g of silochrome. The activity of the immobilized pepsin is determined by the high molecular weight substrate hemoglobin.

It has been established that the activity of the preparation obtained by the proposed method, when it is stored in a wet state at pH 5.0, does not change for several months, and the immobilized enzyme practically

fully retains activity

source of native pepsin.

Claims (1)

Invention Formula The method of immobilization of proteolytic enzymes by mixing at acidic pH values of an enzyme, an activator and a silica-containing carrier pre-treated with an aminoalkylsilane derivative, followed by separation and washing of the immobilized enzyme, characterized in that silochromic, using silochrome, use silochrome as the silica-containing surface, using silochrome as the silica-bearing keller compound, using silochrome, in order to improve the quality of the enzymes, silochrome is used as silochrome, and silochrome using a silochrome, in order to improve the quality of the enzymes, silochrome using a silochrome will use silochrome as a silochrome, and silochrome will use silochrome as a silica gel. lead at a pH of 4.5-5.5 and a portion of the addition of the activator.