RU2294369C2 - Method for preparing immobilized urease - Google Patents

Abstract

FIELD: biochemistry, enzymes, biotechnology.

SUBSTANCE: method involves immobilization of enzyme on inorganic carrier aerosil wherein its surface is modified with casein. Invention provides enhancing specific activity of enzyme, increasing range of thermal stability of immobilized urease, increasing percent of enzyme binding with carrier and to simplify process for immobilizing the enzyme. Invention can be used in development of enzyme electrodes used in treatment of sewage from urea.

EFFECT: improved preparing method of enzyme.

2 cl, 1 tbl, 3 ex

Description

The invention relates to biotechnology, in particular to methods for producing immobilized urease, and may find application in the analytical industry (creation of enzyme electrodes) in the treatment of wastewater from urea.

A known method of obtaining immobilized urease (AC 1373730, publ. 15.02.88), which consists in increasing the stability of the enzyme during storage after binding to oxidized carbon in the presence of steroid glycosides. The disadvantage of this method is a significant increase in the activity of the initial urease (more than 100%), which may be due to the influence of the glycosides themselves on the course of the enzymatic reaction.

The next way to obtain immobilized urease is to bind the enzyme with oxidized activated carbon, modified titanium tetrachloride (AC 1090715, publ. 07.05.84). The disadvantages are the low percentage of binding of the enzyme to the carrier and a short shelf life of the enzyme preparation.

The closest to the expected technical essence and the achieved positive effect is a method of obtaining immobilized urease, which consists in binding the enzyme to an inorganic carrier activated by γ-aminopropyltriethoxysilane and glutaraldehyde (AC 925183, publ. 15.10.82). The disadvantage is the use of harmful substances as modifiers of the surface of the inorganic carrier, the duration and multi-stage process of immobilization, as well as the low specific activity of the immobilized urease with a low range of thermal stability.

The objective of the invention is to increase the specific activity and thermostability range of immobilized urease at optimum pH with a high percentage of binding of the enzyme to the carrier and simplification of the process of immobilization of the enzyme.

The problem is solved by a method involving the immobilization of the urease enzyme on an inorganic carrier - aerosil, the surface of which is modified by a natural high-molecular compound - casein.

The method is as follows. To 5 g of Aerosil add 50 ml of distilled water and mix it with casein, taken in an amount of 3-15 wt.% And dissolved in 10 ml of a 10% sodium hydroxide solution. The resulting suspension was allowed to mature at room temperature for 24 hours. The formation of a rigid skeleton of the carrier is carried out with thorough stirring for 1 hour at a temperature of 110-130 ° C. The resulting carrier is sieved through a sieve with a mesh size of 230 μm and washed five times with 100 ml distilled water to a pH of wash water of 7.0. For immobilization, the urease enzyme isolated from soybeans with a specific activity of 3460 U / g is used. 2.0 ml of the enzyme solution in phosphate-buffered saline pH 7.0 with a urease concentration of 4.0 mg / ml is added to 1 g of a wet carrier. The volume of the enzyme solution was chosen taking into account the complete wettability of the carrier. The concentration of the enzyme is due to the possibility of a more complete process of immobilization. An enzyme solution with a concentration of less than 4.0 mg / ml is not practical to use, because there will be an incomplete load of the enzyme on the carrier. An increase in the concentration of the enzyme above 4.0 mg / ml is unacceptable due to the competition of urease molecules for the right to possess binding sites located on the surface of the carrier. Immobilization is carried out for 24 hours at a temperature of 4 ° C. The immobilization time is due to the process of complete binding of the enzyme to the carrier. The immobilization temperature is selected taking into account the maximum preservation of the activity of the native enzyme. Upon completion of the immobilization process, the carrier with the immobilized enzyme is washed three times with bidistilled water with a volume of 50 ml until the unbound enzyme is completely removed, which is recorded on an SF-26 spectrophotometer at a wavelength of 280 nm against bidistilled water. The activity of the native and immobilized enzyme is evaluated by the photocolorimetric method. As a substrate, a solution of urea in phosphate buffer is used. To establish the optimum pH-activity of immobilized urease, the enzymatic reaction is carried out in phosphate buffer at a pH of 5.4-7.0. The limit of thermal stability of immobilized urease is set according to the values of the highest enzyme activity during the enzymatic reaction in the temperature range from 10 to 60 ° C, since the enzymatic reaction does not proceed below 10 ° C, and above 70 ° C the enzyme loses activity as a result of the denaturation process. The unit of urease activity is the amount of enzyme that results in the reaction of which 1 μmol of ammonia is released with the substrate in 1 minute at 20 ° C.

The method is illustrated by the following examples.

Example 1. To 5 g of Aerosil add 50 ml of distilled water and mix it with casein, taken in an amount of 3 wt.% And dissolved in 10 ml of a 10% sodium hydroxide solution. The resulting suspension was allowed to mature at room temperature for 24 hours. The formation of a rigid skeleton of the carrier is carried out with thorough stirring for 1 hour at a temperature of 110-130 ° C. The resulting carrier is sieved through a sieve with a mesh size of 230 μm and washed five times with 100 ml of distilled water to a pH of wash water of 7.0. For immobilization, the urease enzyme isolated from soybeans with a specific activity of 3460 U / g is used. 2.0 ml of the enzyme solution in phosphate-buffered saline pH 7.0 with a urease concentration of 4.0 mg / ml is added to 1 g of a wet carrier. Immobilization is carried out for 24 hours at a temperature of 4 ° C. Upon completion of the immobilization process, the carrier with the immobilized enzyme is washed three times with bidistilled water with a volume of 50 ml until the unbound enzyme is completely removed. The activity of the immobilized enzyme is evaluated by the photocolorimetric method. A urea solution in phosphate buffer at pH 7.0 is used as a substrate. The enzymatic reaction is carried out at a temperature of 20-60 ° C. An immobilized urease preparation with a specific activity of 3391 U / g of the carrier is obtained, which is 98% of the preservation of the initial activity at 70% binding of the enzyme.

Example 2. Perform analogously to example 1, except that to modify the surface of the media use casein in an amount of 5 wt.%, And as a substrate use a solution of urea in phosphate buffer pH 5.4. The enzymatic reaction is carried out at a temperature of 10-30 ° C, and a preparation with a specific activity of 3460 U / g of carrier is obtained, which is 100% of the preservation of the initial activity with 63% binding of the enzyme.

Example 3. Perform analogously to example 1, except that to modify the surface of the carrier use casein in an amount of 15 wt.%, And as a substrate use a solution of urea in phosphate buffer pH 5.4. The enzymatic reaction is carried out at 10-20 ° C, a preparation is obtained with a specific activity of 3460 U / g of the carrier, which is 100% of the preservation of the initial activity with 83% binding of the enzyme.

The table shows the comparative characteristics of immobilized urease in comparison with the prototype.

Table
Comparative characteristics of immobilized urease The method of obtaining immobilized urease The specific activity of the urease enzyme, E / g of sorbent The percentage of binding of urease,% Preservation of immobilized urease activity,% Optimum pH Optimum thermal stability, ° C Known method 3400 75 88 7.3 eighteen According to example 1 (proposed) 3391 70 98 6.0 20-60 According to example 2 (proposed) 3460 63 one hundred 5,4 10-30 According to example 3 (proposed) 3460 83 one hundred 5,4 10-20

In contrast to the prototype, the proposed method of urease immobilization allows to increase the specific activity of the enzyme and its thermal stability range at optimum pH with a high percentage of binding of the enzyme to the carrier, which is achieved by immobilization on silica - aerosil, the surface of which is modified with casein. In addition, the proposed method eliminates the use of harmful components as modifiers of the surface of the carrier and makes the process of immobilization of the enzyme simpler, since it eliminates a number of stages that can significantly reduce the activity of the immobilized enzyme.

Claims (2)

1. A method of obtaining immobilized urease, providing for the immobilization of the enzyme on a modified inorganic carrier, characterized in that casein modified aerosil is used as the inorganic carrier. 2. The method according to claim 1, characterized in that aerosil is used, modified by mixing with casein, taken in an amount of 3-5 wt.% And dissolved in 10 ml of a 10% sodium hydroxide solution, for 1 h at a temperature of 110 -130 ° C.