SU1070162A1 - Method for purifying urease - Google Patents

Description

This invention relates to the preparation of highly purified and active enzyme preparations that can be widely used in the medical, microbiological, and food industries.

Of the known methods for the purification of enzymes, the most common chromatographic method is based on the use of sorbents til.

The closest to the proposed method is the purification of ureaa, which involves contacting the enzyme solution with a triethylaminoethylcellulose polymer sorbent followed by desorption of the sorbed enzyme with a buffer solution 121. The degree of urease purification is threefold (from 227 to 690 fe / mg protein),

The disadvantage of this method is its multistage and low cVene-ib cleaning.

The purpose of the invention is to simplify the process and increase the degree of purification,.

This goal is achieved according to the urease purification method, which involves contacting the α-enzyme solution with a polymeric sorbent, using macroporous ztirol copolymer and divinylbenzene with a content of the last 15–40 wt.%, Containing diazo groups in the amount of 5-6.7 mM / g as the sorbent. .

Preferably, said copolymer is used, which contains, besides the diazo groups, sulfonic acid groups or alkyl substituted phosphinic acid groups in an amount of 3.2–3.5 mM / g. .

Perfusion solution, urease in potassium phosphate buffer with a pH of 7.6 through a column of sorbent for 4 -. 18 h at 4 ° C. When this happens, sorption on the bollast protein protein sorbent and purification efficiency increases.

The advantages of the proposed method are to carry it out in one stage and to increase the degree of purification by 5-9 times in the case of using a sorbent containing, along with diazo groups, sulfonic acid groups or alkyl substituted phosphinic acid groups in a specified amount.

Pyrometric characteristics of the copolymer composition

a) styrene 05 wt.%, TWO 15 wt. gasoline blowing agent 100% of the monomer mixture. Bulk weight 0.4-0.55 r / cw, total pore volume 0.35-0.75 cmVg, 5). 9-0-130 MVr / U

0.25-0.8 mm (90%),

b) erase 60 wt.%, DVB 40 wt.% Gasoline pore former 100% of the monomer mixture. Bulk weight 0.3-0.36 g / cm total pore volume 0.85-1.1 cm2 / g, 5,275-350, CESD 0.4-1.6 mm (.90%).

Example H. In a column with a diameter of 3.6 mm and a height of 18 cm, 2 g of macroporous styrene copolymer and 40% DVB are placed as 100% gasoline), containing in their structure chemically active diazo groups at a concentration of 5.5 mmM / g. and for 4 h at 4C perfusion 20 ml

5 solution of urease concentration of 1.0 mg / ml in 0.1 m potassium phosphate buffer with a pH of 7.6. After that, the sorbent is filtered off and the urease solution is analyzed for activity.

0 enzyme. The activity of urease is increased 5,5 times. Thus, the specific activity of the initial urease is ф ф / / mg (1.f / mg is micromoles of urea per minute, after purification 5,550 е / mg.

Example 2. A column as described in example 1 is loaded with 2 g of sorbent based on macroporous styrene co-polymer and 15% DVB (100% gasoline is used as a pore-forming agent) containing chemically active diazo groups in its structure at a concentration of 4, 5 mM / g and for 4 hours at 4C, 20 ml of urease solution is perfused. The specific activity of urease increases in

5 times. Initial specific activity

urease is 100 fe / mg; after purification, 500 fe / mg.

Example 3. In a column like

described in example: 1, load 2 g of sorbent based on macroporous styrene copolymer and 15% DVB (use as a blowing agent

100% gasoline containing in

its structure is chemically active diazo groups in concentrations of 6 mM / g and sulphonic acid groups in concentrations of 3.5 mM / g, previously tested with 0.1 m potassium phosphate

buffer to pH 7.4 for 18 hours while perfusing 20 ml of the urease solution. The specific activity of the cuts increases 9 times. The initial specific activity of urease is 100 fe / mg. after cleaning

900 fe / mg.

Example 4. In a column as described in example (No. 1, placed 2 g; sorbent based on macroporous i

copolymer of styrene and 20% DVB {80% n-octane is used as a pore-forming agent, containing in its structure chemically active diazo groups in concentration

6.7 mm / g and the group P (0) fc, (, H,) HE

at a concentration of 3.2 W / g. The sorbent is treated with 0.1 m potassium phosphate buffer to pH 7.4 and perfused with 20 ml of urease solution for 4 hours at 4 ° C. The specific activity of urease is increased by a factor of b. The initial specific activity of urease is 100 fe / mg, after purification, 600 fe / mg.

As can be seen from the above examples, the use of polymeric sorbents with a stable diazo group greatly simplifies the process of urease purification, it is carried out in one stage on typical sorption equipment. Varying a number of sorbents.

containing in its structure simply a chemically active diazo group or a diazo group in combination with sulfo or phosphonic acid groups, it is possible to significantly increase the degree of urease purification by increasing it. 5-9 times.

The efficiency of urease purification is provided by covalent binding of protein impurities on polymeric sorbents with chemically active diazo groups.

The proposed method allows for the production of a highly active level with quality that is higher than the world standards.

Claims (2)

1. A METHOD FOR CLEANING UREASES, involving contacting an enzyme solution with a polymer sorbent, characterized in that, in order to simplify the process and increase the degree of purification of the enzyme, a macroporous copolymer of styrene and divinylbenzene with a content of the latter of 15-40 wt.% Containing diazogroup in the amount of 5-6.7 mmol / g. 2. The method according to π. 1, l and m of a Yusch hours and minutes with the I in that the said copolymer containing, along with the diazo group or sulfonic acid group alkilzame- group _{β-substituted} phosphinic acid in quantitative S ve 3.2-3.5 mmol / g.