SU1353807A1 - Method of cleaning beta-glucanase - Google Patents

Abstract

This invention relates to biotechnology. The purpose of the invention is to accelerate the cleaning process and increase the yield of the target product. The method involves the dissolution of the enzyme preparation / -glucanase in water containing 10 M CaCl, desalting by gel filtration on Sephadex G-25 with a gel filtration rate of 0.4-0.5 ml / cm min, cysteine is added to the resulting solution or glutation in the amount of 0.03-0.08 mg per unit. p-glucanase activity, followed by purification by chromatography on carboxymethyl cellulose, and the eluate is concentrated by ultrafiltration. The specific activity of the enzyme increases by 6-7 times. Output 1.6-1.9 times. 1 tab., 1 Il. with S (L

Description

one

The invention relates to biotechnology, in particular to the enzyme industry of the microbiological industry, and can be used in the production of purified 1,3-1,4- / 3-β-glucanase, free from admixture of concomitant enzymes and other ballast substances, the enzyme can be used for analytical purposes. x to study the structure of glucans.

The purpose of the invention is to accelerate the cleaning process and increase the yield of the target product.

The drawing shows the dependences of the effect of CaCl concentration on the degree of clarification of the enzyme solution and the loss of ft-gluconase during filtration, where 1 is the degree of clarification of the filtrate compared with the initial solution; 2 - loss of enzyme with ballast sediment.

The method consists in the fact that at the stage of preparation of the enzyme solution to be used for. races

- 2,

water is added 10 M of SASTg. At this concentration of CaC, it is possible to achieve maximum clarification of the preparation by separating the ballast proteins with minimal loss of the target product. Replacing dialysis with gel-filtration on Sephadex G-25 at an elution rate of 0.4-0.5 m / cm min allows faster purification and increase the yield of the enzyme. An increase in the yield of the enzyme is also achieved by introducing cysteine or glutathione in an enzyme solution (before chromatography) in an amount of 0.03-0.05 m per unit of jb-glucanase activity. The lack of data on the role of sulfhydryl groups in p-glucanase indicates that the effect of cysteine and glutathione on the yield of the enzyme is not obvious. The use of this set of features allows to speed up the cleaning process by 24-45 hours and increase the yield from 19.4 to 32.8%.

Example 1. To obtain a highly pure preparation of p-glucanase, 50 g of protosubtilin HYuH with glucanase activity of 250 units / g and a protein content of 110 mg / g is dissolved in

500 ml of distilled water containing 10 M CaC. The undissolved precipitate is separated by centrifugation. Transparent enzyme solution containing neutral proteinase, ft-glucanase and oi-amylase activity

ten

15

20

s

- 25

3338072

(i-glucanase, 23.3 U / ml, and a protein content of 10.2 mg / ml) is sent for desalting. For this purpose, gel filtration on Sephadex G-25 loaded in a 50x800 mm column is used. Sephadex is balanced with 10 M phosphate buffer pH 7.0 containing 0.005 M CaC. 500 ml of an enzyme solution was applied to the column at a rate of 0.4 ml / min CM and, after gel filtration, a desalted 2000 ml fraction containing 4.6 units / ml / i-glucanase and 2 mg / ip protein was obtained. The resulting enzyme solution is fed to ion exchange chromatography, which is carried out on carboxymethylcellulose (KM-cellulose) loaded onto a 140x140 mm column.

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The ion exchanger is equilibrated with 10 M phosphate buffer pH 7.0. To improve purification and increase the yield of P-glucanase at the stage of ion-exchange chromatography, cysteine is added to the desalted enzyme solution at a rate of 0.03 mg per unit of enzyme activity. After applying the desalted enzyme solution, the column is washed with 3 liters of initial buffer to completely remove the about-amylase from KM-cellulose. Then the column is washed with 2.5 l of the original buffer containing 7-10 M NaCl. When this occurs, the proteolytic fraction is eluted. Then, elution of the / -glucanases with the initial buffer containing 1M NaCl is carried out; as a result of the last elution, 800 ml of a solution of α-glucanase activity of 7 units / ml and protein content of 0.5 mg / ml B are not detected. Concentration of the eluate / 3-glucanase was carried out by ultrafiltration through cell-cellulose membranes of the UAM-100 type at a pH of 7.0. As a result, a 40 ml fraction with a p-glucanase content of 111 units / mp and protein of 7.4 mg / ml was obtained. The output activity compared with the original drug 35.5%. The specific activity of the enzyme is increased 6.5 times. After lyophilization, a highly purified preparation of p-glucanase was obtained.

The yield of the enzyme at the purification stages is given in the table.

Compared with the known method, the yield of I J -glucanase is increased by 1.8 times (table).

thirty

35

40

45

50

55

PRI mme R 2. The method for obtaining a highly purified preparation of / 3-glucane from protosubtilin HYuh is similar to the method described in Example 1, prior to the stage of ion-exchange chromatography on CM-cellulose. In this case, before deposition on the column with an ion exchanger, glutathione is introduced into the desalted enzyme solution at the rate of 0.07 mg per unit of activity of the j) glucanase. For elution: ai-amylases, proteinases and | 5 -glucanases use the same solutions as in example 1.

carried out with the initial buffer containing 0.8 M NaCl. As a result, 900 ml of solution / 3 tlyuk are obtained,. Nasy 5.5 u / ml and a protein content of 0.4 mg / ml. In this fraction, proteinases and oi-amylase are absent. The concentration of the eluate β-glucanase is carried out by ultrafiltration

10 through cellulose acetate membranes

UAM-100 at a pH of 6; 2; Enzyme Concentrate with a volume of 25 ml has a P-glucanase activity of 160 units / ml

and a protein content of 10.9 mg / ml. Yield Fraction of β-glucanase with a volume of 850 ml 15 by activity compared with the end result has an activity of 7 units / ml and a containing preparation of 32%. Specific active proteins of 0.4 mg / ml. The resulting enzyme is grown 6.4 times, the solution lacks proteinase, and Next, the concentrate is dried for lyophil-6-amylase. Concentration of the eluate but or release of (3-glucanase with acetone). Compared with the known method, the yield of ft-glucanase is increased 1.6 times, and the cleaning process is reduced by 45 hours.

Example 4: To obtain a highly purified preparation / 3-glucanase, 800 g of protosubtilin gux glucanase

I-glucanases are carried out under the conditions described in Example 1. A 25 ml concentrate contains 190 units / mp.

3-glucanases and 12.3 mg / ml protein. The yield of activity compared to the original drug is 38%, the specific activity is increased 6.7 times. Further

activity 400 u / g and protein content 120 mg / g dissolved in 8.0 l

activity 400 u / g and protein content 120 mg / g dissolved in 8.0 l

distilled water containing

one.

concentrate is freeze-dried or glucanase is precipitated with acetone. Compared with the known way out

/ -glucanase increased by 1.9 times, and zoo Yu CACH. The insoluble precipitate the degree of purification of the enzyme is increased separated by centrifugation. Transparent 6,3%. Enzyme solution activity

PRI me R 3. A method for preparing —glucanase 37 U / ml and a content of the highly purified preparation / 3-gluc-protein 11.2 mg / ml is sent to the prostate sublin POC analogue — 5 by gel filtration. in the method described in example 1, to the stage of gel filtration on Sephadex G-25. The column with Sephadex is equilibrated with 2-10 M acetate buffer pH 6.2, containing 0.005 M CaCl.

500 ml of enzyme solution with L-glucanase activity of 23.3 units / ml and protein content of 10.2 mg / ml are applied to the column, the gel filtration rate

Sephadex G-25. loaded into a 200x630 mm column. The desalting conditions are similar to those described in Example 1. The rate of ft-shifter is 40 0.47 ml / cm MIN. After gel filtration, a fraction of 20.0 L is obtained, containing 11.2 U / ml / 3-glucanase and 3.3 mg / ml of protein. Glutathione is introduced into the desalted enzyme solution in 0.5 ml / min. After gel filtration, an amount of 0.05 mg per unit of activity is obtained; 1800 ml of desalted plant / 5-glucanase are obtained and sent to thieves with a 5-glucanase activity of 5 units / ml of a 110x160 cm column, with a loading and protein content of 2 mg / ml. For this KM cellulose solution, cysteine is added to the solution in quantitative ion exchange chromatography. Complete

The gQ removal of the ob-amylase from KM cellulose occurs by washing the column with 20 liters of initial buffer. The column is then washed with 10 L of starting buffer containing 710 M NaCl for elution buffer with oi-amylase. After gg elution of proteinase. As a result of this, the neutral elution is eluted with an initial buffer containing

W NaCl, get 6.0 l of a solution activity of | 3-glucanase 22 u / ml and a protein content of 3.3 mg / ml. The proteve is 0.05 mg per unit of P-glucanase activity and applied to a column with CM cellulose equilibrated with 2-10 M acetate buffer at pH 6.2. Next, the column is washed with 3.5 l of the original

proteinase, passing through the column 3 liters of the original buffer containing 1510 M NaCl. Elution -glucanase

conducted with stock buffer containing 0.8 M NaCl. As a result, 900 ml of a solution of activity / 3 of tlucanase 5.5 U / ml and a protein content of 0.4 mg / ml are obtained. In this fraction, proteinases and oi-amylase are absent. The concentration of the eluate β-glucanase is carried out by ultrafiltration

through cellulose acetate membranes

UAM-100 at a pH of 6; 2; Enzyme Concentrate with a volume of 25 ml has a P-glucanase activity of 160 units / ml

and a protein content of 10.9 mg / ml. The yield of activity compared with the initial preparation is 32%. The specific activity of the enzyme is increased by 6.4 times. Next, the concentrate is dried lyophilically or the 3-glucanase is separated by a acetoactivity of 400 units / g and the protein content of 120 mg / g is dissolved in 8.0 l.

distilled water containing

one.

Yu M CACh. The insoluble precipitates are separated by centrifugation. Clear enzyme solution activity

- β-glucanases of 37 units / ml and a protein content of 11.2 mg / ml are sent for desulfurization by gel filtration on

Sephadex G-25. loaded into a 200x630 mm column. The desalting conditions are similar to those described in Example 1. The rate of ft-shtratsii 0.47 ml / cm MIN. After gel filtrainae and “i-amylase are absent in this fraction. After concentration of the resulting eluate by ultrafiltration through UAM-100 type membranes at a pH of 7.0, a 200 ml concentrate, jb-glucanase activity of 530 units / ml and a protein content of 24.3 mg / ml are obtained. The output of the enzyme compared with the original Yu drug 33.1%. The specific activity of the enzyme increases 6.6 times. After lyophilization or precipitation with acetone, a highly purified preparation of p-glucanase is obtained. Reduce the 15 process to 42 hours.

Froze The method of obtaining a highly purified preparation of β-glucanase from protosubtilin G10x by the analogous method described in Example 1, prior to the stage of ion-exchange chromatography on KM-cellulose. Before this stage, a desalted enzyme solution was added with glutathione in an amount of 0.08 mg per unit of α-glucanase activity. The elution of β-glucanase is carried out under the same conditions as described in Example 1. The 320-β-glucanase fraction has an activity of 7.3 U / ml and a protein content of 0.4 mg / ml. Proteinase and oi-amylase are absent in the resulting eluate. Concentration of the p-glucanase fraction was carried out under the conditions described in Example 1. Concentration 20

It is advisable to add CaC in the concentration of 10 M. This concentration is optimal, since it provides the filtrate with an enzyme solution of the required quality with a relatively small loss of x-glucanase with a ballast sediment (drawing).

An increase in the rate of elution from sephadex G-25 to 0.6 ml / cm min or a decrease to 0.3 ml / cm min leads to a significant increase in the volume of the eluate containing β-glucanase, i.e. to the dilution of the enzyme preparation.

Example 6. I-glucanase was injected as described in Example 1, but

instead of Protosubtilin G10x, a precipitate obtained from the culture fluid by precipitation with ethanol is used as the initial preparation. The output of the enzyme (from the stage of culture liquid 25) 33%.

Claims (1)

Invention Formula Purification method of / 3 -glucanase, including dissolving the starting enzyme preparation in water, desalting it, carboxymethylcellulose chromatography and concentration, which is characterized by the fact that, for the purpose of the 30 ml rat, it contains 160.5 units / ml g of process acceleration purification and above- P-glucanase and 194 mg / ml protein. When the yield of the target product was obtained, the ferment for activity compared to the initial preparation was 38.5%, the specific activity was increased 6.8 times. Next, the concentrate is dried freeze or 40 glucanase precipitated with acetone. Compared to the known method, the yield of fium in glucose solution is added 1.89 times to the enzyme solution. cysteine or glutathione in an amount At the stage of preparation of the enzyme, 0.03-0.08 mg per unit of active solution for coagulation of ballast-P-glucanase. The menth preparation is dissolved in water containing 10 M CaCl1 desalting is carried out by gel filtration on Sephadex G-25 at a rate of 0.4-0.5 ml / cm-min, and before chromatography It is advisable to add CaC in the concentration of 10 M. This concentration is optimal, since it provides the filtrate with an enzyme solution of the required quality with a relatively small loss of x-glucanase with a ballast sediment (drawing). An increase in the rate of elution from sephadex G-25 to 0.6 ml / cm min or a decrease to 0.3 ml / cm min leads to a significant increase in the volume of the eluate containing β-glucanase, i.e. to the dilution of the enzyme preparation. Example 6. I-glucanase was injected as described in Example 1, but instead of Protosubtilin G10x, a precipitate obtained from the culture fluid by precipitation with ethanol is used as the initial preparation. The output of the enzyme (from the stage of the culture fluid) 33%. Invention Formula Purification method of / 3-glucanase, including dissolving the starting enzyme preparation in water, desalting it, carboxymethylcellulose chromatography and concentration, which, in order to speed up the purification process and increase the yield of the target product, ferment cysteine or glutathione is added to the enzyme solution in an amount of 0.03-0.08 mg per unit of P-glucanase activity. The menth preparation is dissolved in water containing 10 M CaCl j desalting is carried out by gel filtration on Sephadex G-25 at a rate of 0.4-0.5 ml / cm-min, and before chromatography Protosubtilin nOx 100 cooking enzyme solution Gel filtration on sephadex G-25 44-45 55-60 59-65 79 80 80 100 92-93 70-73 32-33 40-45 41-47 80 26 32-35 33-38 20 ° / about loss of service ten-. 0.005 0.007 0.01 0.02 Editor N.Rogulich 0.05 Ktz CaCl2, f Compiled In Muronetz Tehred L. Oliynyk Proofreader S. Shekmar Order 5670/24 Circulation 500 Subscription VNIIPI State Committee of the USSR for inventions and discoveries 113035, Moscow, Zh-35, Raushsk nab., 4/5 Production and Printing Enterprise, Uzhgorod, Projecto st., 4 0.02