RU2388821C2 - Method for production of pancreatic ribonuclease - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: method provides for grinding of pancreas. Ground pancreas is treated with solution of sodium chloride of 4.5-6 wt % in the following volume ratio between pancreas and salt solution equal to 1:(1.5-2.0) at the temperature of 25-35°C for 2.5-3.5 hours. Produced cells of pancreas are separated by centrifugation and treated with 95% ethyl alcohol for 15-24 hours in volume ratio of 1:(0.85-1.3) at the temperature of 15-25°C. Pancreas cells are separated by centrifugation with further treatment of these cells with water acidified with hydrochloric acid to pH of 1.0-3.0 for 2.5-4.5 hours at the temperature of 25-35°C in volume ratio of 1:(0.8:1.2). Produced cells of pancreas are separated by centrifugation to produce supernatant. Ribonuclease is extracted from produced supernatant by solutions of sulfuric acid with concentration of 0.15-0.35 for 3-4 hours with volume ratio of 1:(0.8-1.2) at the temperature of 4-6°C. Suspended particles are separated by centrifugation to produce extract that contains ribonuclease. Produced extract is concentrated by ultrafiltration on UPM 10 membrane with further diafiltration. Produced concentrate is cooled down to 4-6°C, and ribonuclease is deposited at pH of 8.0-9.0. Produced residue is washed with ethyl alcohol in volume ratio of 1:5 and dried.

EFFECT: increased yield of ferment.

1 tbl, 1 ex

Description

The invention relates to biotechnology, namely the production of enzymes, and can be used in the medical, chemical and microbiological industry.

A known method of producing ribonuclease (RNAase) [Kunitz M., J. Gen. Physiol, 24, 15 (1940); McDonald M.R., Methods in enzymology, Vol. 2, p. 427 (S.P. Colowick and N. O. Kaplan, Eds.), New York. Academic Press (1955)] by extraction from fresh bovine pancreas 0.25 N. a solution of sulfuric acid in a volume ratio of 1: 2 for 24 hours, followed by precipitation from the extract of trypsinogen, chymotrypsinogen and deoxyribonuclease with ammonium sulfate at 0.6 saturation, then at 0.8 ÷ 0.85 saturation, RNAase is fractionated. An enzyme isolated in this way is usually contaminated with trace amounts of proteolytic enzymes. Cleaning [Tugunov S.S., Konstantinov V.L. and other Factory production of amorphous and crystalline therapeutic preparations of the enzymes trypsin, chymotrypsin and ribonuclease from the pancreatic gland / Proceedings of the Leningrad Chemical-Pharmaceutical Institute, 1967, issue 20, pp. 9-18] of the production effluent of RNA-ase is carried out by re-precipitation of ammonium sulfate. Sludge dehydration RNAse is carried out with ethanol. The drying of crystals of RNAase is carried out under vacuum at a temperature of 50 ° C. The yield of the drug, according to the proposed technology, is 0.1% by weight of the dry pancreas. The disadvantages of this method for the isolation of pancreatic RNAase are the low yield of the product due to the multi-stage process, the duration of the process, the low degree of precipitation of RNAse due to its low concentration in the solution.

A known method of producing pancreatic RNAase from the pancreas of cattle [US Pat. RU 2180918 C1, IPC ⁷ C12N 9/22. A method of obtaining pancreatic ribonuclease. Date of publication: 03/27/2002. Inventor: Krasnoshtanova A.A., Baurina M.M., Kashkina E.A., Krylov I.A. Patent holder: Russian University of Chemical Technology D.I. Mendeleev]. To increase the yield of the drug, the following allocation scheme was proposed. The enzyme is isolated by extraction of 0.25 N. a solution of sulfuric acid in a ratio of 1: 2 for 24 hours. After separation of the suspended particles by centrifugation, the supernatant is subjected to ultraconcentration through a cellulose acetate membrane UPM 450 with a pore diameter of 450 Å at a pressure of 0.2 MPa. The permeate obtained at this stage is used for repeated extraction. Precipitation is carried out at a pH of 7.8 ÷ 8.0. The precipitate of technical RNA-basics is dissolved in distilled water and the solution is heated to a temperature of 85 ÷ 90 ° C, followed by 10 minutes. After that, RNAase is reprecipitated at pH 7.8–8.0. Precipitated crystals of purified RNA-basics are washed with ethanol and dried in a vacuum oven. Obtained in this way 1.2 g of RNAase with 1 kg of pancreas with an activity of 12000-14000 units / ml The yield of the drug according to the proposed technology is 0.6% by weight of the dry pancreas.

The specified method has several disadvantages, the main of which is the duration of the cleaning process — heating, aging and cooling takes 30 minutes, this stage noticeably worsens the quality of the drug — the purified preparation contains about 30% impurity inactivated proteins, while the yield of the drug does not exceed 0.6 % of the mass of dry pancreas.

The objective of the present invention is to increase the yield of pancreatic RNAase under complex processing of the pancreas, with the preliminary extraction of digestive enzymes such as amylase, lipase and protease complex.

The problem is solved in that a method is proposed for extracting pancreatic ribonuclease from the pancreas, which involves grinding, processing the crushed pancreas with a solution of sodium chloride 4.5 ÷ 6 wt.% In the volume ratio of the pancreas: salt solution 1: (1.5 ÷ 2, 0) at a temperature of 25 ÷ 35 ° C for 2.5 ÷ 3.5 hours, the separation of pancreatic cells by centrifugation, treatment of pancreatic cells with 95% ethyl alcohol for 15 ÷ 24 hours in a volume ratio of 1: (0.85 ÷ 1.3) at a temperature of 15 ÷ 25 ° C, adhesive separation approx pancreatic centrifugation; treatment of pancreatic cells with water, acidified with hydrochloric acid to a pH of 1.0 ÷ 3.0, for 2.5 ÷ 4.5 hours at a temperature of 25 ÷ 35 ° C in a volume ratio of 1: (0.8 ÷ 1.2) , separation of pancreatic cells by centrifugation, extraction of ribonuclease with sulfuric acid solutions with a concentration of 0.15 ÷ 0.35 N. within 3 ÷ 4 hours at a volume ratio of 1: (0.8-1.2) at a temperature of 4 ÷ 6 ° С, separation of suspended particles by centrifugation, concentration of the extract by ultrafiltration on a UPM 10 membrane followed by diafiltration, cooling of the extract to 4 ÷ 6 ° C, precipitation of the enzyme at pH 8.0 ÷ 9.0, washing the obtained extract with ethyl alcohol in a volume ratio of 1: 5 and drying. Unlike the prototype, in this isolation method, the extraction time of RNAase from pancreatic cells is reduced, and it is not necessary to inactivate proteins with protease and DNAase activity, since the resulting extract does not contain these enzymes.

The invention is illustrated by the following examples.

Example 1. 1000 g (250 g absolutely dry) of the crushed pancreas is treated with a solution of sodium chloride 4.5 wt.% (Volume ratio of the pancreas: salt solution 1: 1.5), for 2.5 hours at a temperature of 25 ° C, pancreatic cells are separated by centrifugation. Get a supernatant containing amylase 29.6 units / ml, and spent pancreatic cells (weight 770 g, 167 g absolutely dry), which is treated with 95% ethanol for 15 hours in a volume ratio of 1: 0.85 at a temperature 15 ° C, pancreatic cells are separated by centrifugation. Get a supernatant containing a lipase of 8.77 units / ml, and spent pancreatic cells (weight 400 g, 87 g absolutely dry matter), which are treated with water, acidified with hydrochloric acid to pH 1.0, for 2.5 hours, volume ratio of 1: 0.8 at a temperature of 25 ° C. Get a supernatant containing a complex of proteases 1252 units / ml, and spent pancreatic cells (weight 350 g, 72 g absolutely dry matter), which are treated with a solution of sulfuric acid with a concentration of 0.15 N. for 3 hours at a temperature of 4 ° C (volume ratio 1: 0.8). Suspended particles are separated by centrifugation, the result is a supernatant with an RNAse activity of 100 units / ml. The supernatant is subjected to ultraconcentration through an UPM 10 cellulose acetate membrane with a cut-off of 10 kDa at a pressure of 0.2 ÷ 0.4 MPa. The permeate obtained at this stage is reused at the extraction stage, an ultraconcentrate with an RNAse activity of 2100 units / ml is subjected to additional purification using diafiltration. After that, the concentrate is cooled to a temperature of 4 ÷ 6 ° C and the pH of the medium is adjusted to 8.0 ÷ 9.0. The precipitate of RNAse is recrystallized with ethyl alcohol in a ratio of 1: 5. The obtained purified RNA-ase crystals are dried in a vacuum oven. The result is 0.75 g of ribonuclease with an activity of 12000-14000 units / mg. The yield of the drug according to the proposed technology is 0.3% by weight of dry matter of the original pancreas and 1.04% by weight of dry matter at the stage.

The conditions for pretreatment of the pancreas and extraction of RNAase are summarized in the table.

When implementing this method of isolating RNAse from the pancreas, there is an increase in the yield of the enzyme preparation at the stage of RNAse isolation by 1.3–1.7 times in comparison with the analogue, while the activity of the enzyme preparation RNAse is not reduced. The technological scheme is also simplified by eliminating from the technological process the stages of purification of the enzyme preparation from impurity hydrolases and reducing the extraction time.

The calculation of technical and economic indicators of obtaining RNA-basics according to the prototype (in individual production) and in complex production showed that in the first case, the cost of 1 g of the enzyme is 110 rubles, and for complex processing - 13 rubles. Thus, when organizing integrated production, the cost of the drug is reduced by 8.5 times, and the payback period of additional capital investments does not exceed 1.5 years.

Claims (1)

A method of producing pancreatic ribonuclease from the pancreas, comprising crushing, treating the crushed pancreas with a solution of sodium chloride 4.5 ÷ 6 wt.% In the volume ratio of the pancreas: salt solution 1: (1.5 ÷ 2.0) at a temperature of 25 ÷ 35 ° C for 2.5 ÷ 3.5 h, separation of pancreatic cells by centrifugation, treatment of pancreatic cells with 95% ethyl alcohol for 15 ÷ 24 hours in a volume ratio of 1: (0.85 ÷ 1.3) at a temperature of 15 ÷ 25 ° C, the separation of pancreatic cells by centrifugation, treatment of pancreatic cells with water, acidified with hydrochloric acid to a pH of 1.0 ÷ 3.0 for 2.5 ÷ 4.5 hours at a temperature of 25 ÷ 35 ° C in a volume ratio of 1: (0.8 ÷ 1.2), separation of pancreatic cells by centrifugation, extraction of ribonuclease with sulfuric acid solutions with a concentration of 0.15 ÷ 0.35 N. within 3 ÷ 4 hours at a volume ratio of 1: (0.8-1.2) at a temperature of 4 ÷ 6 ° С, separation of suspended particles by centrifugation, concentration of the extract by ultrafiltration on a UPM 10 membrane followed by diafiltration, cooling of the extract to 4 ÷ 6 ° C, precipitation of the enzyme at pH 8.0 ÷ 9.0, washing the obtained extract with ethyl alcohol in a volume ratio of 1: 5 and drying.