Method for preparing collagenase preparation

FIELD: biotechnology, preparative biochemistry, enzymes.

SUBSTANCE: invention relates to a method for preparing the highly purified and highly active enzyme preparation collagenase. Method for preparing the collagenase preparation involves homogenization of hepatopancreas tissue of industrial species crabs in buffer aqueous solutions, isolation of collagenase solution, its purification by successive two-stage ultrafiltration on membranes providing cutting off inert substances and lyophilic drying of the preparation. Ultrafiltration is carried out on poly-fibrous membranes with size of pores 15 kDa and concentrate prepared at the 1-st stage of ultrafiltration is diluted with water in the ratio = 1:(5-10), and concentrate prepared at the 2-d stage of ultrafiltration is subjected for additional successive microfiltration on membranes with size of pore 100 kDa and ultrafiltration on membranes with size of pores 15 kDa. Homogenization of hepatopancreas of industrial species crabs is carried out in acetate-ammonium buffer containing calcium acetate at pH 6.4-6.7 followed by precipitation of collagenolytic enzymes with ammonium sulfate at 60-70% degree of saturation. Invention provides increasing the total collagenolytic activity, specific activity of the preparation and its purity. The collagenase preparation can be used in medicine, veterinary science, biochemistry and food industry and for research aims also.

EFFECT: improved preparing method.

2 cl, 2 ex

 

The invention relates to biotechnology, in particular the production of highly purified and highly active enzyme preparations, which can be used in medicine, veterinary medicine, biochemistry and food industries, as well as for research purposes.

The liver crabs, especially crab, rich in proteolytic enzymes, including collagenases. The liver is available, cheap and non-toxic raw material for the production of enzyme preparations.

A method of obtaining proteolytic complex that includes collagenase activity, from the ground pancreatectomy terrestrial or aquatic animals. Extraction of complex exercise 1 mm solution of calcium chloride containing 0.2% detergent within 12 hours the Precipitate was separated by separation by centrifugation and supernatant removal of the lipid fraction. Clean fine particles are micro filtration on membranes with a pore diameter of 200 μm, the concentration of the obtained solution is performed by ultrafiltration on a membrane with a pore diameter of 15 kDa. A concentrated solution is sterilized by filtration and dried by sublimation. Collagenolytic activity for acid-soluble collagen - 672 u/mg (RF Patent (RU) No. 2034028, C 12 N 9/64, 1995.04.30).

The disadvantages of this method is the use of high con is entrace surfactants, which undoubtedly reduce the activity of enzymes, as well as adversely affect the environment, requiring the development of special methods of disposal of waste. A significant disadvantage is the lack of stage desalting of the preparation and application of freeze-drying instead of freeze. In the specific activity of the obtained product with the activity of the technical preparations.

A method of obtaining an enzyme preparation having collagenolytic activity, releasing enzyme from hepatopancreas commercial crab by tissue homogenization in a buffer solution with a pH of 6-9,5, containing non-ionic detergent in an amount of 0.1-5% and 0.1-2.0 M solution of chloride of an alkali metal. After separation of the precipitate and a layer of lipid solution is purified by filtration on membranes with pore diameter of 0.45 μm, the concentration of the obtained solution is performed by ultrafiltration on a membrane with a pore diameter of 5 kDa. A concentrated solution freeze-dried. Specific proteolytic activity is not lower than 10000 units/mg, a specific activity of individual collagenases significantly less than the specific activity of total drug (EP No. 0402321, C 12 N 9/64, 1990.12.12).

In the specified way to present the same drawbacks as the previous, high ionic strength detergent and salt n is sodium, no stage desalting. Collect the fraction that includes a very broad range of proteins with molecular masses from 15-1200000 kDa, which contains not only collagenase ( 20-25,5 kDa), but also a large number of synthesized proteins, including inhibitors.

A method of obtaining proteolytic complex that includes collagenase activity, from frozen hepatopancreas crab, homogenized by stirring in 0.1-1.0 M solution of alkali metal chloride at room temperature. Then insoluble particles separated by serial filtration through filters with a pore size of 10 to 0.5 and 0.1 mm After separation of the precipitate, the solution is purified by filtration on membranes with pore diameter of 0.45 μm, permeable to proteins with MM 1200 kDa. Removal of emulsified fat, cellular debris and microorganisms concentration of the obtained solution is performed by ultrafiltration on a membrane with a pore diameter of 10 kDa. A concentrated solution freeze-dried. Specific collagenolytic activity is not less than 2000 Mandl units/mg (patent RF №2096456, With 12 N 9/48, 1998. 20.11).

This method is slightly more economical than the previous one, however the product contains a large amount of impurity proteins not related to collagenolytic enzymes, which leads to a small specific collagenases act is vnesti.

The closest in technical essence to the invention is a method for collagenase, as described in the patent of Russian Federation №2039819 (C 12 N 9/48, 1995.20.07). As a raw material for production of collagenase used the liver of commercial crabs, the selection is carried out by mixing the homogenate of hepatopancreas crabs, resulting autolitic hydrolysis by endoprotease with a solution of chitosan pH of 2.5 to 6.5, and the solution obtained after separation of the insoluble material is sequentially subjected to a 2-stage ultrafiltration first on the membrane, cut-off impurity substances with M.M. 100 kDa or more, collecting passes through the membrane solution, and then on the membrane, cut-off substances with MM 30 kDa, collecting the enzyme is not passing through the membrane. The solution containing the enzymatic activity, lyophilizer. The specific activity of collagenase 9800-11200 units/mg protein.

The disadvantages of the method include low purification of the target product. This, apparently, is associated with loss of active collagenase arising at the stage of adding an acidic solution of chitosan (pH of 2.5 to 5.0), which leads to partial inactivation collagenolytic enzymes, and loss of collagenase when using membranes with a pore size of 30 kDa. The recent work on the identification of genes collagenolytic enzymes hepatopancreaticobiliary crab, the Fiddler crab, shrimp, it was shown that their is 21.5 MM-25,0 kDa, therefore, when the pore size of the membrane 30 kDa collagenase largely pass through the membrane and the total activity of the final preparation is reduced.

The objective of the invention is to increase the total collagenolytic activity of the drug, specific activity and purity of the drug.

This object is achieved in that in the method of producing drug collagenase, including homogenization of hepatopancreas commercial crab species in buffered aqueous solutions, the selection of a solution of collagenase, cleaning his sequential 2-stage ultrafiltration on membranes, cut-off impurity substances and freeze-dried preparation, the ultrafiltration is performed on polivolokonnykh membranes with pore size of 15 kDa, and the concentrate obtained in the 1st stage ultrafiltration, diluted with water in ratio of 1:5-10, and the concentrate obtained in the 2nd stage ultrafiltration, optionally sequentially subjected to filtration on membranes with pore size of 100 kDa and ultrafiltration membranes with a pore size of 15 kDa.

Homogenization of hepatopancreas commercial species of crabs are in the acetate-ammonium buffer containing calcium acetate at pH 6.4 and 6.7, with the subsequent deposition collagenolytic enzymes by ammonium sulfate 6-70% of saturation.

The essence of the method consists in the following. As the source of collagenase used the liver of commercial species of crab which is homogenized in 0.1 M ammonium acetate pH 6.4, containing 0.1-1.0 mm calcium acetate, separating the extract by centrifugation, add ammonium sulfate 60-70% saturation, and the precipitate containing collagenase, separated by centrifugation, dissolved in water with addition of 0.1-1.0 mm calcium acetate to obtain the true solution. Concentration, desalting, cleaning is performed as follows. The concentration of the solution is performed on membrane with a total filtration area 2 m2(HLIs, Mytischi) using polivolokonnykh membranes and pore size of 15 kDa. The resulting concentrate is diluted with distilled water 5-10 times and again concentrated. In the 2-stage ultrafiltration occurs preconcentration and desalting complex preparation of collagenases. The resulting solution was further subjected to purification by membrane for microfiltration with pore size of 100 kDa to remove high molecular weight impurities presumably carbohydrate nature, as well as inhibitors. Target product contained in the filtrate, which is again sent to ultrafiltration, and the concentrate was washed with water and freeze-dried.

The result PTS is on an enzyme high collagenolytic activity. Output by activity - 850-110%, determined by the method of Mandl's account. The specific activity of collagenase 48000-56000 Mandl units/mg protein.

Example 1.

20 kg of hepatopancreas homogenized in 0.1m-ammonium acetate pH 6.4, then the precipitate was separated by centrifugation at 10,000 rpm, the supernatant add ammonium sulfate to 60% saturation, and after 20 h and centrifuged again. The precipitate (sulfate paste) is dissolved in 15 liters of distilled water to obtain the true solution. The volume of the resulting solution is brought to 100 liters, mix thoroughly. The concentration of the solution is performed on membrane (HLIs, Mytischi) privalochnoj a membrane with a pore size of 15 kDa and a total filtration area 2 m2. The resulting concentrate by volume of 10 l was adjusted with water to 50 l, and again concentrated to 10 HP Then the concentrate is poured, and the installation is washed twice with distilled water for flushing remaining on the fibers of the collagenase. Wash water combined with the concentrate. As a result of operations occurs desalting and concentration of the original drug collagenase. The resulting solution was further subjected to purification by membrane for microfiltration PELLICON, "Millipore" with a pore size of 100 kDa, the target product is contained in the filtrate. The filtrate is then concentrated as described above, is about 10 l, three times washed with distilled water, combined with leaching waters from privalochnogo apparatus, the liquid is frozen to -40°and dried by freeze-drying TG-50, Germany.

Output by activity 850%, specific activity 48000 Mandl units/mg protein.

Example 2.

500 grams of the drug collagenase production company "Bioprogress" dissolved in 15 liters of distilled water to obtain the true solution. The volume of the resulting solution was adjusted to 50 l, mix thoroughly. The concentration of the solution is performed on membrane (HLIs, Mytischi) privalochnoj a membrane with a pore size of 15 kDa and a total filtration area 2 m2. The resulting concentrate by volume of 10 l was adjusted with distilled water to 50 l, and again concentrated to 10 HP Then the concentrate is poured, and the installation is washed twice with distilled water for flushing remaining on the fibers of the collagenase. Wash water combined with the concentrate. This results in demineralization of the original drug collagenase.

The resulting solution was further subjected to purification by membrane for microfiltration PELLICON, "Millipore" with a pore size of 100 kDa. Target product contained in the filtrate. The filtrate is then concentrated as described above, up to 10 l, three times washed with distilled water, the liquid is frozen to -40°and dried on sublimat the Onna drying TG-50, Germany. Output by activity 1100%, specific activity 56000 Mandl units/mg protein.

Thus, the proposed method through the use of the optimal pore size membranes (15-100 kDa) allows to increase up to 850-1100% yield of the target product by removing inhibiting admixtures to increase the specific activity collagenolytic enzymes 4-6 times compared with the known method. Conducting ultrafiltration membranes with a pore size of 15 kDa allows you to remove low molecular weight impurities, but to fully preserve collagenolytic enzymes and exercise at the same time desalting and concentration of drug that results in concentrated solutions, drying times are significantly reduced. Replacement stage of deposition of insoluble impurities sour expensive solution of chitosan on the salting out of proteins cheap ammonium sulfate at acid and neutral pH promotes the preservation activity collagenolytic enzymes and cheaper process. In addition, the use of acetate and ammonium sulfate for extraction and vysalivaniya collagenolytic enzymes more successful from the point of view of environmental safety of the process, since solutions of ammonium salts when diluted can be used to fertilize plants.

1. The method of producing drug number is agensy, including homogenization of hepatopancreas commercial crab species in buffered aqueous solutions, the selection of a solution of collagenase, cleaning his sequential 2-stage ultrafiltration on membranes, cut-off impurity substances, and freeze-dried preparation, characterized in that the ultrafiltration is performed on polivolokonnykh membranes with pore size of 15 kDa, and the concentrate obtained in the 1st stage ultrafiltration, diluted with water in ratio of 1:5-10, and the concentrate obtained in the 2nd stage ultrafiltration, optionally subjected sequentially filtration on membranes with pore size of 100 kDa and ultrafiltration membranes with a pore size of 15 kDa.

2. The method of producing drug collagenase according to claim 1, characterized in that the homogenization of lead acetate-ammonium buffer containing calcium acetate at pH 6.4 and 6.7, with the subsequent deposition collagenolytic enzymes by ammonium sulfate 60-70% saturation.



 

Same patents:

FIELD: biotechnology, amino acids.

SUBSTANCE: method for preparing taurine involves milling a biological raw, its treatment and isolation of taurine. Waste from meat-processing industry is used as a raw and treatment is carried out with sodium hydroxide solution. Prepared solution is treated successively with protosubtilin, homogenized mass of cattle pancreas or liver. Isolation of taurine from hydrolyzate is carried out by sorption on anion-exchange resin AB-17-2 followed by precipitation of product with 10-fold hydromodulus of ethyl alcohol and the following purification. Method provides increasing yield of taurine in using inedible raw.

EFFECT: improved preparing method, increased yield.

3 ex

FIELD: biotechnology, biochemistry, enzymes.

SUBSTANCE: invention relates to a method for activation of pancreas proteolytic enzymes from slaughtering animals. Proposed method provides elevating the enzymatic activity of pancreas proteolytic enzymes, reducing time and enhancing the effectiveness of activation process. Invention can be used in preparing protein hydrolyzates in meat processing.

EFFECT: improved method for activation.

The invention relates to biotechnology, can be used in medicine, cosmetology, dermatology, biochemistry and food industries, as well as for research purposes
The invention relates to biotechnology, in particular the production of highly purified, highly active enzyme preparations, which can be used in medicine, biochemistry and food industries, as well as for research purposes

the enzyme" target="_blank">

The invention relates to the field of medicine and biotechnology and relates to DNA sequences encoding human proteins TX and The related converging interleukin-1-beta enzyme

by activity" target="_blank">

The invention relates to the production of biologically active IL-1protease using recombinant DNA technology and can be used in medicine

The invention relates to native proteins schemes complement modified such that the protein is able to form stable C3 to Mac
The invention relates to biochemistry, in particular to the allocation method carboxypeptidase from porcine pancreas, and can be used to obtain genetically engineered products in biotechnology

FIELD: medicine.

SUBSTANCE: raw material obtained out of gray substance homogenate should be supplemented with ionol (2.6-di-tret-butyl-4-methylphenol) at quantity necessary to prepare 0.0001 M final solution. Addition of this substance to raw material enables to keep its high activity being equal to 12.5-16.0 sec and enables to prolong its storage terms at (-40 ± 2) C up to 10 wk before carrying out sublimation and prevent the decrease of activity during sublimation process.

EFFECT: higher efficiency.

1 ex, 2 tbl

The invention relates to biotechnology, can be used in medicine, cosmetology, dermatology, biochemistry and food industries, as well as for research purposes
The invention relates to biotechnology, in particular the production of highly purified, highly active enzyme preparations, which can be used in medicine, biochemistry and food industries, as well as for research purposes
The invention relates to biochemistry, in particular to the allocation method carboxypeptidase from porcine pancreas, and can be used to obtain genetically engineered products in biotechnology
The invention relates to biotechnology, can be used in the meat industry for the hydrolysis and modification of collagen containing raw material

The invention relates to a method of stabilizing an aqueous solution or suspension of metalloprotease, as well as to aqueous solution of metalloprotease stabilized to better use, storage and transportation of such enzyme

The invention relates to medicine and concerns the preservation of the activity of the raw materials for the manufacture of soluble thromboplastin

The invention relates to medicine and the receipt of raw materials for the manufacture of soluble thromboplastin

FIELD: medicine.

SUBSTANCE: raw material obtained out of gray substance homogenate should be supplemented with ionol (2.6-di-tret-butyl-4-methylphenol) at quantity necessary to prepare 0.0001 M final solution. Addition of this substance to raw material enables to keep its high activity being equal to 12.5-16.0 sec and enables to prolong its storage terms at (-40 ± 2) C up to 10 wk before carrying out sublimation and prevent the decrease of activity during sublimation process.

EFFECT: higher efficiency.

1 ex, 2 tbl

Up!