Method for Purification of Proteolytic Enzymes

The present invention relates to medical and microbiological industries and, in particular, to the preparation of enzymes and, more specifically, to methods for purification of proteolytic enzymes.

The present invention is useful in the preparation of various proteolytic enzymes of a high purity grade useful in medical practice for the treatment of certain diseases, in medical industry for the production of purified therapeutic and prophylactic serum; and in the food industry for the production of cheese, wine beer. Furthermore, the present invention is suitable for the production of reagents for scientific research.

To produce pure enzymes from naturally-occurring sources such as biological liquids, extracts of organs and tissues, cells of microorganisms, fermentation broth, it is necessary to separate the accompanying proteins including other enzymes, nucleic acids and products of decomposition thereof, low-molecular compounds and salts. Conventional methods of recovery and purification of enzymes are based on differences in physicochemical properties of enzymes and other components of the mixture to be separated such as molecular weight, charge, and solubility. These methods are exemplified by fractional precipitation of proteins by means of salts and organic solvents; gel-filtration, ultra-filtration, on-exchange chromatography. However, each of the prior art methods is insufficiently effective. Satisfactory results are obtained by combination of several methods which, however, is associated with great losses of enzymes and increased production and labor costs. The method of biospecific or affinity chromatography principally differs from the traditional methods in that it is based on the use of the most individualized property of an enzyme, i.e. its ability of selectively reacting with substrates or inhibitors resembling the substrates in the structure thereof.

Addition of a ligand, i.e. substrate or an analogue thereof to an inert carrier provides a specific sorbent which is capable of selectively combining, from a complex mixture of compounds, an enzyme only. After washing the sorbent with a suitable solution for elimination of impurities, the enzyme retained by the sorbent is desorbed by means of a solution which is selected so as to dissociate the enzyme-ligand complex (variation of pH, elevation of the salt concentration, addition of organic solvents). The resulting enzyme product is generally enriched, to a substantial degree, with the desired enzyme and frequently it comprises a pure enzyme. The method makes it possible to substantially increase yields of enzymes and considerably improve quality of purification thereof.

Also known in the art are methods for purification and isolation of proteolytic enzymes which are based on the lon-exchange chromatography. Special mention should be made of the method based on chromatography on modified silochromes. However, this method is insufficiently selective, and is unsuitable for certain technical applications such as recovery of enzymes at a high yield directly from a fermentation broth or gastric juice, i.e. from mixtures with a low concentration of an active enzyme containing a large amount of inorganic and organic impurities including colored ones.

Further known in the art is a method for recovery and purification of neutral and alkaline proteinases from a brewing wort by means of a phenol-formaldehyde sulphocationite Duolite 10. The purification process involves 6 successive stages in addition to dialysis and lyophilization, i.e. double precipitation of the protein fraction with acetone, followed by centrifugation, double chromatography on a DEAE-cellulose, purification on Duolite 10 and precipitation by means of ammonium sulphate.

Duolite 10 in this case is used for the removal of colored impurities from the mixture, not for a direct separation of individual enzymes. The final object of using it is separation of a mixture of neutral and alkaline proteinases with a yield ranging from 33 to 67%.

In this method for recovering enzymes, biospecific sorbents are most extensively used which are prepared from agarose activated by bromocyane and covalently bonded with compounds specific for this particular class of enzymes, i.e. ligands.

In the prior art methods for purification of enzymes use is made of biospecific sorbents prepared by interaction of Sepharose 4B activated with cyanogen bromide with such peptide ligands as methyl ether and amide of .epsilon.-aminocapronyl-D-phenylalanine, methyl ether of .epsilon.-aminocapronyl-L-phenylalanine-D-phenylalanine; N-2,4-dinitrophenylhexamethylene diamine and an antibiotic, i.e. Gramicidin S. This method, however, has certain disadvantages. The main disadvantage resides in that the above-mentioned ligands (except Gramicidin S) can be obtained only by an extensive multi-stages synthesis, whereby the preparation of sorbents is hampered.

Also known in the art is a method for purification of enzymes with the use, as biospecific sorbents, of Sepharose 4B covalently bonded with an antibiotic Gramicidin S (cf. "Biokhimija" (Biochemistry), vol. 41, 2, 294; 1976).

A sorbent containing, as a ligand, a known antibiotic Gramicidin S is more readily available, since the latter is prepared in a relatively simple manner. At present, Gramicidin C, however, finds limited application in medicine and is produced in small quantities. Disadvantages of a sorbent based on Gramicidin S-Sepharose 4B may be exemplified by difficulties encountered in the synthesis of the sorbent owing to a very low solubility of Gramicidin S. Reaction of Gramicidin S with Sepharose 4B activated with bromocyane is conducted in solutions with a high content of dimethylformamide which might partly destroy the structure of Sepharose. In the sorbent synthesis Gramicidin S settles, thus reducing the final product yield and hindering the product purification. Furthermore, the range of application of Gramicidin S-Sepharose 4B is limited to certain classes of proteinases and does not involve practically important serine proteinases.

It is an object of the present invention to overcome the above-mentioned disadvantages.

It is an object of the present invention to provide such a method which makes it possible to selectively recover and purify proteolytic enzymes from naturally-occurring sources.

It is another object of the present invention to provide a simplified method for purification of proteolytic enzymes.

These objects are accomplished by the purification of proteolytic enzymes is performed by way of a biospecific sorption of dissolved proteolytic enzymes on a sorbent, i.e. a derivative of agarose chemically combined with a ligand, followed by elution of the sorbed enzymes with salt buffers. In accordance with the present invention biospecific sorption of proteolytic enzymes is effected using a sorbent prepared by a covalent addition, to agarose, of a naturally-occurring polypeptide bacitracin.

The method according to the present invention is especially efficient for recovering enzymes directly from a fermentation broth containing colored impurities. A colorless enzyme is recovered by means of bacitracin-sepharose; in doing so, a substantial effect is obtained in a single-stage process of its purification owing to the sorbent selectivity. Moreover, the novel biospecific sorbent according to the present invention can be utilized for operation with solutions within a wide range of pH values (of from 1.8 to 8.0) wich enables its use for recovering proteolytic enzymes of different classes including carboxyl, thiol, serine, metal-proteinsases and exopeptidases.

By using the novel biospecific sorbent according to the present invention, it is possible to recover, with a yield of from 74 to 95%, various proteolytic enzymes with an increase of activity of 2 to 500 times depending on purity of the starting compound.

The present invention makes it possible to enlarge the range of applications of the sorbent using a pH interval most suitable for the stability and activity of the enzyme being recovered; besides, there is no necessity in correction of pH of a biological liquid or an extract, wherefrom the enzyme is recovered. Thus, an individual enzyme subtilisine can be now recovered from a coloured culture liquid of Bacillus subtilis in a single stage with a product yield of 90%.

In accordance with the present invention it is advisable that the biospecific sorption be effected using a solution of carboxyl proteinases with a pH value within the range of from 1.8 to 5.6 or from a solution of serine proteinases with a pH within the range of from 6 to 7.5.

In certain cases it is desirable to wash the sorbent with concentrated solutions of salts to remove non-specific sorbed proteins; thus in the purification of pig and horse peps in it is advisable to wash to sorbent with a 1M solution of sodium chloride in an acetate buffer at a pH of 5.0.

Other objects and advantages of the present invention will now become more fully apparent from the following detailed disclosure of the method for purification of proteolytic enzymes and examples illustrating the same.

The method according to the present invention makes it possible to recover and purify proteolytic enzymes from partially purified commercial preparations of enzymes, commercial complex preparations of process enzymes, biological liquids, extracts of organs and tissues, cells and cultural liquids of microorganisms.

The method according to the present invention is based on a biospecific sorption of dissolved proteolytic enzymes on a sorbent, viz. a derivative of agarose chemically, e.g. covalently, combined with a ligand. It is advisable to use, as the ligand in accordance with the present invention, an antiobiotic bacitracin comprising a naturally-occurring cyclododecapeptide containing three D-aminoacids. It is a non-competing inhibitor of the proteinase series.

It is likely that this is responsible for a specific interaction of the novel sorbent with proteolytic enzymes of different classes. Bacitracin is readily soluble in water. A great number of r660000000000000000000000000000000000000000000000000000000000000000