EXTRACELLULAR HYALURONIDASE FROM Streptomyces koganeiensis Russian patent published in 2015 - IPC C12N9/26 A61K38/49 C12R1/465 

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of biochemistry and biotechnology. Claimed hyaluronodase from Streptomyces koganeiensis ATCC 31394, including the N-terminal amino acid sequence AGENGATTTFDGPVA and having a molecular weight of 21.6 kDa, an isoelectric point (pI) in the range from 4.4 to 4.8 and enzymatic activity higher than 40000 IU/mg and lower than 50000 IU/mg. Described pharmaceutical or veterinary composition, containing the said hyaluronidase in a mixture with at least one suitable auxiliary substance and/or a carrier for treating diseases or states, for which decomposition of hyaluronic acid in tissues and organs is required or useful. The application of the said hyaluronidase as a reagent for the quantitative/qualitative determination of hyaluronic acid is claimed. The method of obtaining hyaluronic acid in accordance with the claimed invention includes the following stages: a) concentration by ultrafiltration through a filter with 10 kDA cut-off, dialysis of the supernatant, obtained after the fermentation of Streptomyces koganeiensis ATCC 31394, chromatography on a weak cation-exchanger by the elution of the column with a sodium-acetate buffer at pH 4.5 and separation of a protein fraction with hyaluronidase activity; b) diafiltration and chromatography of the protein fraction with hyaluronidase activity from stage a) on a strong anion-exchanger, separation of the protein fraction with hyaluronidase activity by the elution of the column with the buffer TRIS-HC1 and NaCl at pH 8 and separation of the protein fraction with hyaluronidase activity; c) chromatography of the protein fraction with hyaluronidase activity from stage b) on a strong cation-exchanger by the elution with a sodium phosphate buffer at pH 4.8 and separation of the protein fraction with hyaluronidase activity; d) chromatography of the protein fraction with hyaluronidase activity from stage C on a strong anion-exchanger after bringing pH to 5 and elution of the column with a sodium acetate buffer, with the further reduction of pH to 4 and elution of the column with the buffer solution of sodium acetate with obtaining in this way of two different protein fractions with different hyaluronidase activity, and selection of one of them, demonstrating higher hyaluronidase activity.

EFFECT: invention makes it possible to obtain the separated protein of hyaluronidase, demonstrating higher hyaluronidase activity.

11 cl, 9 dwg, 5 tbl, 3 ex