EXTRACELLULAR YALURONIDASE FROM STREPTOMYCES KOGANEIENSIS
The invention relates to Streptomyces koganeiensis ATCC 31394 hyaluronidase having molecular weight of 21.6 kDalton, which has hyaluronidase activity and stability markedly higher than those of the hyaluronidase obtained from such microorganism to date. The invention further relates to a process for the isolation and purification of said hyaluronidase and its use for the preparation of pharmaceutical compositions or as an analytical reagent.
1 . Hyaluronidase from Streptomyces koganeiensis ATCC 31394 comprising the N-terminal amino acid sequence shown in SEQ ID No. 1.
2 . Hyaluronidase according to claim 1 having a molecular weight of 21.6 kDa, an isoelectric point (pI) ranging from 4.4 to 4.8 and an enzymatic activity equal to or higher than 40,000 I.U./mg.
3 . A process for the preparation of the hyaluronidase according to claim 1 comprising the following steps:
a) submitting the supernatant obtained from the fermentation of Streptomyces koganeiensis ATCC 31394 to weak cation-exchange chromatography and isolating the protein fraction with hyaluronidase activity;
b) submitting the protein fraction with hyaluronidase activity from step a) to diafiltration and strong anion-exchange chromatography and isolating the protein fraction with hyaluronidase activity;
c) submitting the protein fraction with hyaluronidase activity from step b) to strong cation-exchange chromatography and isolating the protein fraction with hyaluronidase activity;
d) submitting the protein fraction with hyaluronidase activity from step c) to strong anion-exchange chromatography and isolating the protein fraction with hyaluronidase activity.
4 .- 7 . (canceled)
8 . A pharmaceutical or veterinary compositions containing the hyaluronidase of claim 1 in admixture with suitable excipients and/or carriers.
9 . The composition according to claim 8 in the form of injectable preparations or of topical preparations for epidermal, transdermal or ophthalmic application.
10 . The compositions according to claim 8 further containing one or more active principles selected from: steroidal and non-steroidal antinflammatories, antitumor agents, allergens, local anesthetics, antibiotics, monoclonal antibodies, cytokines, enzymes and sulphated hyaluronic acid.
11 . The compositions according to claim 10 in which the active principle is sulphated hyaluronic acid and the topical composition for epidermal application is in the form of cream, gel, ointment or spray.
12 . A reagent in biochemical assays for the quali/quantitative determination of hyaluronic acid, said reagent comprising the hyaluronidase according to claim 1 .
13 . The composition according to claim 8 for use in the treatment of diseases, in which treatment degradation of hyaluronic acid in the affected tissues or organs is advantageous or desirable.
14 . The composition according to claim 8 wherein the disease is selected from edema, inflammation, chilblain, solid tumors, IgE-mediated allergies, oral diseases and spontaneous vitreous haemorrhages.
15 . The composition according to claim 8 wherein the disease is bovine mastitis.
16 . A pharmaceutical or veterinary composition containing the hyaluronidase of claim 2 in admixture with suitable excipients and/or carriers
17 . A method for treating a disease selected from edema, inflammation, chilblain, solid tumors, IgE-mediated allergies, oral diseases and spontaneous vitreous haemorrhages, comprising administering the hyaluronidase according to claim 1 to a subject in need thereof.
18 . The method according to claim 17 wherein the disease is bovine mastitis.