Claims for Patent: 5,252,481

Summary for Patent: 5,252,481

Title:	Mutant of bacterium Clostridium histolyticum, a process for the obtaining thereof, and its use in the production of clostripain-free collagenase
Abstract:	A mutant of bacterium Clostridium histolyticum, designated Clostridum histolyticum K26 clo 88 of the following characteristics: the lack of a motility system which is a fundamental characteristic of the parenteral strain; and, the inability to biosynthesize the accompanying enzyme clostripain. A process of obtaining the above mutant and its use in a process for the production of clostripain-free collagenase are also provided.
Inventor(s):	Holjevac; Milan (Zagreb, YU), Udovicic; Ivan (Zagreb, YU), Cizmek; Sonja (Zagreb, YU), Sojak-Derkos; Vlasta (Zagreb, YU), Gamulin; Stjepan (Zagreb, YU), Delic; Vladimir (Zagreb, YU)
Assignee:	Pliva Farmaceutska, Kemijska (YU)
Application Number:	07/733,764
Patent Claims:	1. A biologically pure, non-motile culture of Clostridum histolyticum, NCAIM B(P) 001145, which produces clostripain-free collagenase. 2. A process of obtaining a biologically pure, non-motile culture of Clostridium histolyticum, NCAIM B(P) 001145 which produces clostripain-free collagenase wherein said process comprises treating spores of said Clostridium histolyticum NACIM B(P) 001145 with the mutagenic agent, N-methyl-N'-nitro-N-nitrosoguanidine, for 45 to 90 minutes, at about room temperature, then separating the spores by centrifugation, suspending the spores in a physiological solution, inoculating the spores into a solid medium, and incubating at 37 degrees for about 3 days under anaerobic conditions to obtain said strain. 3. A process for the production of clostripain-free collagenase, which comprises cultivating Clostridium histolyticum NCAIM B(P) 001145 in a liquid nutrient medium containing proteose-peptone, enzymatically hydrolyzed proteins of casein and soy, a vitamin solution, a reducing agent, and nitrogen, for 10 to 24 hours at a temperature ranging from 28 degrees celsius to 37 degrees celsius and a pH of 7.2 to 8.9, under anaerobic submersed conditions, and with a combination of precipitants isolating purified clostripain-free collagenase. 4. The process of claim 3 which further comprises stabilizing said purified clostripain-free collagenase by adding to said purified collagenase, carbohydrate in a concentration ranging from 5.times.10.sup.-3 M to 2.5.times.10.sup.-2 M and soluble protein in a concentration of 10 to 50 mg/ml of said purified collagenase. 5. The process of claim 3 wherein said vitamin solution contains riboflavin. 6. The process of claim 3 wherein said reducing agent is Na-thioglycolate. 7. The process of claim 5 wherein said combination of precipitants contains calcium chloride, acetone, ammonium sulfate and calcium phosphate gel.

Details for Patent 5,252,481

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2010-10-12