1992 Fiscal Year Final Research Report Summary

PRODUCTION AND SUPPRESSION MECHANISMS OF COLLAGENASE PRODUCED BY Bacteroides speci

Research Project

Project/Area Number	02670862
Research Category	Grant-in-Aid for General Scientific Research (C)
Allocation Type	Single-year Grants
Research Field	Conservative dentistry
Research Institution	HYOGO COLLEGE OF MEDICINE
Principal Investigator	YANAGISAWA Takamichi HYOGO COLLEGE OF MEDICINE, FACULTY OF MEDICINE, LECTURER, 医学部, 講師 (50118585)
Project Period (FY)	1990 – 1992
Keywords	Bacteroides gingivalis / Collagenase gene / Molecular cloning
Research Abstract	Bacteroides gingivalis ATCC 33277 was growing quite well anaerobically ( 85% N_2, 10% H_2, 5% CO_2 ) in GAM broth ( Nissui Pharmaceutical Co.) supplemented with 5 mug/ml hemin and 10 mug/ml menadione. B. gingivalis which was cultivated anaerobically at 37 ﾟC for 4-5 days had the highest collagenolytic activity in the membrane fractions. collagenolytic activity of B. gingivalis was stimulated by Ca^<2+>. Chromosomal DNA from B. gingivalis ATCC 33277 was isolated by the procedure of Maniatis et al. The DNA was particully digested with S_<au> 3A1. Only DNA fragments of 5-10 kilobases were ligated to pUC 18. E.coli JM103 expression vector, and a gene library was constructed. Collagenase positive trans-formants were identified by the collagenolytic and gelatinolytic activity screening methods, however, colonies carrying the Bacteroides collagenase gene were not selected with enzyme activity. Therefore In order to serect colonies with the conventional rabbit anti-collagenase antibody, I tried to purified the collagenase from B. gingivalis. I attempted to purify the collagenase by gel filtration on ephacryl S-300HR followed by Gelatin Sepharose affinity chromatography, but failed. Collagenolytic activity that was the indicator of collagenase from B. gingivalis were decreasing in process of purification. It was suggested the possibility that enzyme activity of collagenase removed from menbrane is unstable exceedingly for the reason that collagenase from B. gingivalis is the membrane bound em-zyme.