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Purification and characterization of a streptomycete collagenase
Author(s) -
Chakraborty R.,
Chandra A. L.
Publication year - 1986
Publication title -
journal of applied bacteriology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 0021-8847
DOI - 10.1111/j.1365-2672.1986.tb04294.x
Subject(s) - chemistry , biochemistry , collagenase , enzyme , iodoacetamide , sephadex , mastoparan , enzyme assay , size exclusion chromatography , cysteine , streptomyces , bacteria , biology , receptor , g protein , genetics
A soil streptomycete designated as Streptomyces sp. A 8 produced an extracellular collagen hydrolysing enzyme that appeared to be ‘true collagenase’as it degraded native collagen under physiological conditions and cleaved the synthetic hexapeptide 4‐phenylazobenzyloxycarbonyl‐L‐prolyl‐L‐leucyl‐glycyl‐L‐prolyl‐D‐arginine into two tripeptides. The enzyme was purified by diethyl aminoethyl cellulose chromatography and Sephadex G‐150 gel filtration. The purified enzyme had an apparent molecular weight of about 75000 by SDS‐polyacrylamide gel electrophoresis. Treatment with lithium chloride did not dissociate it into subunits. A strong inhibition was observed with chelating agents such as α‐α‐dipyridyl and 8‐hydroxyquinoline. Ethylene diamine tetracetate completely inhibited the enzyme activity. Among the cations tested only Ca 2 + and Mg 2 + enhanced the collagenase activity. Heavy metal ions like Pb 2 + , Ag + , Cu 2 + and Zn 2 + strongly inhibited the enzyme. The EDTA inhibition could be reversed with Ca 2 + . Cysteine and reduced glutathione caused significant reduction in enzyme activity. Parachloromercuribenzoate and iodoacetamide had no effect on the collagenase. Amino acid analysis revealed the absence of cysteine and tyrosine. Many of the properties were the same as collagenases of Clostridium histolyticum and Vibrio alginolyticus.