Novel thermostable serine collagenase from Thermoactinomyces sp. 21E: purification and some properties.
A thermophilic actinomycete strain Thermoactinomyces sp. 21E producing a highly thermostable serine collagenase was isolated from Bulgarian soil. The collagenase, produced extracellular by Thermoactinomyces sp. 21E, was purified to homogeneity by heat treatment, ultrafiltration, saturation with ammonium sulfate and gel filtration chromatography with a 101-fold increase in specific activity and 58% recovery. The collagenase has a relative molecular mass of 50000 by SDS-PAGE. The optimum temperature for the enzyme activity was 60-65 degrees C in the absence of Ca(2+) and 70-75 degrees C in the presence of Ca(2+). About 40% of the original activity remaining after incubation at 85 degrees C for 30 min in the presence of Ca(2+). The optimum pH for the enzyme activity was 9.0-9.5 and the enzyme was stable for 1h at 70 degrees C in the pH range from 7.5 to 12.5. The collagenase was strongly inhibited by active-site inhibitors of serine protease PMSF and DFP, which indicated that the enzyme is serine protease. The enzyme activity was completely inhibited by Hg(2+), Cu(2+) and Fe(2+). However, Ca(2+ )strongly activated the collagenase activity. The collagenase from Thermoactinomyces sp. 21E showed high activity toward type I collagen, acid-soluble collagen, gelatin and Pz-PLGPR. However, elastin for collagenase was inert as substrate. The properties of the collagenase from strain 21E suggest that this enzyme is a new collagenolytic protease that differs from the collagenases and serine proteases reported so far.
Petrova
Detelina Hristova
DH
Laboratory of Virology, University of Sofia St. Kliment Ohridski, Faculty of Biology, Boulevard Dragan Tzankov 8, 1164 Sofia, Bulgaria. detyp@yahoo.com
Shishkov
Stoyan Angelov
SA
Vlahov
Stoyan Stefanov
SS
D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D017364:Collagenases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D061965:Matrix Metalloproteinase Inhibitors; D008670:Metals / Q000494:pharmacology; D008848:Micromonosporaceae / Q000201:enzymology*; D012694:Serine / Q000378:metabolism*; D012988:Soil Microbiology; D013379:Substrate Specificity; D013696:Temperature
D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D061965:Matrix Metalloproteinase Inhibitors; D008670:Metals; D012694:Serine; D017364:Collagenases