Intracellular alkaline proteases produced by thermoacidophiles: detection of protease heterogeneity by gelatin zymography and polymerase chain reaction (PCR).
In this study 24 thermoacidophilic archeal and bacterial strains isolated from hot-springs and hot-soils were screened for their ability to produce intracellular alkaline proteases. The protease activities of the strains, based on azocasein hydrolysis, showed a variation from 0.6 to 5.1 U. The cell extracts of three most potent producers were further examined and it was found that their proteases exhibited maximum activity at 60-70 degrees C and showed a pH optimum over a range of pH 7.0-8.5. Gelatin zymography revealed that two of the selected archeal strains produced multiple active SDS-resistant proteases. On the other hand, PCR amplification of alkaline serine protease gene sequences of total DNA from all isolates yielded four distinct amplification fragments of 650, 450, 400 and 300 bp, which might have been derived from different serine protease genes.
Department of Biological Sciences, Middle East Technical University, Ankara, Turkey. biosemra@metu.edu.tr
Semra
S
Kocabiyik
Bilge
B
Erdem
D019641:DNA, Archaeal / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D019605:Euryarchaeota / Q000145:classification / Q000201:enzymology* / Q000235:genetics*; D005780:Gelatin; D019848:Gene Expression Regulation, Archaeal; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D020539:Sequence Analysis, Protein; D012697:Serine Endopeptidases / Q000145:classification / Q000235:genetics*; D013045:Species Specificity; D013696:Temperature
D019641:DNA, Archaeal; D005780:Gelatin; D012697:Serine Endopeptidases