Intracellular alkaline proteases produced by thermoacidophiles: detection of protease heterogeneity by gelatin zymography and polymerase chain reaction (PCR)

• Published in: Bioresource technology 2002-08, Vol.84 (1), p.29-33
• Main Authors: Kocabıyık, Semra, Erdem, Bilge
• Format: Article
• Language: English
• Subjects: Alkaline serin protease; Archaea; Bacteria; Biological and medical sciences; Biotechnology; Cells; DNA; DNA, Archaeal - genetics; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Enzyme engineering; Euryarchaeota - classification; Euryarchaeota - enzymology; Euryarchaeota - genetics; Fermentation; Fundamental and applied biological sciences. Psychology; Gelatin; Gene Expression Regulation, Archaeal; Gene Expression Regulation, Enzymologic; Hydrogen-Ion Concentration; Hydrolysis; Methods. Procedures. Technologies; pH effects; Polymerase Chain Reaction; Production of selected enzymes; Sensitivity and Specificity; Sequence Analysis, Protein; Serine Endopeptidases - classification; Serine Endopeptidases - genetics; Species Specificity; Temperature; Thermoacidophiles
• ISSN: 0960-8524; 1873-2976
• DOI: 10.1016/S0960-8524(02)00019-6

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 °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.