Abstract
Amylase zymography was carried out for the detection of amylases produced by a Geobacillus stearothermophilus strain isolated from the Thermal Center “Las Trincheras” in Venezuela. Zymography is an electrophoretic technique used to study hydrolases by means of thin gels containing copolymerized-specific substrates, under nonreducing conditions. In this study, 0.1% starch was incorporated into the gel as substrate. The formation of clear zones against a dark background in the gel stained with iodine indicated the presence of amylolytic activity. The thermophilic bacteria released several extracellular amylases to a selective growth medium supplemented with 1% soluble starch at 55 °C after 40 h incubation. The amylolytic enzymes showed an optimum temperature of 60 °C and an optimum pH at 6.0. The amylases were partially purified by cold acetone precipitation followed by two chromatographic techniques. These purified amylases showed different molecular masses which were determined by sodium dodecyl sulfate gel electrophoresis and confirmed by zymography.
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Acknowledgments
This work was supported by Project Pem 2001002268 from the Venezuelan Fondo Nacional de Ciencia, Tecnología e Innovación.
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Andrades, A., Contreras, L.M. (2017). Amylase Zymography. In: Wilkesman, J., Kurz, L. (eds) Zymography. Methods in Molecular Biology, vol 1626. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7111-4_29
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DOI: https://doi.org/10.1007/978-1-4939-7111-4_29
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