Heat Stable Alkaline Phosphatase from Thermophiles

Enzymes used as the label for most non-radioactive assays and in biosensors, are the most unstable component of these systems. As immunoassays and biosensors move from the tightly controlled laboratory environment to demanding field conditions, the need for a more stable enzyme becomes critical. Alkaline phosphatase is the most widely used enzyme for these types of applications. A heat stable enzyme would not only allow use over a range of temperatures but would also increase the shelf life of these detection systems. Alkaline phosphatase is currently obtained from mammalian sources and is not stable at elevated temperatures. Thermophilic microorganisms producing heat stable alkaline phosphatase have been isolated by J. K. Research. The objective of this proposed research is to select a thermophile producing heat stable alkaline phosphatase and characterize the enzyme. Media will be developed to enhance enzyme production. A purification scheme will be optimized for maximum recovery of purified alkaline phosphatase. Sufficient heat stable enzyme will be produced to determine kinetic parameters and the most suitable reaction conditions.