The isolation, identification, characterization and regulation of alpha-amylase in Sulfolobus solfataricus

Sulfolobus solfataricus is a hyperthermophilic acidophile. It is an obligate aerobe and member of the crenarchaeota. It uses both chemolithoautotrophic and heterotrophic growth and grows on both simple and complex carbohydrates. It produces a variety of glycosyl hydrolases one of which is a secreted alpha-amylase. This alpha-amylase was isolated, identified and characterized. The enzyme was identified as being low abundance and transcriptionally regulated with a second level of regulation characterized by partitioning to the cell envelope.; It was necessary to develop methods for large scale cultivation and genetic manipulation of S. solfataricus as well as purification techniques for this low abundance enzyme. Assays for detection of amylase activity were optimized for both culture supernatant and concentrated fractions. The lack of sensitivity of the assay even after optimization required the production of antibodies. These antibodies made it possible to localize active amylase to the supernatant and inactive amylase to the cell envelope.; Attempts to express a recombinant form of the enzyme in S. solfataricus and Escherichia coli were unsuccessful. Bioinformatic analysis was unable to identify either a DNA binding site for a proposed positive regulator identified as Car or S-layer homology domains that are known to mediate binding of proteins to the S-layer proteins of bacteria and archaea. A mechanism that makes use of hydrophobic regions for possible membrane tethering is a part of the tethering model. Production of the S. solfataricus alpha-amylase is transcriptionally regulated while activity is regulated by partitioning to the cell envelope prior to release into the culture media. Both methods of regulation appear to require an inducer such as starch.