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The alpha-glucosidase of Sulfolobus solfataricus: Characterization of the gene and its activity
Abstract
Sulfolobus solfataricus is a prokaryotic thermoacidophile and is phylogenetically classified within the crenarchaeotal subdivision of the archaea. It has the ability to grow both lithoautotrophically utilizing sulfur and carbon dioxide as the energy and carbon source respectively or chemoheterotrophically on a wide variety of sugars as the sole carbon and energy source. The $\alpha$-glucosidase of S. solfataricus was chosen to be a model enzyme for an investigation of chemoheterotrophic growth in S. solfataricus. To accomplished this the $\alpha$-glucosidase was characterized and its gene, malA, was cloned and characterized. In the work described here the role of $\alpha$-glucosidase in the heterotrophic growth of S. solfataricus was examined. The first step in the investigation was to purify the $\alpha$-glucosidase. The $\alpha$-glucosidase from S. solfataricus is a homotetramer with an apparent subunit mass of 80 kDa. The enzyme liberates glucose from maltose and maltooligomers at a rate that decreases with increasing substrate length. Maximal activity of the purified enzyme was reached at 105$\sp\circ$C, it exhibited half lives of 11 hours at 85$\sp\circ$C, 3.0 hours at 95$\sp\circ$C and 2.75 hours at 100$\sp\circ$C, and a pH optima of 4.5. The enzyme exhibited novel chromatographic properties requiring pretreatment with 6 M guanidine hydrochloride to electrophorese as a monomer. At reduced temperatures, only the fully denatured form of the enzyme was protease sensitive. $\alpha$-Glucosidase inhibitors demonstrated carbon source specific inhibition of S. solfataricus growth indicative of a vital role for $\alpha$-glucosidase in the growth of S. solfatavicus on maltose and starch. The $\alpha$-glucosidase gene, malA, is 2083 bp and encodes a protein of 693 amino acids. It is flanked on the 5$\sp\prime$ side by an unusual 1 kb intergenic region. Phylogenetic analysis indicate the closest S. solfaruricus $\alpha$-glucosidase homologs are of mammalian origin. Characterization of the recombinant enzyme purified from E. coli revealed that glycogen is a substrate for the recombinant enzyme. Unlike maltose, glycogen hydrolysis is optimal at the intracellular pH of the organism. These results implicate a unique role for the S. solfataricus $\alpha$-glucosidase in carbohydrate metabolism. The $\alpha$-glucosidase being responsible for the catabolism of both extracellular and intracellular glucose polymers.
Subject Area
Microbiology
Recommended Citation
Rolfsmeier, Michael Lee, "The alpha-glucosidase of Sulfolobus solfataricus: Characterization of the gene and its activity" (1997). ETD collection for University of Nebraska-Lincoln. AAI9815906.
https://digitalcommons.unl.edu/dissertations/AAI9815906