Characterization of the thermophilic isoamylase from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092

Isoamylase catalyzes the hydrolysis of alpha- 1,6-glucosidic linkages of starch and related polysaccharides. In this study, the treX gene (GenBank accession no. AE006815 REGION: 9279... 11435) encoding the thermophilic isoamylase was PCR-cloned from the genomic DNA of Sulfolobus solfataricus ATCC 35092 to an expression vector with a T7lac promoter. Both wild-type and His-tagged isoamylases were expressed in Escherichia coli. The wild-type isoamylase was purified sequentially using heat treatment, nucleic acid precipitation, ion-exchange chromatography, and gel filtration chromatography while the His-tagged isoamylase was purified from the cell-free extract directly by metal chelating chromatography. Both enzymes were active only under their homo-trimer forms. In the absence of NaCl, both enzymes became inactive monomers. In addition, both enzymes were more stable when being stored at room temperature than at 4 degrees C. They had an apparent optimal pH of 5 and an optimal temperature at 75 degrees C. The enzyme activities remained unchanged after a 2 h incubation at 80 and 75 degrees C for the wild-type and His-tagged enzymes, respectively. These thermophilic isoamylases showed a potential to be used in industry to degrade the branching points of starch at a high temperature. (c) 2005 Elsevier B.V. All rights reserved.