A thermoactive glucoamylase with biotechnological relevance from the thermoacidophilic Euryarchaeon Thermoplasma acidophilum.

A gene encoding an intracellular glucoamylase was identified in the genome of the extreme thermoacidophilic Archaeon Thermoplasma acidophilum. The gene taGA, consisting of 1,911 bp, was cloned and successfully expressed in Escherichia coli. The recombinant protein was purified 22-fold to homogeneity using heat treatment, anion-exchange chromatography, and gel filtration. Detailed analysis shows that the glucoamylase, with a molecular weight of 66 kDa per subunit, is a homodimer in its active state. Amylolytic activity was measured over a wide range of temperature (40-90 degrees C) and pH (pH 3.5-7) and was maximal at 75 degrees C and at acidic condition (pH 5). The recombinant archaeal glucoamylase uses a variety of polysaccharides as substrate, including glycogen and amylose. Maximal activity was measured towards amylopectin with a specific activity of 4.2 U/mg and increased almost threefold in the presence of manganese. Calcium ions have a pronounced effect on enzyme stability; in the presence of 5 mM CaCl(2), the half-life increased from 15 min to 2 h at 80 degrees C.

Isolation and molecular analysis of six taproot expressed genes from sugar beet.

The taproot from sugar beet (Beta vulgaris L.) undergoes a specific developmental process to function as a food storage organ. Suppression Subtractive Hybridization (SSH) was utilized for the isolation of cDNA fragments for taproot expressed genes. Isolation and molecular analysis of six cDNAs encoding the complete gene product revealed that these genes comprise homologues of a drought-inducible linker histone, a homologue of a major latex-like protein, a phosphoenolpyruvate carboxylase kinase, a putative vacuolar processing enzyme, a thaumatin-like protein and an alanine- and glutamic acid-rich protein. All genes are transcribed in taproots while transcription in leaves is low or undetectable.