Novel thermoactive glucoamylases from the thermoacidophilic Archaea Thermoplasma acidophilum, Picrophilus torridus and Picrophilus oshimae

The thermoacidophilic Archaea Thermoplasma acidophilum (optimal growth at 60degreesC and pH 1-2), Picrophilus torridus and Picrophilus oshimae (optimal growth at 60degreesC and pH 0.7) were able to utilize starch as sole carbon source. During growth these microorganisms secreted heat and acid-stable glucoamylases into the culture fluid. Applying SDS gel electrophoresis activity bands were detected with appearent molecular mass (Mw) of 141.0, 95.0 kDa for T. acidophilum, 133.0, 90.0 kDa for P. torridus and 140.0, 85.0 kDa for P. oshimae. The purified enzymes were incubated with various polymeric substrates such as starch, pullulan, panose and isomaltose. The product pattern, analyzed by HPLC, showed that in all cases glucose was formed as the sole product of hydrolysis. The purified glucoamylases were optimally active at pH 2.0 and 90degreesC and have an isoelectric points (pI) between 4.5 and 4.8. Enzymatic activity was detected even at pH 1.0 and 100degreesC. The glucoamylases were thermostable at elevated temperature with a half-life of 24 h at 90degreesC for both P. torridus and T. acidophilum, and 20 h at 90degreesC for P. oshimae. The enzyme system of T. acidophilum has a lower K-m value for soluble starch (1.06 mg/ml) than the enzymes from P. oshimae and P. torridus (4.35 mg/ml and 2.5 mg/ml), respectively. Enzyme activity was not affected by Na+, Mg++, Ca++, Ni++, Zn++, Fe++, EDTA and DTT.