Characterization of a Novel Glucoamylase from the Thermoacidophilic Archaeon Picrophilus torridus Heterologously Expressed in E. coli



There is an interest in the production of heat- and acid-stable enzymes due to their potential application in various industrial fields, particularly in the food, brewery, and textile industries. Microorganisms living in extreme habitats are a good source for such enzymes as they enable to perform biotransformation reactions under non-conventional conditions. After the complete genome analysis of the thermoacidophilic archaeon Picrophilus torridus (optimal growth at pH 0.7 and 60 °C) a number of genes were identified that encode for amylolytic enzymes, proteases, and esterases. A gene encoding an intracellular glucoamylase from P. torridus was cloned and successfully expressed in E. coli. The recombinant enzyme was purified to homogeneity with a yield of 37 % by heat treatment, anion exchange, and gel filtration chromatography. As revealed by non-denaturating PAGE, the active enzyme forms a homotetramer (73 kDa/subunit). The recombinant glucoamylase shows activity between 30 °C and 65 °C and a pH of between 4.5 and 6.5. Interestingly, the enzyme shows unique substrate specificity compared to already known glucoamylases. In addition to the hydrolysis of branched and linear α-glucans, the purified enzyme preferentially attacks maltotriose. The Vmax for maltotriose (10 U/mg) is even higher than the Vmax for starch (8 U/mg). The high maltotriose preference of this archaeal enzyme is unique among all glucoamylases described so far.