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.
Dock
Christiane
C
Institute of Technical Microbiology, Hamburg University of Technology, Kasernenstr 12, 21073, Hamburg, Germany.
Hess
Matthias
M
Antranikian
Garabed
G
D000687:Amylopectin / Q000378:metabolism; D000688:Amylose / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D019281:Dimerization; D020536:Enzyme Activators / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006003:Glycogen / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature; D013822:Thermoplasma / Q000201:enzymology* / Q000235:genetics
D020536:Enzyme Activators; D006003:Glycogen; D000688:Amylose; D000687:Amylopectin; D005087:Glucan 1,4-alpha-Glucosidase; D002118:Calcium