A Thermostable Cyclodextrin Glycosyltransferase from Thermoanaerobacter sp. 5K
Nancy N. Nichols,
Badal C. Saha,
Sarah E. Frazer,
Michael A. Cotta,
Cyclodextrin glycosyltransferase (CGTase) from the thermophilic anaerobe Thermoanaerobacter sp. 5K was
purified and characterized. The enzyme was purified with ammonium sulfate precipitation followed by α-CD-bound,
epoxy-activated Sepharose 6B affinity chromatography. Molecular weight of the purified enzyme was 70.6 kDa. The
enzyme had optimal activity at 80-90°C and retained greater than 90% activity between 75°C and 95°C. Optimal pH
activity was observed at 7.0, with at least 50% activity between pH 4.0 and 9.0. It was highly stable at elevated
temperature, with no loss of activity after incubation at 80°C for 4 hours or at 90°C for 30 min. Km and Vmax values were
0.222 mg/mL and 0.206 mg β-CD/mL/min, respectively, with soluble starch. Amino acid composition of the enzyme was
deduced from the sequence of the cloned CGTase gene. The mature enzyme has a deduced molecular weight of 75.63 kDa
and contains residues conserved in the CGTase class of amylase enzymes.
Keywords: Anaerobe; cyclodextrin glycosyltransferase, cyclodextrin, Thermoanaerobacter, thermophilic extremozyme.
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