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A Thermostable Cyclodextrin Glycosyltransferase from Thermoanaerobacter sp. 5K

Ayse Avci, Nancy N. Nichols, Badal C. Saha, Sarah E. Frazer, Michael A. Cotta, Sedat Donmez
Current biotechnology 2014 v.3 no.4 pp. 305-312
Thermoanaerobacter, affinity chromatography, agarose, amino acid composition, ammonium sulfate, amylases, chemical precipitation, cyclomaltodextrin glucanotransferase, enzyme activity, genes, molecular weight, pH, starch, temperature, thermal stability
Cyclodextrin glycosyltransferase (CGTase) from the thermophilic anaerobe hermoanaerobacter 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.