Cloning and characterization of thermostable-deoxy-D-ribose-5-phosphate aldolase from <i>Hyperthermus butylicus</i>

Abstract

The 2-deoxy-D-ribose-5-phosphate aldolase gene from Hyperthermus butylicus was subcloned, overexpressed in Escherichia coli and purified to apparent homogeneity. Analysis of the sequence of gene revealed an open reading frame (ORF) of 672 base pairs encoding 237 amino acids predicted to yield a protein of molecular mass 26.4 kDa. The encoded protein was overexpressed in E. coli and purified to apparent homogeneity. The enzyme activity is optimal at pH 5.5 and 80°C. For 2-deoxyribose-5-phosphate, the apparent Km was calculated to be 0.15 ± 0.01 mM. The recombinant
protein was heat stable; no activity loss was observed even after incubation at 90°C for 10 min. In addition, the thermophilic enzyme also showed a remarkable resistance to acetaldehyde; it retained more than 70% activity after exposure for 8 h to 300 mM acetaldehyde at 25°C.