Abstract

The 2-deoxy-D-ribose-5-phosphate aldolase gene from Hyperthermus butylicuswas 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 K_m 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.

Key words: 2-Deoxy-D-ribose-5-phosphate aldolase, thermophiles, aldol condensation, acetaldehyde resistance.

Abbreviation

DERA, 2-Deoxy-D-ribose-5-phosphate aldolase; DRP, 2-deoxy-D-Ribose-5-Phosphate; ORF, open reading frame; TPI, triose-phosphate isomerase; GDP, glycerol-3-phosphate dehydrogenase; DERAHbu, Hyperthermus butylicus 2-deoxy-D-ribose 5-phosphate aldolase; LB, lysogeny broth; IPTG, isopropyl-β-D-thiogalactopyranoside; PEG, polyethylene glycol; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; DMSO, dimethyl sulfoxide.