African Journal of
Biotechnology

  • Abbreviation: Afr. J. Biotechnol.
  • Language: English
  • ISSN: 1684-5315
  • DOI: 10.5897/AJB
  • Start Year: 2002
  • Published Articles: 12193

Full Length Research Paper

Cloning and characterization of thermostable-deoxy-D-ribose-5-phosphate aldolase from Hyperthermus butylicus

Qiuyan Wang1, Rong Chen1, Pengfei Du1, Huili Wu1, Xiaolin Pei1, Bing Yang1, Lihua Yang1, Lifeng Huang1, Jinhua Liu2* and Tian Xie1*
  1Center for Biomedicine and Health, Hangzhou Normal University; Hangzhou 310012, P. R. China. 2College of Material, Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou 310012, P. R. China.
Email: [email protected]

  •  Accepted: 26 April 2010
  •  Published: 17 May 2010

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 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.

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.