Cloning, nucleotide sequence, and efficient expression of the gene coding for thermostable aldehyde dehydrogenase from Bacillus stearothermophilus, and characterization of the enzyme

Abstract

The aldhT gene coding thermostable aldehyde dehydrogenase (ALDH-T) from Bacillus stearothermophilus SIC1 was cloned by colony hybridization using Escherichia coli JM109 and pUC19 as a host-vector system. Nucleotide sequence analysis showed that the aldhT gene was composed of 1,464 bp (488 amino acid residues) which can encode a protein with molecular weight of 52,912. The aldhT gene was highly expressed in E. coli carrying the recombinant plasmid, pUALD27R, which contains a native promoter of the aldhT gene. The enzyme accumulated as a soluble form and not an inclusion body in E. coli, and the concentration was about 10% of total cytoplasmic protein. ALDH-T was purified to homogeneity with a specific activity of 36.3 U/mg protein by heat treatment of the cell extract, ammonium sulfate precipitation, gel filtration and finally ion-exchange chromatography. The molecular weight of the native ALDH-T was estimated to be 220 kDa by gel filtration, whereas subunit molecular weight determined by SDS-PAGE was 53 kDa, suggesting a tetrameric enzyme structure. ALDH-T was considerably stable after heating at 65°C for 30 min, and the optimum temperature for the enzyme reaction was 55 to 60°C. The enzyme was capable of oxidizing several aliphatic aldehydes, particularly C 6-aliphatic aldehyde and hexanal, but did not oxidize benzaldehyde, an aromatic aldehyde. The deduced amino acid sequence of ALDH-T exhibited 45% homogeneity to that of the human cytoplasmic aldehyde dehydrogenase sequence. Two amino acid residues believed to be implicated in the active site, Cys-289 and Glu-255, were conserved among the different aldehyde dehydrogenases.