Clone the Alcohol Dehydrogenase and L-lactate Dehydrogenase of Bio-hydrogen Producing Bacteria Ethanoligenens harbinense B49 and Its Sequence Analysis

Abstract:

Designed the degenerate primers of alcohol dehydeogenase and L-lactate dehydrogenase to augment Ethanoligenens harbinense B49 genomic DNA, and obtained about 780 bp and 610 bp PCR product respectively. Augmented flank sequences of the two PCR fragments with the Cassette PCR method. Similarity alignment showed that the products of the cloned DNA were very high similar to those of alcohol dehydrogenase genes and L-lactate dehydrogenase genes respectively. One of the two sequences was 1902 bp long, and the ORF of adh was 1101 bp long and encoded 366 amino acids. Its putative molecular weight was about 39.71 kD, its calculational isoionic point was pH 5.93. The maximal identity and positive was 51% and 73% with Clostridium thermocellum ATCC 27405 adh. The other one was 2490 bp long, and the ORF of adh was 951 bp long and encoded 316 amino acids. Its putative molecular weight was 34.23 kD, its calculational isoionic point was pH 6.09. The maximal identity and positive was 55% and 74% with Bacillus megaterium L-ldh. Successfully cloning these two genes would not only enrich the gene resources of L-lactate dehydrogenase and alcohol dehydrogenase genes, but also give the scientific warrant for the metabolic engineering research and the construction of the gene-engineering bacteria.