Characterization of the ectC gene and its expression product in Halomonas sp. Nj223 from Antarctica deep-sea sediment

Abstract:

A moderately halophilic bacterium Halomonas sp. NJ223 was isolated from Antarctica deep-sea sediment. This bacterium accumulates ectoine as the main compatible solute in response to severe osmotic stress. Ecoine synthase catalyzes circulation of γ-N-acetyl-α，γ-diaminobutyric acid (ADABA) to ectoine in the last step of the three enzymatic steps. The gene of ectoine synthase from this strain was amplified by PCR and the DNA sequence of a 393-bp segment was sequenced. The amino acid sequences of this enzyme present high homology to the known sequence. The significance of this gene was proved by the expression in Escherichia coli. Thus, the amplified fragment was cloned into the expression vector pET-his. The insert position, the size and the reading frame were identified by PCR, restriction digestion and the sequence analysis of the recombinant plasmids. SDS-PAGE shows that the relative molecular mass of the expression product was 15kDa as predicted, which indicated that the recombinant plasmids could express the gene of ectoine synthase. The biosynthetic pathway of ectoine was partially elucidated by renaturation and enzyme activity detection of purified ectoine synthase in vitro. Determination of effect of pH and temperature on enzyme activity shows that the optimal reaction condition of pH was 8.0 and temperature was 25℃.