Cloning and Characterization of Fructose-1,6-Bisphosphate Aldolase from Euphausia superba

Fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) is a highly conserved enzyme that is involved in glycolysis and gluconeogenesis. In this study, we cloned the fructose-1,6-bisphosphate aldolase gene from Euphausia superba (EsFBA). The full-length cDNA sequence of EsFBA is 1098 bp long and encodes a 365-amino-acid protein. The fructose-1,6-bisphosphate aldolase gene was expressed in Escherichia coli (E. coli). A highly purified protein was obtained using HisTrap HP affinity chromatography and size-exclusion chromatography. The predicted three-dimensional structure of EsFBA showed a 65.66% homology with human aldolase, whereas it had the highest homology (84.38%) with the FBA of Penaeus vannamei. Recombinant EsFBA had the highest activity at 45 degrees C and pH 7.0 in phosphate buffer. By examining the activity of metal ions and EDTA, we found that the effect of metal ions and EDTA on EsFBA's enzyme activity was not significant, while the presence of borohydride severely reduced the enzymatic activity; thus, EsFBA was confirmed to be a class I aldolase. Furthermore, targeted mutations at positions 34, 147, 188, and 230 confirmed that they are key amino acid residues for EsFBA.

Automated summary

Fructose-1,6-bisphosphate aldolase is a conserved enzyme involved in glycolysis and gluconeogenesis. In this study, the gene encoding aldolase was cloned from Euphausia superba and expressed in Escherichia coli. The purified protein showed high homology to aldolases from other species. It exhibited optimal activity at 45 degrees C and pH 7.0 and was sensitive to the presence of borohydride. Specific amino acid residues were identified as key for the enzyme's functionality.