Molecular characterization of the levansucrase gene from Pseudomonas aurantiaca S-4380 and its expression in Escherichia coli

Abstract

DFA IV is di-D-fructose-2,6′:6,2′-dianhydride, consisting of two fructose residues. It can be enzymatically synthesized from levan by levan fructotransferase, and can be used for mineral absorption. Understanding of the structure and composition of levan is important to obtain high-level production of DFA IV. A bacterial strain, Pseudomonas aurantiaca S-4380, was identified to produce low-branched levan, and the levansucrase gene (lscA) from this bacterium was found to be composed of 1,275 bp coding for a protein of 424 amino acids, with an estimated molecular weight of 47 kDa. The bacterial levansucrase gene was expressed in Escherichia coli DH5α by its own promoter and lac promoter. The recombinant levansucrase was produced in soluble form with 170U of levansucrase activity from 1-ml E. coli structure broth. The expressed enzyme from the clone showed similar biochemical properties, such as size of active levansucrase, degree of branching, and optimum temperature, with P. aurantiaca S-4380 levansucrase.