Expression, purification and characterization of a recombinant levan fructotransferase

A 1.6 kb DNA fragment including the lftM gene, encoding a levan fructotransferase (LFTase) of Microbacterium sp. AL-210, was subcloned into a high-expression vector, pET-29b, and the recombinant enzyme was overexpressed in Escherichia coli. Most of the LFTase activity was detected in the cytoplasmic fraction after induction with isopropyl b-D-thiogalactoside. The recombinant enzyme with a tag of six histidine residues at the C-terminus was purified 132-fold by affinity and gel-filtration chromatography. Analysis of the N-terminal amino acid sequence revealed that the first 42 amino acids were post-translationally cleaved off. The molecular mass of the purified LftM was approx. 54 kDa as determined by SDS/PAGE, which corresponded well with a predicted size from the nucleotide sequence of the lftM gene lacking 42 amino acids. The enzyme converted levan into difructose anhydride IV (DFA IV) with a Km of 2 mg/ml and a Vmax of 40.6 µmol/min at pH 7.0 and 40 °C. The pH-dependence study of the enzyme for DFA IV production showed that LftM had a broad pH optimum (5.0–8.0) and the pKa values obtained were 4.5 and 8.9 at 40 °C. These results suggest that the acidic residues at the active site may play important roles for the catalytic mechanism of the LFTase.