Cloning of inulin fructotransferase (DFA III-producing) gene from Arthrobacter globiformis C11-1

A gene encoding an inulin fructotransferase (DFA III-producing) [EC 2.4.1.9 3] from Arthrobacter globiformis C11-1 was cloned and the nucleotide sequen ce was determined. The cloned fragment contained a 1353 bp open reading fra me. The initiation codon was estimated to be an unusual codon, GTG. The gen e encoded a signal peptide (40 amino acid residues) for secretion. The mole cular mass of the native enzyme was calculated as 43,400 Da from the sequen cing data. The deduced amino acid sequence of the enzyme had 74.0% homology with that of inulin fructotransferase (DFA III-producing) from Arthrobacte r sp. H65-7, It also had 45.1% homology with that of inulin fructotransfera se (DFA I-producing) [EC 2.4.1.200] from Arthrobacter globiformis S14-3. Th e enzyme produced in the culture supernatant of an Escherichia coli clone w as purified to the electrophoretically homogeneous stage. The N-terminal am ino acid sequence of the cloned enzyme secreted in the broth was the same a s that of the native enzyme from A. globiformis C11-1. Therefore, on this e nzyme, it is estimated that the cleavage sites by the signal peptidase for secretion of A. globiformis C11-1 and E. coli JM109 are the same.