MOLECULAR-CLONING OF LEVAN FRUCTOTRANSFERASE GENE FROM ARTHROBACTER NICOTINOVORANS GS-9 AND ITS EXPRESSION IN ESCHERICHIA-COLI

The gene encoding an extracellular levan fructotransferase, designated the lft gene, was cloned from the genomic DNA of Arthrobacter nicotin ovorans GS-9, and expressed in Escherichia coli. It was found that a s ingle open reading frame consisted of 1554 base pairs that encoded a p olypeptide composed of a signal peptide of 33 amino acids and a mature protein of 484 amino acids (M-r 53,152), and it was also found that a putative ribosome-binding site was present in the upstream from the O RF. The primary structure had no significant similarity with those of inulin fructotransferases, but had low similarity to the catalytic reg ions of other fructosylhydrolases. The expression of the lft gene was increased on a plasmid, pLFT-BB1, in which the lft gene was fused with alpha-peptide of the lacZ gene of pUC18. An E. coli transformant carr ying pLFT-BB1 expressed six times as much activity of levan fructotran sferase as that of the original strain, A. nicotinovorans GS-9.