MOLECULAR CHARACTERIZATION OF A FRUCTANASE PRODUCED BY BACTEROIDES-FRAGILIS BF-1

The Bacteroides fragilis BF-1 fructanase-encoding gene (fruA) was clon ed and expressed in Escherichia coli from the recombinant plasmid pBS1 00. The fruA gene consisted of 1,866 bp encoding a protein of 622 amin o acids with a calculated M(r) of 70,286. The apparent M(r) of the fru ctanase, determined by in vitro cell-free transcription-translation an d sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, was approximately 71,500. An alignment of the amino acid sequences of the B. fragilis BF-1 fructanase and the Bacillus subtilis levanase rev ealed that 45.5% of the amino acids were identical. The fruA gene was expressed in E. coli from its own promoter; however, no E. coli promot er-like sequence was evident upstream from the gene. A major E. coli t ranscription start point and a single B. fragilis BF-1 transcription s tart point were located. Expression of the fruA gene was constitutive in E. coli(pBS100) and B. fragilis BF-1. The ratio of sucrase activity to inulinase activity (S/I ratio) was constant for enzyme preparation s from E. coli(pBS100), indicating that both activities were associate d with the fructanase. For B. fragilis BF-1, the S/I ratio varied cons iderably depending on the carbon source used for growth, suggesting th at a separate sucrase is produced in addition to the fructanase in B. fragilis BF-1. Localization experiments and TnphoA mutagenesis indicat ed that the fructanase was exported to the periplasm. Sequence analysi s of the N-terminal region of the fructanase revealed a putative 30-am ino-acid signal peptide. The enzymatic properties of the purified fruc tanase were investigated. The enzyme was able to hydrolyze sucrose, ra ffinose, inulin, and levan but not melezitose, indicating that it was a beta-D-fructofuranosidase which was able to hydrolyze beta(2-->1)-li nked and beta(2-->6)-linked fructans.