Blatch, Gregory ORCID: 0000-0003-0778-8577 and Woods, David R (1993) Molecular characterization of a fructanase produced by Bacteroides fragilis BF-1. Journal of Bacteriology, 175 (10). pp. 3058-3066. ISSN 0021-9193

Abstract

The Bacteroidesfragilis BF-1 fructanase-encoding gene (fruA) was cloned and expressed in Escherichia coli from the recombinant plasmid pBS100. ThefruA gene consisted of 1,866 bp encoding a protein of 622 amino acids with a calculated Mr of 70,286. The apparent Mr of the fructanase, determined by in vitro cell-free transcription-translation and 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 Bacilus subtilis levanase revealed that 45.5% of the amino acids were identical. ThefruA gene was expressed in E. coli from its own promoter; however, no E. coli promoter-like sequence was evident upstream from the gene. A major E. coli transcription start point and a single B. fragilis BF-1 transcription start point were located. Expression of the fiuA 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 preparations from E. coli(pBS100), indicating that both activities were associated with the fructanase. For B. fragilis BF-1, the S/I ratio varied considerably depending on the carbon source used for growth, suggesting that a separate sucrase is produced in addition to the fructanase in B. fragilis BF-1. Localization experiments and TnphoA mutagenesis indicated that the fructanase was exported to the periplasm. Sequence analysis of the N-terminal region of the fructanase revealed a putative 30-amino-acid signal peptide. The enzymatic properties of the purified fructanase were investigated. The enzyme was able to hydrolyze sucrose, raffinose, inulin, and levan but not melezitose, indicating that it was a 13-D-fructofuranosidase which was able to hydrolyze 10(2-4)-linked and 0(2-6)-linked fructans.

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