Molecular characterization of a fructanase produced by Bacteroides fragilis BF-1.
The Bacteroides fragilis BF-1 fructanase-encoding gene (fruA) was cloned and expressed in Escherichia coli from the recombinant plasmid pBS100. The fruA gene consisted of 1,866 bp encoding a protein of 622 amino acids with a calculated M(r) of 70,286. The apparent M(r) 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 Bacillus subtilis levanase revealed 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 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 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 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 beta-D-fructofuranosidase which was able to hydrolyze beta(2-->6)-linked fructans.
Department of Microbiology, University of Cape Town, Rondebosch, South Africa.
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Blatch
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Woods
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001441:Bacteroides fragilis / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000145:classification* / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D014176:Protein Biosynthesis; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000201:enzymology; D013393:Sucrase / Q000145:classification / Q000235:genetics; D014158:Transcription, Genetic; D043324:beta-Fructofuranosidase
D001426:Bacterial Proteins; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; D013393:Sucrase; C021512:levanase; C023086:inulinase