title: Nonomuraea sp. ID06-A0189 inulin fructotransferase (DFA III-forming): gene cloning, characterization and conservation among other Nonomuraea species.
abstract: The inulin fructotransferase (DFA III-forming)(EC 4.2.2.18) gene in Nonomuraea sp. ID06-A0189 was amplified from genomic DNA, sequenced and expressed in Escherichia coli. The 1326-bp gene, designated as Nsp-ift, encodes a protein composed of a putative 37-amino-acid signal peptide and 404-amino-acid mature protein. A putative ribosomal binding sequence was identified 12 bases upstream from the start codon. However, a typical bacterial promoter could not be found by in silico analysis. The deduced amino-acid sequence of the enzyme was most similar to that of inulin fructotransferase (DFA I-forming) in Frankia sp. EAN1pec. Phylogenetic analysis of deduced amino-acid sequences indicated that Nonomuraea sp. ID06-A0189 and Frankia sp. EAN1pec inulin fructotransferases formed a distinct clade from those from Arthrobacter sp. H65-7, A. globiformis and Bacillus sp. snu-7 that showed 57, 56 and 56% identity to that of Nsp-ift, respectively. The Nsp-ift without a putative signal peptide was successfully expressed in E. coli and partially purified using His-tag affinity chromatography. The recombinant enzyme displayed optimum temperature between 65 and 70 °C, optimum pH between 5.5 and 6.0 and remained stable up to 70 °C. The properties were identical to those of the original enzyme. Of 10 Nonomuraea species tested by Southern hybridization, enzyme activity measurements and PCR, only Nonomuraea sp. ID06-A0189 has the Nsp-ift gene, suggesting that Nsp-ift is not highly conserved in this genus.
authors.affiliation: Research Centre for Chemistry, Indonesian Institute of Sciences, Jl. Sangkuriang, Bandung, Indonesia.
authors.forename: Sri
authors.initials: S
authors.lastname: Pudjiraharti
authors.affiliation: Laboratory of Applied Microbiology, Graduate School of Agriculture, Hokkaido University, Sapporo, Hokkaido, Japan.
authors.forename: Midori
authors.initials: M
authors.lastname: Ohtani
authors.affiliation: Laboratory of Applied Microbiology, Graduate School of Agriculture, Hokkaido University, Sapporo, Hokkaido, Japan.
authors.forename: Nanami
authors.initials: N
authors.lastname: Takano
authors.affiliation: Laboratory of Applied Microbiology, Graduate School of Agriculture, Hokkaido University, Sapporo, Hokkaido, Japan.
authors.forename: Ayumi
authors.initials: A
authors.lastname: Abe
authors.affiliation: Research Centre for Biotechnology, Indonesian Institute of Sciences, Jl. Raya Bogor KM 46, Cibinong, Bogor, Indonesia.
authors.forename: Puspita
authors.initials: P
authors.lastname: Lisdiyanti
authors.affiliation: Laboratory of Applied Microbiology, Graduate School of Agriculture, Hokkaido University, Sapporo, Hokkaido, Japan.
authors.forename: Michiko
authors.initials: M
authors.lastname: Tanaka
authors.affiliation: Laboratory of Applied Microbiology, Graduate School of Agriculture, Hokkaido University, Sapporo, Hokkaido, Japan.
authors.forename: Teruo
authors.initials: T
authors.lastname: Sone
authors.affiliation: Laboratory of Applied Microbiology, Graduate School of Agriculture, Hokkaido University, Sapporo, Hokkaido, Japan.
authors.forename: Kozo
authors.initials: K
authors.lastname: Asano
mesh_terms: D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000145:classification* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial / Q000235:genetics; D006602:Hexosyltransferases / Q000737:chemistry* / Q000145:classification* / Q000235:genetics; D010802:Phylogeny; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006602:Hexosyltransferases; C097478:inulin fructotransferase (DFA I-producing)