title: Cloning and heterologous expression of the ftfCNC-2(1) gene from Weissella confusa MBFCNC-2(1) as an extracellular active fructansucrase in Bacillus subtilis.
abstract: Fructan-exopolysaccharides (fructan-EPS) (inulin and levan) and their oligosaccharides (fructooligosaccharides, FOS) have drawn considerable interest in the food and pharmaceutical industries. EPS-producing lactic acid bacteria have been reported to produce β-fructans (inulin and levan), as well as α-glucans, by the function of sucrase enzymes, i.e., fructansucrase and glucansucrase. A fructansucrase ftfCNC-2(1) gene from Weissella confusa strain MBFCNC-2(1) was previously cloned in Escherichia coli. In this study, we aimed to express the ftf[CNC-2(1)] gene in Bacillus subtilis to obtain the active form of the extracellular recombinant protein FTF[CNC-2(1)]. This cloning was achieved by inserting the gene in-fusion with the signal sequence of the B. subtilis subtilisin E. SDS-polyacrylamide gel electrophoresis analysis and in situ activity assay with Periodic Acid-Schiff staining revealed that the recombinant FTF[CNC-2(1)] was successfully expressed as an extracellular protein from B. subtilis DB403 in its active form, which was confirmed using sucrose and raffinose.
authors.affiliation: Pharmaceutical Biotechnology Research Group, Faculty of Pharmacy, Universitas Indonesia, Kampus UI Depok, Depok 16424, Indonesia. Electronic address: amarila.malik@ui.ac.id.
authors.forename: Amarila
authors.initials: A
authors.lastname: Malik
authors.affiliation: Pharmaceutical Biotechnology Research Group, Faculty of Pharmacy, Universitas Indonesia, Kampus UI Depok, Depok 16424, Indonesia.
authors.forename: Maria Tyas
authors.initials: MT
authors.lastname: Hapsari
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), 8916-5 Takayama, Ikoma, Nara 630-0101, Japan.
authors.forename: Iwao
authors.initials: I
authors.lastname: Ohtsu
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), 8916-5 Takayama, Ikoma, Nara 630-0101, Japan.
authors.forename: Shu
authors.initials: S
authors.lastname: Ishikawa
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), 8916-5 Takayama, Ikoma, Nara 630-0101, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Takagi
mesh_terms: D001412:Bacillus subtilis / Q000166:cytology* / Q000235:genetics*; D003001:Cloning, Molecular; D005110:Extracellular Space / Q000201:enzymology*; D005630:Fructans / Q000378:metabolism; D007444:Inulin / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics; D011887:Raffinose / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013381:Subtilisins / Q000235:genetics; D013393:Sucrase / Q000235:genetics* / Q000378:metabolism*; D013395:Sucrose / Q000378:metabolism; D058836:Weissella / Q000201:enzymology* / Q000235:genetics*
chemical_list: D005630:Fructans; D009844:Oligosaccharides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C116580:fructooligosaccharide; D013395:Sucrose; D007444:Inulin; C072599:levan; D013393:Sucrase; D013381:Subtilisins; D011887:Raffinose
keywords: Bacillus subtilis; Fructansucrase; Fructosyltransferase; Inulosucrase; Weissella confusa; ftf