title: Production, Purification, and Gene Cloning of a β-Fructofuranosidase with a High Inulin-hydrolyzing Activity Produced by a Novel Yeast Aureobasidium sp. P6 Isolated from a Mangrove Ecosystem.
abstract: After screening of over 300 yeast strains isolated from the mangrove ecosystems, it was found that Aureobasidium sp. P6 strain had the highest inulin-hydrolyzing activity. Under the optimal conditions, this yeast strain produced an inulin-hydrolyzing activity of 30.98 ± 0.8 U/ml after 108 h of a 10-l fermentation. After the purification, a molecular weight of the enzyme which had the inulin-hydrolyzing activity was estimated to be 47.6 kDa, and the purified enzyme could actively hydrolyze both sucrose and inulin and exhibit a transfructosylating activity at 30.0 % sucrose, converting sucrose into fructooligosaccharides (FOS), indicating that the purified enzyme was a β-D-fructofuranosidase. After the full length of a β-D-fructofuranosidase gene (accession number KU308553) was cloned from Aureobasidium sp. P6 strain, a protein deduced from the cloned gene contained the conserved sequences MNDPNGL, RDP, ECP, FS, and Q of a glycosidehydrolase GH32 family, respectively, but did not contain a conserved sequence SVEVF, and the amino acid sequence of the protein from Aureobasidium sp. P6 strain had a high similarity to that of the β-fructofuranosidase from any other fungal strains. After deletion of the β-D-fructofuranosidase gene, the disruptant still had low inulin hydrolyzing and invertase activities and a trace amount of the transfructosylating activity, indicating that the gene encoding an inulinase may exist in the Aureobasidium sp. P6 strain.
authors.affiliation: College of Marine Life Sciences, Ocean University of China, Yushan Road, No. 5, Qingdao, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Jiang
authors.affiliation: College of Marine Life Sciences, Ocean University of China, Yushan Road, No. 5, Qingdao, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Ma
authors.affiliation: College of Marine Life Sciences, Ocean University of China, Yushan Road, No. 5, Qingdao, China.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Chi
authors.affiliation: College of Marine Life Sciences, Ocean University of China, Yushan Road, No. 5, Qingdao, China.
authors.forename: Guang-Lei
authors.initials: GL
authors.lastname: Liu
authors.affiliation: College of Marine Life Sciences, Ocean University of China, Yushan Road, No. 5, Qingdao, China. zhenming@sdu.edu.cn.
authors.forename: Zhen-Ming
authors.initials: ZM
authors.lastname: Chi
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017753:Ecosystem; D005285:Fermentation; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D015870:Gene Expression; D006868:Hydrolysis; D007444:Inulin / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D004718:Saccharomycetales / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism; D053833:Wetlands; D043324:beta-Fructofuranosidase / Q000235:genetics / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; C116580:fructooligosaccharide; D013395:Sucrose; D007444:Inulin; D043324:beta-Fructofuranosidase
keywords: Aureobasidium sp; Inulinase; Mangrove ecosystems; Transfructosylase; β-Fructofuranosidase