title: Purification, cloning and functional differences of a third fructan 1-exohydrolase (1-FEHw3) from wheat (Triticum aestivum).
abstract: A third fructan exohydrolase isoform (1-FEHw3) was purified from wheat stems by a combination of ammonium sulfate precipitation, ConA affinity and ion-exchange chromatography. Homogeneity of the preparation was indicated by the presence of a single band (70 kDa) after SDS-PAGE. The enzyme hydrolyzed mainly beta2-1 linkages in fructans and was inhibited by sucrose. A cDNA could be obtained after reverse transcriptase polymerase chain reaction (RT-PCR)-based strategies and screening of a cDNA library. Functionality tests of the cDNA performed after heterologous expression in the yeast Pichia pastoris showed that the encoded protein has essentially the same characteristics as the native enzyme. Homology with previously described 1-FEH isoforms from wheat was high (97% identity), and the enzyme showed minor differences to the previously published enzymes. The relative abundance of 1-FEH transcripts in different tissues was investigated by using quantitative RT-PCR.
authors.affiliation: Laboratory of Molecular Plant Physiology, Department of Biology, Botanical Institute, K.U. Leuven, Kasteelpark Arenberg 31, B-3001 Leuven, Belgium. lieswheat@yahoo.com
authors.forename: Liesbet
authors.initials: L
authors.lastname: Van Riet
authors.forename: Denise
authors.initials: D
authors.lastname: Altenbach
authors.forename: Rudy
authors.initials: R
authors.lastname: Vergauwen
authors.forename: Stefan
authors.initials: S
authors.lastname: Clerens
authors.forename: Akira
authors.initials: A
authors.lastname: Kawakami
authors.forename: Midori
authors.initials: M
authors.lastname: Yoshida
authors.forename: Wim
authors.initials: W
authors.lastname: Van den Ende
authors.forename: Andres
authors.initials: A
authors.lastname: Wiemken
authors.forename: André
authors.initials: A
authors.lastname: Van Laere
mesh_terms: D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010802:Phylogeny; D010843:Pichia / Q000201:enzymology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013395:Sucrose / Q000494:pharmacology; D014908:Triticum / Q000201:enzymology* / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D013395:Sucrose; D006026:Glycoside Hydrolases; C044118:fructan beta-fructosidase