Purification, cloning and functional differences of a third fructan 1-exohydrolase (1-FEHw3) from wheat (Triticum aestivum).
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.
Van Riet
Liesbet
L
Laboratory of Molecular Plant Physiology, Department of Biology, Botanical Institute, K.U. Leuven, Kasteelpark Arenberg 31, B-3001 Leuven, Belgium. lieswheat@yahoo.com
Altenbach
Denise
D
Vergauwen
Rudy
R
Clerens
Stefan
S
Kawakami
Akira
A
Yoshida
Midori
M
Van den Ende
Wim
W
Wiemken
Andres
A
Van Laere
André
A
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*
D018076:DNA, Complementary; D013395:Sucrose; D006026:Glycoside Hydrolases; C044118:fructan beta-fructosidase