Premium
Purification, cloning and functional differences of a third fructan 1‐exohydrolase (1‐FEHw3) from wheat ( Triticum aestivum )
Author(s) -
Van Riet Liesbet,
Altenbach Denise,
Vergauwen Rudy,
Clerens Stefan,
Kawakami Akira,
Yoshida Midori,
Van den Ende Wim,
Wiemken Andres,
Van Laere André
Publication year - 2008
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.2008.01070.x
Subject(s) - fructan , complementary dna , yeast , biochemistry , biology , pichia pastoris , enzyme , ammonium sulfate precipitation , gene isoform , microbiology and biotechnology , cdna library , sucrose , gene , recombinant dna , size exclusion chromatography
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 β2‐1 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.