Cloning and functional analysis of a high DP fructan:fructan 1-fructosyl transferase from Echinops ritro (Asteraceae): comparison of the native and recombinant enzymes.
Inulin-type fructans are the simplest and most studied fructans and have become increasingly popular as prebiotic health-improving compounds. A natural variation in the degree of polymerization (DP) of inulins is observed within the family of the Asteraceae. Globe thistle (Echinops ritro), artichoke (Cynara scolymus), and Viguiera discolor biosynthesize fructans with a considerably higher DP than Cichorium intybus (chicory), Helianthus tuberosus (Jerusalem artichoke), and Dahlia variabilis. The higher DP in some species can be explained by the presence of special fructan:fructan 1-fructosyl transferases (high DP 1-FFTs), different from the classical low DP 1-FFTs. Here, the RT-PCR-based cloning of a high DP 1-FFT cDNA from Echinops ritro is described, starting from peptide sequence information derived from the purified native high DP 1-FFT enzyme. The cDNA was successfully expressed in Pichia pastoris. A comparison is made between the mass fingerprints of the native, heterodimeric enzyme and its recombinant, monomeric counterpart (mass fingerprints and kinetical analysis) showing that they have very similar properties. The recombinant enzyme is a functional 1-FFT lacking invertase and 1-SST activities, but shows a small intrinsic 1-FEH activity. The enzyme is capable of producing a high DP inulin pattern in vitro, similar to the one observed in vivo. Depending on conditions, the enzyme is able to produce fructo-oligosaccharides (FOS) as well. Therefore, the enzyme might be suitable for both FOS and high DP inulin production in bioreactors. Alternatively, introduction of the high DP 1-FFT gene in chicory, a crop widely used for inulin extraction, could lead to an increase in DP which is useful for a number of specific industrial applications. 1-FFT expression analysis correlates well with high DP fructan accumulation in vivo, suggesting that the enzyme is responsible for high DP fructan formation in planta.
Van den Ende
Wim
W
KU Leuven, Laboratory of Molecular Plant Physiology, Kasteelpark Arenberg 31, B-3001 Leuven, Belgium. wimvandenende@bio.kuleuven.ac.be
Clerens
Stefan
S
Vergauwen
Rudy
R
Boogaerts
David
D
Le Roy
Katrien
K
Arckens
Lutgarde
L
Van Laere
André
A
D000064:Acclimatization; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D031182:Echinops Plant / Q000201:enzymology*; D005630:Fructans / Q000096:biosynthesis; D006602:Hexosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010802:Phylogeny; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment
D018076:DNA, Complementary; D005630:Fructans; D011993:Recombinant Fusion Proteins; D006602:Hexosyltransferases; C114852:1,2-beta-fructan 1(F)-fructosyltransferase