Cloning and functional characterization of a fructan 1-exohydrolase (1-FEH) in edible burdock (Arctium lappa L.)

Abstract

Background: We have previously reported on the variation of total fructooligosaccharides (FOS), total inulooligosaccharides (IOS) and inulin in the roots of burdock stored at different temperatures. During storage at 0°C, an increase of FOS as a result of the hydrolysis of inulin was observed. Moreover, we suggested that an increase of IOS would likely be due to the synthesis of the IOS by fructosyltransfer from 1-kestose to accumulated fructose and elongated fructose oligomers which can act as acceptors for fructan:fructan 1-fructosyltransferase (1-FFT). However, enzymes such as inulinase or fructan 1-exohydorolase (1-FEH) involved in inulin degradation in burdock roots are still not known. Here, we report the isolation and functional analysis of a gene encoding burdock 1-FEH.

Results: A cDNA, named aleh1, was obtained by the RACE method following PCR with degenerate primers designed based on amino-acid sequences of FEHs from other plants. The aleh1 encoded a polypeptide of 581 amino acids. The relative molecular mass and isoelectric point (pI) of the deduced polypeptide were calculated to be 65,666 and 4.86. A recombinant protein of aleh1 was produced in Pichia pastoris, and was purified by ion exchange chromatography with DEAE-Sepharose CL-6B, hydrophobic chromatography with Toyopearl HW55S and gel filtration chromatography with Toyopearl HW55S. Purified recombinant protein showed hydrolyzing activity against β-2, 1 type fructans such as 1-kestose, nystose, fructosylnystose and inulin. On the other hand, sucrose, neokestose, 6-kestose and high DP levan were poor substrates.The purified recombinant protein released fructose from sugars extracted from burdock roots. These results indicated that aleh1 encoded 1-FEH.

Figure 1

Structures of inulin type fructan; 1-kestose (A), nystose(B) and inulin(C).

Figure 2

Comparison of deduced amino-acid sequence of aleh1 with those of other Asteraceous 1-FEH. AlEH1, deduced amino-acid sequence of aleh1 (AB611034); Chi_1-FEH2, Cichorium intybus 1-FEH IIa (AJ295033); Vh_1-FEH, Vernonia herbacea (AM231149) and Chi_1-FEH1, Cichorium intybus 1-FEH I (AJ242538) are aligned. Potential N-glycosylation sites in the sequence are underlined. DPNG, RDP and EC motifs are shaded in gray. The three carboxylic acids that are thought to be crucial for enzyme activity are indicated by black inverted triangle. Consensus line: asterisks (*) indicated identical residues; colons (:) indicated conserved substitutions; and periods (.) indicated semi-conserved substitutions. Putative N-terminal amino acid of AlEH1 is indicated with an arrow.

Figure 3

SDS-PAGE of recombinant AlEH1. SDS-PAGE was conducted on a 10% (w/v) polyacrylamide gel, and gel was stained with CBB. Lane A: purified recombinant protein treated with PNGase F, Lane B: purified recombinant protein, lane M: protein marker (APRO Life Science, JAPAN). The molecular sizes of protein (a) and (b) were estimated to be about 80,000 and 65,000, respectively. The protein (c) is PNGase F.

Figure 4

Time courses of saccharides formed from 100 mM 1-kestose (A), nystose (B) or 5% inulin (C) by purified recombinant AlEH1. Symbols of circle, square and triangle indicate fructose, sucrose and 1-kestose, respectively. Symbols of diamond and inverted triangle indicate nystose and fructosylnystose, respectively. Data are means of 3 replications. Standard deviation indicated with bars.

Figure 5

High performance anion-exchange chromatograms of the saccharides formed from burdock roots extract by purified recombinant AlEH1. Reaction mixtures containing purified recombinant enzyme were incubated with sugar extract from burdock roots stored in soil for six months for 0, 1 and 24 h at 30°C. G, glucose; F, fructose; S, sucrose; 1-K, 1-kestose; Nys, nystose; F-Nys, fructosylnystose; 6a, 1^F(1-β-D-fructofuranosyl)₄ sucrose; 7a, 1^F(1-β-D-fructofuranosyl)₅ sucrose; 8a, 1^F(1-β-D-fructofuranosyl)₆ sucrose; F2, inulobiose; F3, inulotriose; F4, inulotetraose; F5, inulopentaose.