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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>A cDNA, named <it>aleh1</it>, was obtained by the RACE method following PCR with degenerate primers designed based on amino-acid sequences of FEHs from other plants. The <it>aleh1 </it>encoded a polypeptide of 581 amino acids. The relative molecular mass and isoelectric point (<it>pI</it>) of the deduced polypeptide were calculated to be 65,666 and 4.86. A recombinant protein of <it>aleh1 </it>was produced in <it>Pichia pastoris</it>, 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.</p> <p>The purified recombinant protein released fructose from sugars extracted from burdock roots. These results indicated that <it>aleh1 </it>encoded 1-FEH.</p

Effect of temperatures on inulobiose and inulooligosaccharides in burdock roots during storage

The variation of inulobiose, inulotrose and other higher inulo-n-oses in burdock roots stored at 0, 15 and 20℃ during six weeks was investigated. Results showed that at 15 and 20℃, inulobiose appeared after three day storage and then it increased progressively to 2.03 and 2.72 mg/g dry weight (DW) after 42 days, while inulotriose, inulotetraose, inulopentaose, inulohexaose and inuloheptaose appeared after one week and increased also progressively, and were 1.81 and 2.30, 1.92 and 2.12, 1.52 and 1.89, 1.18 and 2.14, and 1.45 and 3.75 mg/g DW, respectively. At 0℃, the different inulooligosaccharides (IOSs) increased sharply after the second week but inulobiose and inulotriose decreased slightly after five weeks. However, their contents were much higher than those observed at 15 and 20℃. This suggests that low temperature induced strongly the hydrolysis of inulin and formation of IOSs in burdock root suggesting that this reaction would not be the result of a putative endo-inulinase but results from the activity of fructan:fructan 1-fructosyltransferase (1-FFT)

ごぼう (Arctium lappa L.) 由来フルクタン : フルクタン1-フルクトシルトランスフェラーゼの精製，クローニングと機能解析

A fructan:fructan 1-fructosyltransferase (1-FFT) was purified edible for the first time, and a cDNA encoding 1-FFT was isolated from the plant. Two 1-FFTs named 1-FFTa and 1-FFTb were purified from extract of edible burdock by sulfate precipitation, followed by chromatographies on DEAE-Sepharose CL-6B, Toypearl HW-55S and Sehadex G-100 columns. Inulin-type fructan such as nystose and frustooligosaccharides with higher DP were produced from 1-kestose by purified 1-FFTs. The general properties of both purified enzymes were very similar to each other. The purified 1-FFTa and 1-FFTb showed a single band by native PAGE and two bands, relative molecular messes of about 46,000 and 17,500 for 1-FFTa and 46,000 and 17,000 for 1-FFTb, by SDS-PAGE, respectively. The N-terminal sequences of the 46,000 peptides of both enzymes were the same, and those of the 17,000 and the 17,000 peptides were also identical. Based on the sequences, the 1-FFT cDNA named alft1 was cloned. The alft1 encoded a polypeptide of 617 amino acids. The relative molecular mass and pI of the mature protein region of deduced polypeptide were calculated to be 60,213 and 4.89, respectivety. The characteristics of recombinant protein produced by Pichia pastoris closely resembled those of the native 1-FFTs purified from edible burdock. In this study, we demonstrated that alft1 encoding edible burdock 1-FFT was involved in the elongation of fructosyl chains of fructooligosaccharides in edible burdock. ごぼうから初めてフルクタン:フルクタン1-フルクトシルトランスフェラーゼ(1-FFT)を精製し，それをコードするcDNAをクローニングした。1- FFTaおよび1-FFTbと名付けた2種の1-FFTが硫安分画，DEAE-Sepharose CL-6B, Toyopearl HW-55SおよびSephadex G-100の各種クロマトグラフィーを行うことによりごぼう抽出液から精製された。ニストースや高い重合度をもつフルクトオリゴ糖などのイヌリン型フルクタンが1-ケストースから精製1-FFTにより生成された。両精製酵素の一般性質はよく似ていた．1-FFTaおよび1-FFTb両精製酵素はネイティブ PAGEにより単一バンドを示したが，SDS-PAGEでは1-FFTaでは分子量が約46,000と17,500の位置に，1-FFTbでは約 46,000と17,000の位置に，それぞれ2本のバンドを示した。両酵素の46,000のペプチドのN末端配列は同じ配列であり，両酵素の 17,500と17,000のペプチドのN末端配列もまた同じ配列であった。これらの配列に基づいてalft1と名付けた1-FFT cDNAをクローニングした。alft1は617アミノ酸からなるポリペプチドをコードしていた。推定されるアミノ酸配列の成熟タンパク質領域の分子量と等電点は60,213および4.89と計算された。Pichia pastorisで生産された組み換えタンパク質の性質はごぼうから精製した1-FFTとよく似ていた。本研究において，われわれはごぼう由来1-FFTとそれをコードするalft1がごぼうのフルクトオリゴ糖のフルクトシル鎖の伸長に関与することを示した

Isolation and structural determination of reducing fructooligosaccharides newly produced in stored edible burdock

Fresh edible burdock roots were stored in soil of 1 m depth underground from November to May. Three fructooligosaccharide derivatives without a terminal glucose residue, designated saccharides 1, 2 and 3, were generated in the stored burdock roots. They were purified from the sugar extract using carbon-Celite column chromatography. Saccharides 1, 2 and 3 have R-sucrose values (retention time of sucrose = 1) of 1.55, 2.15 and 2.73 by HPAEC, reducing terminal, molar ratios (reducing sugar to D-fructose) of 0.50, 0.33 and 0.25 and degrees of polymerization of 2, 3 and 4 by TOF-MS, respectively. Analyses by GLC and NMR confirmed the three different following structures: first was inulobiose [β-D-fructofuranosyl-(2→1)-β-D-fructopyranose], and the two others were inulotriose [β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl-(2→1)-β-D-fructopyranose] and inulotetraose [β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl-(2→1)-β-D-fructopyranose]. The NMR spectra showed that 70 to 80 % of the terminal fructose residue of the three saccharides is pyranosyl form, while 20 to 30 % is furanosyl form. The ^C_- and ^1H-signals were also assigned by 2D-NMR including COSY, HSQC, HSQC-TOCSY and HMBC. These saccharides could be synthesized by purified burdock fructan:fructan 1-frustosyltransferase from 1-kestose to free D-fructopyranose giving inulobiose and sucrose, while elongation of fructoranosyl units occurred at this trasferred fructofuranosyl residue to produce inuloolisaccharides having one, two and more additional units of fructofuranose. 新鮮ごぼう根を地下1mの土中で11月から翌年の5月まで6カ月間貯蔵した。この貯蔵ごぼうに末端グルコース残基を有しないフルクトオリゴ糖が新しく生成された。このごぼうから抽出した糖液について電気化学検出器付陰イオン交換高速液体クロマトグラフィー(HPAEC)分析を行ったところ，糖1は1- kestose (3a)のあとに溶出され，糖2，3はnystose (4a), fructosylnystose (5a)のあとにそれぞれ溶出された。この糖抽出液について活性炭-セライトカラムクロマトグラフィーを行うことにより糖1，2，3を単離した。糖 1，2，3はHPAECによる相対保持時間(sucrose = 1)，1.55，2.15，2.73を有し，還元末端をそれぞれもっていた。また，糖1，2，3のフルクトースに対する還元糖の比は 0.50，0.33，0.25であることとTOF-MS分析の結果から糖1，2，3の重合度は2，3，4であることがわかった。これらの結果と各糖の NMR分析，糖メチル誘導体のGLC分析結果から，糖1，2，3はそれぞれβ-D-fructofuranosyl-(2→1)-β-D-fructopyranose (inulobiose)，β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl-(2→1)-β-D-fructopyranose (inulotriose)，β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl-(2→1)-β-D-fructo-pyranose (inulotetraose)と同定された。13C-NMR分析により各糖の還元末端フルクトース残基の70-80％がピラノース構造をとることがわかった。それぞれの糖の13C-，1H- シグナルの完全帰属も初めて行った。さらに上述のごぼう貯蔵中に生成されたinulobiose，inulotriose，inulotetraoseと同様の糖が，ごぼうから精製したfructan fructan 1-fructosyltransferase (1-FFT)のフルクトシル転移作用により1-kestoseとD-フルクトピラノースから合成されることを初めて見出した