Ueno Keiji, Ishiguro Yojiro, Yoshida Midori, Onodera Shuichi, Shiomi Norio
Department of Food and Nutrition Sciences, Graduate School of Dairy Science Research, Rakuno Gakuen University, 582 Bunkyodai Midorimachi, Ebetsu, 069-8501, Japan.
Chem Cent J. 2011 Apr 5;5(1):16. doi: 10.1186/1752-153X-5-16.
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.
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.
我们之前报道过牛蒡根在不同温度下储存时，总低聚果糖（FOS）、总低聚菊粉（IOS）和菊粉的变化情况。在0°C储存期间，观察到由于菊粉水解导致FOS增加。此外，我们认为IOS的增加可能是由于1-蔗果三糖的果糖基转移至积累的果糖和可作为果聚糖：果聚糖1-果糖基转移酶（1-FFT）受体的延长的果糖寡聚物而合成IOS。然而，参与牛蒡根中菊粉降解的酶，如菊粉酶或果聚糖1-外切水解酶（1-FEH）仍不清楚。在此，我们报道了一个编码牛蒡1-FEH基因的分离和功能分析。
通过基于其他植物FEH氨基酸序列设计的简并引物进行PCR后，采用RACE方法获得了一个名为aleh1的cDNA。aleh1编码一个581个氨基酸的多肽。推导的多肽的相对分子质量和等电点（pI）经计算分别为65,666和4.86。aleh1的重组蛋白在毕赤酵母中产生，并通过用DEAE-琼脂糖CL-6B进行离子交换色谱、用Toyopearl HW55S进行疏水色谱和用Toyopearl HW55S进行凝胶过滤色谱进行纯化。纯化的重组蛋白对β-2,1型果聚糖如1-蔗果三糖、蔗果四糖、果糖基蔗果四糖和菊粉具有水解活性。另一方面，蔗糖、新蔗果三糖、6-蔗果三糖和高聚合度的左聚糖是较差的底物。纯化的重组蛋白从牛蒡根中提取的糖中释放出果糖。这些结果表明aleh1编码1-FEH。
