以內切木聚醣酶結合液相層析串聯質譜法建立阿拉伯木聚醣分子特徵

Abstract

阿拉伯木聚醣為穀物主要半纖維素組成，複雜的側支結構是影響其吸水性、黏度、保氣性及益生質功能的因素，更是分析上的挑戰。本研究利用內切木聚醣酶將櫸木 (BWX)、燕麥 (OSX)與小麥 (WAX)三種來源水溶性同質與異質木聚醣水解出保有側支的寡醣，再以液相層析串聯軌道阱質譜 (LC-Orbitrap-MS/MS)進行結構解析，鑑別骨幹與側支的鍵結資訊。單醣組成及甲基化醣基鍵結分析顯示，BWX為同質木聚醣，而OSX與WAX為異質阿拉伯木聚醣，含有阿拉伯糖醣基分支。本研究選擇真菌Trichoderma viride 之glycoside hydrolase (GH) 11 family 內切木聚醣酶釋出寡醣，所建立液相層析串聯軌道阱質譜法解析出直線型木寡醣及分支型阿拉伯木寡醣共17種，分布在三至七醣聚合度範圍。解析結果顯示所選擇的三種木聚醣均以(1→4)-鍵結之木糖醣基為骨幹，BWX為單純的直線型，OSX樣品之分支率為15%，包含 (1→3)-鍵結單取代10%及 (1→2;1→3)-鍵結雙取代5%阿拉伯糖側支。WAX分支率較高為20%，包含 (1→3)-鍵結單取代17%、連續 (1→3)-鍵結單取代2%以及 (1→2;1→3)-鍵結雙取代1%。所建立分析平台同時可以由水解寡醣產物解析結果，推測此glycoside hydrolase (GH) 11 family的內切木聚醣酶具有-3至+4共七個受質結合位，在結合位-3、-2、+2及+3上可容納單取代及雙取代形式的醣基側支修飾。本研究建立之分析平台能夠辨別木聚醣骨幹上阿拉伯糖醣基取代基鍵結及取代形式的差異，為以木糖為骨幹的聚醣及寡醣產品提供便捷的品質管制平台，並可延伸發展為木寡醣益生質之酵素來源選擇與產品品質的監測工具。

Arabinoxylan is one of the major hemicellulose components in cereal's cell walls. The complex structural characteristics of arabinoxylan alter its water-absorbing, viscosity, gas retention capacity, prebiotic properties and pose a challenge to chemical analysis. This study developed an effective analytical tool based on enzymatic digestion combining a high-resolution liquid-chromatography-orbitrap tandem mass spectrometry (LC-Orbitrap-MS/MS) to reveal the structural characteristics of three water-soluble xylans from beechwood (BWX), oat spelts (OSX), and wheat (WAX). Results of sugar composition and permethylation linkage analyses indicated that BWX was a homogenous xylan, and OSX and WAX were heterogenous arabinoxylans with arabinosyl branches. Glycoside hydrolase family 11 endoxylanase from Trichoderma viride was selected to release oligomers from the hemicellulose samples. The platform could characterize 17 oligosaccharides, including linear and branched structures in the degree of polymerization range of 3 ~ 7. The structural characterizing results indicate that (1→4)-linked xylan was the backbone for all three polysaccharides. BWX was a linear xylan. The degree of substitution of the OSX was 15%, including 10% of (1→3)-linked monosubstituted and 5% of (1→2;1→3)-linked disubstituted arabinosyl branches; the degree of substitution of the WAX was 20%, higher than OSX, including 17% of (1→3)-linked monosubstituted, 2% of continuously (1→3)-linked monosubstituted and 1% of (1→2;1→3)-linked disubstituted arabinosyl branches. The information of released oligosaccharides also could induce that the active sites of the glycoside hydrolase (GH) 11 family endoxylanase contained seven binding subsites. The binding subsites could be labeled from -3 to +4, where the point of glycosidic bond cleavage between subsites -1 and +1. The enzyme can also accommodate monosubstituted and disubstituted arabinosyl side groups at the -3, -2, +2, and +3 subsites. This developed analytical platform can differentiate substituted linkages and patterns of branches on xylans. It can be an effective tool for quality control of arabinoxylans and applied to search for enzyme sources for prebiotic development from xylo-oligosaccharides.