A novel type of arabinoxylan arabinofuranohydrolase isolated from germinated barley -: Analysis of substrate preference and specificity by nano-probe NMR
A novel type of arabinoxylan arabinofuranohydrolase isolated from germinated barley -: Analysis of substrate preference and specificity by nano-probe NMR
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DOI：
10.1046/j.1432-1327.2000.01758.x
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发表时间：
2000-11-01
期刊：
影响因子：
--
通讯作者：
Thomsen, KK
中科院分区：
文献类型：
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作者：
Ferré, H;Broberg, A;Thomsen, KK
从大麦芽中分离得到一种阿拉伯木聚糖阿拉伯呋喃水解酶。酶制剂Ara 1含有两种多肽，其表观分子量约为60和66 kDa，pI为4.55，N-末端氨基酸序列几乎相同。以对硝基苯基α-L-阿拉伯呋喃糖苷（pNPA）为底物，Ara 1的Km为0.5 mm，Vmax为6.7 μ mol·min（-1）。(mg蛋白质（-1）最大活性显示在pH 4.2和60 degreesC，并且，在这些条件下，酶的半衰期为8 min。Ara 1制剂显示对对硝基苯基α-L-阿拉伯吡喃糖苷或对硝基苯基β-D-吡喃木糖苷没有活性。使用纯寡糖并通过TLC和纳米探针NMR分析研究底物偏好和特异性。Ara 1从高分子量阿拉伯木聚糖和阿拉伯聚糖衍生的寡糖中释放阿拉伯糖，但对直链或支链阿拉伯聚糖无活性。阿拉伯糖容易从单取代和双取代的低聚木糖中释放出来。而单一的2-O-连接和3-O-连接的阿拉伯糖取代基上的非还原性末端木糖以相似的速率释放，有一个明确的偏好2-O-连接的阿拉伯糖内部木糖残基。当Ara 1作用于具有双取代的非还原性末端木糖的寡糖时，3-O-连接的阿拉伯糖基团优选作为起始攻击点。具有双取代内部木糖的寡聚木糖是不良底物，并且不能确定偏好。这里描述的酶是第一个报道的阿拉伯木聚糖阿拉伯呋喃水解酶，其能够从单取代和双取代的木糖中释放阿拉伯糖，并且其以与对寡糖底物观察到的速率相似的速率水解对硝基苯基α-L-阿拉伯呋喃糖苷。
An arabinoxylan arabinofuranohydrolase was isolated from barley malt. The enzyme preparation, Ara 1, contained two polypeptides with apparent molecular masses of approximate to 60 and approximate to 66 kDa, a pI of 4.55 and almost identical N-terminal amino-acid sequences. With p-nitrophenyl alpha -L-arabinofuranoside (pNPA) as substrate, Ara 1 exhibited a K-m of 0.5 mm and a V-max of 6.7 mu mol.min(-1).(mg of protein)(-1). Maximum activity was displayed at pH 4.2 and 60 degreesC, and, under these conditions, the half-life of the enzyme was 8 min. The Ara 1 preparation showed no activity against p-nitrophenyl alpha -L-arabinopyranoside or p-nitrophenyl beta -D-xylopyranoside. Substrate preference and specificity were investigated using pure oligosaccharides and analysis by TLC and nano-probe NMR. Ara 1 released arabinose from high-molecular-mass arabinoxylan and arabinoxylan-derived oligosaccharides but was inactive against linear or branched-chain arabinan. Arabinose was readily released from both singly and doubly substituted xylo-oligosaccharides. Whereas single 2-O-linked and 3-O-linked arabinose substituents on non-reducing terminal xylose were released at similar rates, there was a clear preference for 2-O-linked arabinose on internal xylose residues. When Ara 1 acted on oligosaccharides with doubly substituted, non-reducing terminal xylose, the 3-O-linked arabinose group was preferred as the initial point of attack. Oligosaccharides with doubly substituted internal xylose were poor substrates and no preference could be determined. The enzyme described here is the first reported arabinoxylan arabinofuranohydrolase which is able to release arabinose from both singly and doubly substituted xylose, and it hydrolyses p-nitrophenyl alpha -L-arabinofuranoside at a rate similar to that observed for oligosaccharide substrates.