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首页> 外文期刊>Journal of the Institute of Brewing >Purification of an Arabinofuranosidase and Two β-Xylopyranosidases from Germinated Wheat
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Purification of an Arabinofuranosidase and Two β-Xylopyranosidases from Germinated Wheat

机译：从发芽小麦中纯化阿拉伯呋喃糖苷酶和两种β-木吡喃糖苷酶

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摘要

Arabinosidase and β-xylosidase activities were detected in germinated wheat grain, and both increased over seven days of germination, under malting conditions. Arabinosidase was partially purified by anion exchange chromatography, chromatofocusing and gel filtration chromatography. The pH optimum of the partially purified enzyme was 4.2 and the K_M was 1.90 mM p-NP-Ara. Edman degradation, MALDI-TOF mass spectrometry and nano-ESI mass spectrometry were used to identify the two major proteins in the partially purified arabinosidase mixture. The two proteins were a β-amylase with an amino acid sequence partially homologous to a barley β-amylase, and three wheat serine protease inhibitors. Further purification, by affinity chromatography and hydrophobic interaction chromatography, removed the identified contaminating proteins. At this point an 80 kDa protein was detected by SDS-PAGE. No identity could be assigned to this protein by MALDI-TOF mass spectrometry by reference to electronic protein databases. Similarly, the β-xylosidase was partially purified by anion exchange chromatography followed by chromatofocusing and gel filtration chromatography. The first step separated the mixture into two distinct fractions with K_M values of 3.35 and 4.01 mM p-NP-Xyl and pH optima of 4.5. The latter fraction also displayed xylanase activity against RBB-xylan.
机译：在发芽条件下，在发芽的小麦籽粒中检测到阿拉伯糖苷酶和β-木糖苷酶活性，并且在发芽的七天中它们均增加。阿拉伯糖苷酶通过阴离子交换色谱，色谱聚焦和凝胶过滤色谱法部分纯化。部分纯化的酶的最适pH为4.2，K_M为1.90 mM p-NP-Ara。使用Edman降解，MALDI-TOF质谱和纳米ESI质谱鉴定部分纯化的阿拉伯糖苷酶混合物中的两种主要蛋白质。这两种蛋白是一种与大麦β-淀粉酶部分同源的氨基酸序列的β-淀粉酶，以及三种小麦丝氨酸蛋白酶抑制剂。通过亲和色谱和疏水相互作用色谱进一步纯化，除去了鉴定出的污染蛋白。此时，通过SDS-PAGE检测到80kDa的蛋白质。 MALDI-TOF质谱无法通过参考电子蛋白质数据库为该蛋白质分配任何身份。类似地，β-木糖苷酶通过阴离子交换色谱，随后的色谱聚焦和凝胶过滤色谱来部分纯化。第一步将混合物分为两个不同的馏分，K_M值为3.35和4.01 mM p-NP-Xyl，pH最适值为4.5。后一部分也显示出抗RBB-木聚糖的木聚糖酶活性。

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