College of Food Science and Engineering, Shandong Agricultural University, No. 61 Daizong Street, Tai'an 271018, China.
Molecules. 2020 Mar 29;25(7):1572. doi: 10.3390/molecules25071572.
In this study, an endo-1,4-β-xylanase was purified from wheat malt following the procedures of ammonium sulfate precipitation, cation-exchange chromatography, and two-step anion-exchange chromatography. The purified endo-1,4-β-xylanase had a specific activity of 3.94 u/mg, demonstrating a weight average molecular weight (Mw) of approximately 58,000 Da. After LC-MS/MS (Liquid chromatography-tandem mass spectrometry) identification, the purified enzyme had the highest matching degree with a GH10 (Glycoside Hydrolase 10) domain-containing protein from wheat, there were 23 match peptides with a score above the threshold and the prot-cover was 45.5%. The resulting purified enzyme was used to investigate its degradation ability on high viscosity wheat-derived water-extractable arabinoxylan (WEAX). Degradation experiments confirmed that the purified enzyme was a true endo-acting enzyme, which could degrade large WEAX into smaller WEAX. The average degree of polymerization (avDP) and the viscosity of WEAX decreased with the increasing reaction time. The enzyme could degrade a small amount of WEAX into arabinoxylan-oligosaccharides (AXOS) with a degree of polymerization of 2-6, but no monosaccharide was produced. The degradation occurred rapidly in the first 3.5 h and decreased with the further prolongation of reaction time.
在这项研究中，采用硫酸铵沉淀、阳离子交换层析和两步阴离子交换层析的方法，从麦芽中纯化出内切 1,4-β-木聚糖酶。纯化后的内切 1,4-β-木聚糖酶比活为 3.94u/mg，分子量约为 58000Da。经 LC-MS/MS（液相色谱-串联质谱）鉴定，该纯化酶与小麦中 GH10（糖苷水解酶 10）结构域含有的蛋白具有最高的匹配度，有 23 条匹配肽段的得分超过阈值，prot-cover 为 45.5%。用所得的纯化酶研究其对高粘度小麦源可提取阿拉伯木聚糖（WEAX）的降解能力。降解实验证实，该纯化酶是一种真正的内切酶，可将大分子量的 WEAX 降解成小分子量的 WEAX。WEAX 的平均聚合度（avDP）和黏度随反应时间的延长而降低。该酶能将少量 WEAX 降解成聚合度为 2-6 的阿拉伯木聚糖低聚糖（AXOS），但不产生单糖。降解反应在前 3.5 小时迅速发生，随着反应时间的进一步延长而降低。
