Shannon Applied Biotechnology Centre, Department of Applied Science, Limerick Institute of Technology, Moylish Park, Limerick, Ireland,
Appl Biochem Biotechnol. 2014 Feb;172(4):1747-62. doi: 10.1007/s12010-013-0640-1. Epub 2013 Nov 22.
This work reports the first investigation of Remersonia thermophila hemicellulosic hydrolytic enzyme production, with subsequent purification of an extracellular endo-β-1,4-xylanase (RtXyl) and its application in bread making. The research describes RtXyl purification from sorghum-induced submerged liquid cultures of this moderately thermophilic, aerobic, ascomycete fungus. The purified enzyme is a single subunit protein with a molecular mass of 42 kDa and exhibits glycosyl hydrolase family-10-like activity over a broad pH and temperature range. Optimal activity was measured at pH 6.0 and 65 °C respectively, which is suitable for bread making applications. Substrate specificity studies revealed that RtXyl is purely xylanolytic with no side-activities against other plant polysaccharides. The RtXyl catalytic efficiency (K cat/K m) was highest with oats spelt xylan (810.90 mg mL(-1) s(-1)), wheat arabinoxylan (809.52 mg mL(-1) s(-1)) and beechwood xylan (417.40 mg mL(-1) s(-1)) with less efficiency towards insoluble oats spelt xylan (236.40 mg mL(-1) s(-1)). Hydrolysis products analysed by thin layer chromatography yielded a range of xylosaccharides, predominantly xylotriose and xylobiose. RtXyl application in a basic wheat bread recipe at low dosages (0.297 XU/g) showed its suitability to increase loaf volume by 8.0 % compared with the control bread. RtXyl increased loaf softness by 19.6 % while reducing bread staling by 20.4 % up to 4 days of storage.
这项工作首次报道了嗜热 Remersonia thermophila 半纤维素水解酶的生产研究，随后对一种胞外内切-β-1,4-木聚糖酶（RtXyl）进行了纯化，并将其应用于面包制作。该研究描述了从高粱诱导的这种中温需氧子囊菌的液体深层发酵液中纯化 RtXyl。纯化的酶是一种具有 42 kDa 分子量的单一亚基蛋白，在较宽的 pH 和温度范围内表现出糖苷水解酶家族 10 样活性。最佳活性分别在 pH 6.0 和 65°C 下测量，这适合于面包制作应用。底物特异性研究表明，RtXyl 是纯粹的木聚糖酶，对其他植物多糖没有侧活性。RtXyl 的催化效率（K cat/K m）以燕麦 spelt 木聚糖（810.90 mg mL(-1) s(-1)）、小麦阿拉伯木聚糖（809.52 mg mL(-1) s(-1)）和山毛榉木聚糖（417.40 mg mL(-1) s(-1)）最高，对不溶性燕麦 spelt 木聚糖（236.40 mg mL(-1) s(-1)）的效率较低。薄层层析分析水解产物得到一系列木聚糖，主要是木三糖和木二糖。在低剂量（0.297 XU/g）下将 RtXyl 应用于基本小麦面包配方中，与对照面包相比，其适合将面包体积增加 8.0%。RtXyl 增加了面包的柔软度 19.6%，同时减少了面包老化 20.4%，直到储存 4 天。
