Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Bangkok, Thailand.
J Biosci Bioeng. 2011 May;111(5):528-36. doi: 10.1016/j.jbiosc.2010.12.024. Epub 2011 Jan 26.
A thermophilic xylan-degrading Actinomadura sp. S14 was isolated from compost in Thailand. Hemicellulase activities such as endo-1,4-β-xylanase, β-xylosidase and α-arabinofuranosidase were induced with xylan-containing agriculture wastes and oat spelt xylan. The gene encoding xylanase consisting of 687bp was cloned from Actinomadura sp. S14. The deduced amino acid sequence contained a signal peptide of 41 amino acids and a probable mature xylanase of 188 amino acids. An open reading frame (xynS14) corresponding to a mature xylanase was expressed in Escherichia coli and Pichia pastoris. The specific activity of purified XynS14 (P. pastoris) was 2.4-fold higher than XynS14 (E. coli). Both XynS14s showed the same basic properties such as optimal pH and temperature (pH 6.0 and 80°C) and stability in a broad pH range (pH 5.0-11.0) and at high temperatures up to 80°C. Both XynS14s showed approximately the same substrate specificity and K(m) values toward various xylans, but XynS14 (P. pastoris) showed higher V(max) and K(cat) than XynS14 (E. coli). Higher specific activities of XynS14 (P. pastoris) may be due to protein-folding in the host. Purified XynS14 showed more endo-1,4-β-xylanase activity on xylan and xylooligosaccharides than on xylotriose.
从泰国堆肥中分离到一株嗜热木聚糖降解放线菌 Actinomadura sp. S14。该菌可以利用含木聚糖的农业废弃物和燕麦 spelt 木聚糖诱导产生半纤维素酶活性，如内切-1,4-β-木聚糖酶、β-木糖苷酶和α-阿拉伯呋喃糖苷酶。从 Actinomadura sp. S14 中克隆得到编码木聚糖酶的基因，长 687bp。推测的氨基酸序列含有 41 个氨基酸的信号肽和 188 个氨基酸的成熟木聚糖酶。与成熟木聚糖酶相对应的开放阅读框（xynS14）在大肠杆菌和毕赤酵母中表达。与 XynS14（大肠杆菌）相比，纯化的 XynS14（毕赤酵母）的比酶活提高了 2.4 倍。两种 XynS14 均具有相同的基本性质，如最适 pH 和温度（pH6.0 和 80°C）以及在较宽的 pH 范围（pH5.0-11.0）和高达 80°C 的高温下的稳定性。两种 XynS14 对各种木聚糖的底物特异性和 K(m)值相近，但 XynS14（毕赤酵母）的 V(max)和 K(cat)值均高于 XynS14（大肠杆菌）。XynS14（毕赤酵母）具有更高的比酶活可能是由于在宿主中蛋白质折叠的原因。纯化的 XynS14 对木聚糖和木寡糖的内切-1,4-β-木聚糖酶活性高于对木三糖的活性。
