Yan Q J, Wang L, Jiang Z Q, Yang S Q, Zhu H F, Li L T
Bioresource Utilization Laboratory, College of Engineering, China Agricultural University, Beijing 100083, China.
Bioresour Technol. 2008 Sep;99(13):5402-10. doi: 10.1016/j.biortech.2007.11.033. Epub 2008 Jan 3.
An extracellular beta-xylosidase from the thermophilic fungus Paecilomyces thermophila J18 was purified 31.9-fold to homogeneity with a recovery yield of 2.27% from the cell-free culture supernatant. It appeared as a single protein band on SDS-PAGE with a molecular mass of approx 53.5 kDa. The molecular mass of beta-xylosidase was 51.8 kDa determined by Superdex 75 gel filtration. The enzyme was a glycoprotein with a carbohydrate content of 61.5%. It exhibited an optimal activity at 55 degrees C and pH 6.5, respectively. The enzyme was stable in the range of pH 6.0-9.0 and at 55 degrees C. The purified enzyme hydrolyzed xylobiose and higher xylooligosaccharides but was inactive against xylan substrates. It released xylose from xylooligosaccharides with a degree of polymerization ranging between 2 and 5. The rate of xylose released from xylooligosaccharides by the purified enzyme increased with increasing chain length. It had a K(m) of 4.3mM for p-nitrophenol-beta-d-xylopyranoside and was competitively inhibited by xylose with a K(i) value of 139 mM. Release of reducing sugars from xylans by a purified xylanase produced by the same organism increased markedly in the presence of beta-xylosidase. During 24-hour hydrolysis, the amounts of reducing sugar released in the presence of added beta-xylosidase were about 1.5-1.73 times that of the reaction employing the xylanase alone. This is the first report on the purification and characterization of a beta-xylosidase from Paecilomyces thermophila.
从嗜热真菌嗜热拟青霉J18中分离得到一种胞外β-木糖苷酶，经纯化后纯度提高了31.9倍，达到同质，从无细胞培养上清液中的回收率为2.27%。该酶在SDS-PAGE上呈现为一条单一蛋白带，分子量约为53.5 kDa。通过Superdex 75凝胶过滤法测定，β-木糖苷酶的分子量为51.8 kDa。该酶是一种糖蛋白，碳水化合物含量为61.5%。它分别在55℃和pH 6.5时表现出最佳活性。该酶在pH 6.0 - 9.0范围内和55℃时稳定。纯化后的酶能水解木二糖和更高聚合度的木寡糖，但对木聚糖底物无活性。它能从聚合度在2至5之间的木寡糖中释放出木糖。纯化后的酶从木寡糖中释放木糖的速率随链长增加而提高。它对对硝基苯酚-β-D-吡喃木糖苷的K(m)值为4.3 mM，被木糖竞争性抑制，K(i)值为139 mM。在β-木糖苷酶存在的情况下，同一生物体产生的纯化木聚糖酶从木聚糖中释放还原糖的量显著增加。在24小时水解过程中，添加β-木糖苷酶时释放的还原糖量约为仅使用木聚糖酶反应的1.5 - 1.73倍。这是关于嗜热拟青霉β-木糖苷酶纯化和特性的首次报道。
