Drug Development/Diagnostics & Biotechnology Division, Indian Institute of Chemical Biology, 4, Raja S C Mullick Road, Kolkata 700032, India.
Appl Biochem Biotechnol. 2010 Sep;162(2):373-89. doi: 10.1007/s12010-009-8763-0. Epub 2009 Sep 1.
A low molecular weight endo-xylanase (EC 3.2.1.8) was purified from an edible mushroom Termitomyces clypeatus grown in submerged medium with oat spelt xylan. Xylanase was purified to apparent homogeneity by ammonium sulfate fractionation and gel filtration chromatography. Its molecular weight was determined by gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 12 kDa. The enzyme was found to be most active at 50 degrees C and pH 5.0, being most stable at pH 6.5. The K(m) for oat spelt xylan was determined to be 10.4 mg/ml. The specificities of the enzyme was observed to be highly specific towards oat spelt xylan and was inhibited by mercuric chloride (HgCl(2)), N-bromosuccinimide, and trans-1,2-diaminocyclohexane-N',N',N',N'-tetraacetic acid strongly. The inhibitory action of N-bromosuccinimide on enzyme confirmed the presence of one tryptophan residue in its substrate-binding site. Amino acid analysis for xylanase showed the presence of high amount of hydrophobic serine, glycine, threonine, and alanine residues. The N-terminal sequencing study for the previously purified and characterized 56 kDa xylanolytic amyloglucosidase reveal the presence of 33.30% identity with glucoamylase chain A from Aspergillus awamori. The N-terminal sequence analysis of the present 12 kDa enzyme showed highest similarity (72.22% identity) towards xylanase from Neurospora crassa.
一种低分子量内切木聚糖酶（EC 3.2.1.8）从在浸没培养基中生长的食用蘑菇 Termitomyces clypeatus 中被纯化出来，该培养基使用燕麦斯佩尔特木聚糖作为碳源。木聚糖酶通过硫酸铵分级沉淀和凝胶过滤层析被纯化到近乎均一的状态。其分子量通过凝胶过滤层析和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳确定为 12 kDa。该酶在 50°C 和 pH 5.0 时表现出最大活性，在 pH 6.5 时最稳定。确定燕麦斯佩尔特木聚糖的 K(m)为 10.4 mg/ml。该酶对燕麦斯佩尔特木聚糖具有高度特异性，并被氯化汞（HgCl(2)）、N-溴代丁二酰亚胺和反式-1,2-二氨基环己烷-N',N',N',N'-四乙酸强烈抑制。N-溴代丁二酰亚胺对酶的抑制作用证实了其底物结合位点存在一个色氨酸残基。木聚糖酶的氨基酸分析表明，其存在大量的疏水性丝氨酸、甘氨酸、苏氨酸和丙氨酸残基。先前纯化和表征的 56 kDa 木聚糖裂解淀粉酶的 N 末端测序研究表明，其与 Aspergillus awamori 的葡糖淀粉酶链 A 具有 33.30%的同源性。目前 12 kDa 酶的 N 末端序列分析显示，其与 Neurospora crassa 的木聚糖酶具有最高的相似性（72.22%的同源性）。
