Sinma Kanokkorn, Khucharoenphaisan Khwanchai, Kitpreechavanich Vichien, Tokuyama Shinji
Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka, Japan.
Biosci Biotechnol Biochem. 2011;75(10):1957-63. doi: 10.1271/bbb.110353. Epub 2011 Oct 7.
An extracellular thermostable xylanase produced by Saccharopolyspora pathumthaniensis S582 was purified 167-fold to homogeneity with a recovery yield of 12%. The purified xylanase appeared as a single protein band on SDS-PAGE, with a molecular mass of 36 kDa. The optimal temperature and pH of the xylanase were 70 °C and 6.5. The enzyme was stable within a pH range of 5.5-10.0. It retained its activity after incubation at 50 °C for 2 h. Its half lives at temperatures of 60 and 70 °C were 180 and 120 min respectively. Hydrolysis of beechwood xylan by the xylanase yielded xylobiose and xylose as major products. The enzyme acted specifically on xylan as an endo-type xylanase, and exhibited a K(m) value of 3.92 mg/mL and a V(max) value of 256 µmol/min/mg. Enzyme activity was completely inhibited by Hg(2+), and was stimulated by Rb(+) and Cs(+). The xylanase gene was cloned from genomic DNA of Saccharopolyspora pathumthaniensis S582 and sequenced. The ORF consisted of 1,107 bp and encoded 368 amino acid residues containing a putative signal peptide of 23 residues. This xylanase is a new member of family (GH) 10 that shows highest identity, of 63.4%, with a putative xylanase from Nocardiopsis dassonvillei subsp. dassonvillei.
由泰国糖多孢菌S582产生的一种胞外耐热木聚糖酶被纯化了167倍，达到了均一性，回收率为12%。纯化后的木聚糖酶在SDS-PAGE上呈现为单一蛋白条带，分子量为36 kDa。该木聚糖酶的最适温度和pH分别为70℃和6.5。该酶在pH 5.5-10.0范围内稳定。在50℃孵育2小时后仍保留其活性。其在60℃和70℃的半衰期分别为180分钟和120分钟。该木聚糖酶水解山毛榉木聚糖产生的主要产物是木二糖和木糖。该酶作为一种内切型木聚糖酶，对木聚糖具有特异性作用，其K(m)值为3.92 mg/mL，V(max)值为256 µmol/min/mg。酶活性被Hg(2+)完全抑制，被Rb(+)和Cs(+)激活。从泰国糖多孢菌S582的基因组DNA中克隆并测序了木聚糖酶基因。开放阅读框由1107 bp组成，编码368个氨基酸残基，其中包含一个23个残基的假定信号肽。这种木聚糖酶是糖基水解酶（GH）10家族的一个新成员，与达松维尔诺卡氏菌达松维尔亚种的一种假定木聚糖酶具有最高的同源性，为63.4%。
