Takao Makoto, Akiyama Kana, Sakai Takuo
Department of Food and Nutrition, Faculty of Agriculture, Kinki University, 3327-204 Naka-machi, Nara 631-8505, Japan.
Appl Environ Microbiol. 2002 Apr;68(4):1639-46. doi: 10.1128/AEM.68.4.1639-1646.2002.
A strain of a thermophilic bacterium, tentatively designated Bacillus thermodenitrificans TS-3, with arabinan-degrading activity was isolated. It produced an endo-arabinase (ABN) (EC 3.2.1.99) and two arabinofuranosidases (EC 3.2.1.55) extracellularly when grown at 60 degrees C on a medium containing sugar beet arabinan. The ABN (tentatively called an ABN-TS) was purified 7,417-fold by anion-exchange, hydrophobic, size exclusion, and hydroxyapatite chromatographies. The molecular mass of ABN-TS was 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point was pH 4.5. The enzyme was observed to be more thermostable than known ABNs; it had a half-life of 4 h at 75 degrees C. The enzyme had optimal activity at 70 degrees C and pH 6.0. The enzyme had apparent K(m) values of 8.5 and 45 mg/ml and apparent V(max) values of 1.6 and 1.1 mmol/min/mg of protein against debranched arabinan (alpha-1,5-arabinan) and arabinan, respectively. The enzyme had no pectin-releasing activity (protopectinase activity) from sugar beet protopectin, differing from an ABN (protopectinase-C) from mesophilic Bacillus subtilis IFO 3134. The pattern of degradation of debranched arabinan by ABN-TS indicated that the enzyme was an endo-acting enzyme and the main end products were arabinobiose and arabinose. The results of preliminary experiments indicated that the culture filtrate of strain TS-3 is suitable for L-arabinose production from sugar beet pulp at high temperature.
分离出了一株具有阿拉伯聚糖降解活性的嗜热细菌菌株，暂命名为嗜热脱硝芽孢杆菌TS-3。当该菌株在含有甜菜阿拉伯聚糖的培养基中于60℃培养时，可胞外产生一种内切阿拉伯聚糖酶（ABN）（EC 3.2.1.99）和两种阿拉伯呋喃糖苷酶（EC 3.2.1.55）。通过阴离子交换、疏水、尺寸排阻和羟基磷灰石色谱法，将ABN（暂称为ABN-TS）纯化了7417倍。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定，ABN-TS的分子量为35 kDa，等电点为pH 4.5。观察发现该酶比已知的ABN更耐热；在75℃下其半衰期为4小时。该酶在70℃和pH 6.0时具有最佳活性。该酶对脱支阿拉伯聚糖（α-1,5-阿拉伯聚糖）和阿拉伯聚糖的表观K(m)值分别为8.5和45 mg/ml，表观V(max)值分别为1.6和1.1 mmol/min/mg蛋白质。该酶对甜菜原果胶无果胶释放活性（原果胶酶活性），这与嗜温性枯草芽孢杆菌IFO 3134的ABN（原果胶酶-C）不同。ABN-TS对脱支阿拉伯聚糖的降解模式表明该酶是一种内切作用酶，主要终产物是阿拉伯二糖和阿拉伯糖。初步实验结果表明，TS-3菌株的培养滤液适合在高温下从甜菜浆中生产L-阿拉伯糖。
