Sakka Makiko, Goto Masayuki, Fujino Tsuchiyoshi, Fujino Emi, Karita Shuichi, Kimura Tetsuya, Sakka Kazuo
Graduate School of Bioresources, Mie University, Tsu, Japan.
Biosci Biotechnol Biochem. 2010;74(10):2077-82. doi: 10.1271/bbb.100458. Epub 2010 Oct 7.
A cellulase gene cluster of Clostridium josui was sequenced, and was found to encode 11 proteins responsible for cellulosome (cellulolytic complex) formation, viz., cipA, cel48A, cel8A, cel9A, cel9B, orfX, cel9C, cel9D, man5A, cel9E, and cel5B, in order from the upstream side. All the predicted enzymes had a dockerin module, suggesting that these proteins are members of the C. josui cellulosome. Among these genes, the man5A gene encoding β-mannanase was expressed in Escherichia coli and the recombinant enzyme (rMan5A) was characterized. rMan5A showed strong activity toward carob galactomannan and low activity toward guar gum, suggesting that it prefers non-galactosylated mannan to galactomannan. This enzyme hydrolyzed ivory nut mannan to produce mainly mannotriose and larger mannooligosaccharides, and was not active toward mannotriose. An antiserum raised against the recombinant enzyme detected Man5A in the culture supernatants of C. josui, which was grown on either ball-milled cellulose or glucose as a carbon source.
对约氏梭菌的一个纤维素酶基因簇进行了测序，发现它编码11种负责纤维小体（纤维素分解复合体）形成的蛋白质，即从上游开始依次为cipA、cel48A、cel8A、cel9A、cel9B、orfX、cel9C、cel9D、man5A、cel9E和cel5B。所有预测的酶都有一个锚定蛋白模块，表明这些蛋白质是约氏梭菌纤维小体的成员。在这些基因中，编码β-甘露聚糖酶的man5A基因在大肠杆菌中表达，并对重组酶（rMan5A）进行了特性分析。rMan5A对角豆半乳甘露聚糖表现出较强活性，对瓜尔豆胶活性较低，这表明它更喜欢非半乳糖基化的甘露聚糖而非半乳甘露聚糖。该酶水解象牙果甘露聚糖主要产生甘露三糖和更大的甘露寡糖，且对甘露三糖无活性。用针对重组酶制备的抗血清在以球磨纤维素或葡萄糖作为碳源生长的约氏梭菌培养上清液中检测到了Man5A。
