Wonganu Benjamaporn, Pootanakit Kusol, Boonyapakron Katewadee, Champreda Verawat, Tanapongpipat Sutipa, Eurwilaichitr Lily
Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakhorn-Pathom 73170, Thailand.
Protein Expr Purif. 2008 Mar;58(1):78-86. doi: 10.1016/j.pep.2007.10.022. Epub 2007 Nov 7.
Endoglucanase is a major cellulolytic enzyme produced by Syncephalastrum racemosum (BCC18080). Preliminary results showed that this endoglucanase is thermotolerant as it retained more than 50% of its activity after incubation at 80 degrees C for an hour. As this property may be of industrial use, we have cloned the full-length BCC18080 endoglucanase gene of 1020 nucleotides. Sequence analysis suggested that it belonged to the glycosyl hydrolase family 45. N-terminal sequencing and analysis by SignalP program suggested that the first 32 amino acid residues encoded the signal peptide. Expression of the recombinant clones with and without its own signal peptide in Pichia pastoris demonstrated that P. pastoris produced active 55 and 30 kDa secreted proteins. N-terminal sequencing suggested that the 55 kDa band was the mature protein while the 30 kDa band was the truncated protein. Glycoprotein analysis showed that the 55 kDa protein was glycosylated; while the smaller protein was not. All recombinant endoglucanases showed optimal temperature of 70 degrees C and optimal pH of 5-6. They retained more than 50% activity for 4h at 70 degrees C. In addition, high k(cat) and low apparent K(m) of these recombinant proteins indicated good properties of this enzyme against carboxylmethylcellulose.
内切葡聚糖酶是总状共头霉（BCC18080）产生的一种主要纤维素分解酶。初步结果表明，这种内切葡聚糖酶具有耐热性，因为在80℃孵育1小时后仍保留超过50%的活性。由于这一特性可能具有工业用途，我们克隆了1020个核苷酸的全长BCC18080内切葡聚糖酶基因。序列分析表明它属于糖基水解酶家族45。N端测序和SignalP程序分析表明，前32个氨基酸残基编码信号肽。在毕赤酵母中表达带有和不带有自身信号肽的重组克隆，结果表明毕赤酵母产生了活性分别为55 kDa和30 kDa的分泌蛋白。N端测序表明55 kDa条带是成熟蛋白，而30 kDa条带是截短蛋白。糖蛋白分析表明55 kDa蛋白被糖基化，而较小的蛋白未被糖基化。所有重组内切葡聚糖酶的最适温度为70℃，最适pH为5 - 6。它们在70℃下4小时内保留超过50%的活性。此外，这些重组蛋白的高催化常数（k(cat)）和低表观米氏常数（K(m)）表明该酶对羧甲基纤维素具有良好的性质。
