Construction of cellobiohydrolase from filamentous fungus with endo-type cleavage fashion using protein engineering

利用蛋白质工程以内型切割方式构建丝状真菌纤维二糖水解酶

• 批准号: 10660078
• 负责人: MORIKAWA Yasushi
• 金额: $ 0.77万
• 依托单位: Nagaoka University of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10660078/
• 关键词: Trichoderma reesei; cellobiohydrolase II; Thermmonospora fusca; endoglucanase E2; cellulase; tertiary structure; protein engineering; エキソ型セルラーゼ; エンド型セルラーゼ; 構造活性相関; 遺伝子発現; エンドグルカナーゼ E2

Cellobiohydrolase II (CBH II) from Trichoderma reesei, an exoglucanase, has two long loops (the N-loop and C-loop) covering the substrate binding cleft, which are deleted or shortened in endoglucanase E2 from Thermomonospora fusca. The presence or length of these loops is thought to explain the distinction between endo- and exo-cleavage fashions of cellulases.To enhance the ability of CBH II to degrade crystalline cellulose and to lead to allowing CBH II to possess an endoglucanase activity, we tried to construct chimera CBH II mutants by substituting its loops to the E2 short loops. When the chimera CBH II mutants were expressed in Schizosaccharo-myces pombe and Aspergillus oryzae transformed with each of the vectors carrying their genes, their amounts decreased to a great extent copared to that of native CBH II, or they could not be detected by Western blotting. Furthermore, even the chimera mutants slightly expressed had been breakdown to the truncated ones by proteases. The tertiary structure of the mutant with the C-loop of E2 was analysed by computor modelling to be similar as those of the postulated chimera and the native CBH II. From these results, it was assumed that many difficulties arose in the folding and secretion of the mutants after the transcription of the genes in S. pombe and A. oryzae.As a second trial, to cause CBH II to have an endoglucanase activity, we carried out the site-directed mutagenesis of Glu399 and Asp137 presumed to block the non-reducing end of cellulose to pass through the tunnel of the substrate binding site. Although these mutants also decreased in the amounts expressed in S. pombe and A. oryzae, their detailed characteristics are now analysing. We are constructing the T. reesei transformation system to express the mutants, because the difficulties in the secretion and folding recognized in S. pombe and A. oryzae would be able to minimize in the system.

来自里氏木霉的纤维二糖水解酶II（CBH II）是一种外切葡聚糖酶，具有覆盖底物结合裂缝的两个长环（N环和C环），其在来自褐色热单孢菌的内切葡聚糖酶E2中缺失或缩短。这些环的存在或长度被认为可以解释纤维素酶内切和外切方式的区别。为了增强CBH II降解结晶纤维素的能力并导致CBH II具有内切葡聚糖酶活性，我们试图通过将其环替换为E2短环来构建嵌合体CBH II突变体。当嵌合体CBH II突变体在用携带其基因的每种载体转化的裂殖酵母和曲霉中表达时，它们的量在很大程度上与天然CBH II的量相当，或者它们不能被Western印迹检测到。此外，即使是轻微表达的嵌合体突变体也被蛋白酶降解为截短的突变体。通过计算机模拟分析，具有E2的C环的突变体的三级结构与假定的嵌合体和天然CBH II的三级结构相似。从这些结果可以推测，在S. pombe和A.作为第二次试验，为了使CBH II具有内切葡聚糖酶活性，我们进行了Glu399和Asp137的定点诱变，推测其阻断纤维素的非还原端通过底物结合位点的通道。虽然这些突变体在S. pombe和A.目前正在分析其详细特征。我们正在构建T。reesei转化系统表达突变体，因为S. pombe和A.在系统中的最小化。