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

里氏木霉(Trichoderma Reesei)纤维素酶II(CbH II)是一种外切葡聚糖酶，在底物结合裂隙上有两个长环(N环和C环)，这两个环在青海热单胞菌的内切葡聚糖酶E2中缺失或缩短。这些环的存在或长度被认为解释了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。将嵌合体Cbh II分别转化到裂殖酵母和米曲霉中，表达产物的量与天然Cbh II相当，或用Western blotting检测不到。此外，即使是轻微表达的嵌合体突变体也被蛋白酶分解为截短的嵌合体突变体。通过计算机模拟分析了带有E2 C环的突变体的三级结构，认为它与假设的嵌合体和天然的CBH II相似。根据这些结果，推测在庞贝和稻曲霉的基因转录后，突变体的折叠和分泌出现了许多困难。作为第二次试验，我们对Glu399和Asp137进行了定点突变，推测Glu399和Asp137可以阻断纤维素的非还原末端通过底物结合部位的隧道。虽然这些突变体在S.pombe和A.oryzae中的表达数量也减少了，但它们的详细特征现在正在分析中。我们正在构建里赛酵母转化系统来表达突变体，因为在庞氏链霉菌和米曲霉中识别的分泌和折叠方面的困难将能够在该系统中最小化。

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.