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

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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.