In vitro expression of Penicillium janthinellum cellobiohydrolase I gene in a coupled transcription-translation system

An enzymatically-active fungal cellobiohydrolase I (CBH I) was first synthesized in a coupled reticulocyte lysate system lacking of glycosylation modification by the template DNA(Chh1) in the presence of T7 RNA polymerase. The synthesized CBH I had the expected size (57 kDa) and catalyzed the substrate of p-nitrophenyl-beta-D-cellobioside (pNPC), and had no activity against carboxymethyl cellulose (CMC-Na). The K-m and V-max values of the CBH I for pNPC were 0.82 mmol and 0.067 mu mol min(-1) per mu g enzyme, respectively. The results indicated that glycosylation may not be necessary for enzymatic activity of fungal cellulases. (C) 2000 Elsevier Science B.V. All rights reserved.