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 synthe sized in a coupled reticulocyte lysate system lacking of glycosylation modi fication by the template DNA(Chh1) in the presence of T7 RNA polymerase. Th e synthesized CBH I had the expected size (57 kDa) and catalyzed the substr ate of p-nitrophenyl-beta-D-cellobioside (pNPC), and had no activity agains t carboxymethyl cellulose (CMC-Na). The K-m and V-max values of the CBH I f or pNPC were 0.82 mmol and 0.067 mu mol min(-1) per mu g enzyme, respective ly. The results indicated that glycosylation may not be necessary for enzym atic activity of fungal cellulases. (C) 2000 Elsevier Science B.V. All righ ts reserved.