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

Abstract

An enzymatically-active fungal cellobiohydrolase I (CBH I) was first synthesized in a coupled reticulocyte lysate system lacking of glycosylation modification by the template DNACbh1 in the presence of T7 RNA polymerase. The synthesized CBH I had the expected size (57 kDa) and catalyzed the substrate of p-nitrophenyl-β-d-cellobioside (pNPC), and had no activity against carboxymethyl cellulose (CMC-Na). The Km and Vmax values of the CBH I for pNPC were 0.82 mmol and 0.067 μmol min−1 per μg enzyme, respectively. The results indicated that glycosylation may not be necessary for enzymatic activity of fungal cellulases.