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(Cbh1) 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 micromol min(-1) per microg enzyme, respectively. The results indicated that glycosylation may not be necessary for enzymatic activity of fungal cellulases.
Wang
J
J
Veterinary Institute, Changchun University of Agricultural Animal Sciences, 130062, Changchun, PR China.
Gao
P
P
D002474:Cell-Free System; D002480:Cellulase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase; D010407:Penicillium / Q000201:enzymology / Q000235:genetics*; D014176:Protein Biosynthesis; D012156:Reticulocytes; D013698:Templates, Genetic; D014158:Transcription, Genetic
D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase