Screening and Identification of Anti-Glucose Repression Proteins in Cellulase Production Fungus Trichoderma orientalis EU7-22

摘要

The Cys 2 His 2 -type transcription factor CreI/CreA mediates carbon catabolite repression by binding to the promoter region of target genes to inhibit protein expression. The aim of this study was to identify anti-glucose repression proteins from cellulase producing fungus Trichoderma orientalis having the potential to influence the promoter-mediated expression of industrially useful proteins in a glucose-rich medium. Among the four cellulose inducers, wheat bran, pretreated Miscanthus cellulose, Avicel, and sodium carboxymethyl cellulose, pretreated Miscanthus cellulose was the most effective inducer for cellulase production, based on the results of the cellulase activity and the secreted proteins concentration. Conversely, glucose mixed with pretreated Miscanthus cellulose obviously repressed cellulase production in T. orientalis; the higher the concentration of glucose, the lower the cellulase activity and the concentration of secreted proteins. The cellulase activity and secreted protein content was reduced by 96.9 and 71.95%, respectively, with 3% glucose as inhibitor compared to when glucose was absent. Moreover, SDS-PAGE analysis revealed that certain proteins were stably secreted, indicating anti-glucose repression. These proteins were identified by MALDI-TOF. One was an aspartic protease while two were predicted proteins. The gene encoding the aspartic protease was cloned by PCR and analyzed. This showed an efficient method to obtain the target protein.