[Cloning and expression of the endo-beta-glucanase III cDNA gene from Trichoderma viride AS3.3711]

To study the construction of yeast bioengineering strain which can degrade cellulosic waste, an endo-beta-glucanase III (EG III) cDNA gene of Trichoderma viride AS3.3711 was isolated with RT-PCR protocol. After sequencing it was constructed on S. cerevisiae induceable expression vector pYES2. A L9 (3(4)) orthogonal design was used to optimize yeast sonication assistant transformation. The expression of EG III gene was induced by 2% beta-D-glactose, the transcription and expression of it was detected by Northern blotting and Congo Red method respectively. The endo-beta-glucanase activity was assayed as CMCase activity with CMC-Na as a substrate. The results show that the ORF of EG III was 1254 bp, encoding 418 aa, deducing molecular weight 44.1 x 10(3), group 5 (sonication treat time 60 s, incubate 40 min, SS-DNA 150 microg, heat shock 5 min) was the optimum one of the orthogonal experiment, and EG III transformants can produced clear hydrolysis halos on the Congo-Red-CMC plate. The measure of the enzyme activity show that the expression product can be expressed in active forms and secreted to the medium. The enzyme activity was approached the highest level (0.041 U/mL) when the culture time was 60 h. The optimized enzyme reaction temperature was 50 degrees C and the optimized pH was 5.8.