Abstract:

The expression of the cbhⅡ (cellobiohydrolase) gene from Trichoderma viride was studied by using Lactobacillus
expression system. Primers were designed according to T. viride cbhⅡ gene (GenBank accession number: M55080) and
the cbhⅡ gene cDNA was obtained by PCR method with a sequence length of approximately 1 441 bp. An 80 bp signal
peptide sequence was inserted into the upstream gene for the secretory expression of the target protein. Then, the gene was
optimized according to the codon usage of lactic acid bacteria for the synthesis of the whole cbhⅡ gene. The prokaryotic
expression vector pMG36e-S-cbhⅡ was constructed through connection between the target gene and the shuttle vector
pMG36e. Electrotransformation was used to obtain recombinant lactic acid bacteria in competent cells of Lactococcus lactis
NZ3900. The recombinant protein displayed a molecular weight of approximately 53 kD as determined by sodium dodecyl sulfatepolyacrylamide
gel electrophoresis, which is consistent with the expected size. The cellulase activity was 16.7 U/mL in culture
supernatants by 3,5-dinitrosalicylic acid assay, and there was almost no activity in cell lysate precipitates or supernatants.
These results showed that the signal peptide sequence was correctly identified by Lactococcus lactis and extracellular
expression was achieved successfully.

Key words: Trichoderma viride, cellulase, cbhⅡ gene, Lactococcus lactis, cellubiohydrolase activity