Expression of the cellobiohydrolase gene of Aspergillus fumigatus in Escherichia coli

In this study, the genomic DNA of Aspergillus fumigatus was used as a template to construct a genetically engineered cellulase strain with high efficiency. The cbh gene was amplified by PCR, ligated into the prokaryotic expression vector pET-28a, and transformed into Escherichia coli BL21(DE3) to induce the expression of cellobiohydrolase (CBH) protein. The enzyme activity of the recombinant strain was determined by Congo red staining and 3,5-Dinitrosalicylic acid (DNS). The results showed that the expression vector pET-28a::cbh was constructed successfully. The final concentration of isopropyl-β-D-1-thiogalactopyranoside (IPTG) was 1 mmol·L⁻¹, the induction temperature was 28 ℃ and induction time was 14 h, and the purified protein of 75 kDa was obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The pET-28a::cbh/E.coli hydrolysis loops were detected by the Congo red plate with carboxymethyl cellulose-Na (CMC-Na). The results showed that the obtained recombinant strain had enzyme activity of 0.0563 U·mL⁻¹. The recombinant cellulase strain constructed in this study can lay a foundation to improve the quality of forage silage and production performance of ruminants.