Improved production and characterization of Volvariella volvacea Endoglucanase 1 expressed in Pichia pastoris.

Endoglucanase 1 (EG1) isolated from the straw mushroom has great potential in the textile and paper industries. Improving EG1 expression level will add to its value for industrial applications. In this study, we employed two combined strategies to enhance the expression quantity of EG1, which are increase the copy number of EG1 and enhance the folding and secretion efficiency of EG1 in the endoplasmic reticulum by overexpress HAC1. Multiple plasmids, which contains four copies of EG1, were constructed by isocaudamers, resulted a recombinant strain with EG1 activity up to 39.6 U/mL, 262% higher than that measured in the strain containing only a single copy. A significant increase in activity (151%) was found when eight copies of EG1 was introduced into a different host, compared with a host harboring four copies. Further overexpression of the HAC1 transcription factor in the host harboring eight EG1 copies led to activity of 91.9 U/mL, which is 619% higher than that measured in the original strain. Finally, EG1 activity of 650.1 U/mL was achieved in a 3-L scaled-up fed-batch fermenter and the protein yield was 4.05 g/L. The characteristics of recombinant EG1 were also investigated, the optimal values for enzyme activity were 60 °C and pH 5.0, which yielded a catalytic efficiency of 312.9 mL mg-1 min-1 using carboxymethyl cellulose(CMC) as the substrate.