Recombinant Amylase and Cellulase Production

The amylase genes amyE1 (lacking the N-terminal sequence encoding the signal peptide, LNSP) and celE1 (LNSP) were cloned into the pET28a vector and then were transferred into E. coli DE3 cells. The transformants of DE3 were incubated in the liquid LB medium (100 μg/L of ampicillin) at 37°C to an OD₆₀₀ value of about 0.6–0.8 and were transferred into an incubator supplemented with IPTG (0.01%, w/v) at 16°C overnight to induce gene expression. The harvested cells were decomposed and homogenized by sonication (Scientz-II D, Ningbo). The recombinant proteins were purified by using Ni-NTA columns according to the previous reported protocol (Dai et al., 2019 (link)). The activities of amylase and cellulase were assayed by using Cellulase (CL) Assay Kit and α-amylase Assay Kit (Beijing Solarbio Science & Technology Co., Ltd). One unit (U) of enzyme activity was quantified as the amount of enzyme that synthesized against 1 μmol of glucose per minute under the assay conditions. The enzyme concentrations were measured by using a total protein assay kit (Jiancheng Biotech, Nanjing, China). Primers used in this study are listed in Supplementary Table S1.