To protect the enzymes during fed-batch cellulase production by means of partial enzyme recovery at regular intervals.Extracellular enzymes were partially recovered at the intervals of 1, 2, or 3 days. Mycelia were also removed to avoid contamination. Increases in the total harvested cellulase (24–62%) and β-glucosidase (22–76%) were achieved. In fermentor cultivation when the enzymes were recovered every day with 15% culture broth. The total harvested cellulase and β-glucosidase activity increased by 43 and 58%, respectively, with fungal cell concentration maintained at 3.5–4.5 g l−1.Enzyme recovery at regular intervals during fed-batch cellulase cultivation could protect the enzyme in the culture broth and enhance the enzyme production when the fungal cell concentration is maintained in a reasonable range.

•Mesoporous TiO2 (M-TiO2) and mercaptopropyl-functionalized M-TiO2 (SH-M-TiO2) as the carriers of β-glucosidase (BG).•High BG activity recovery, 92.8%, was achieved of the BG immobilized on SH-M-TiO2 (BG-SH-M-TiO2).•The conversion rates of cellobiose in 10 batches were around 90% by BG-SH-M-TiO2.Mesoporous titanium dioxide (M-TiO2) and mercaptopropyl-functionalized M-TiO2 (SH-M-TiO2) were prepared as carriers for immobilization of β-glucosidase (BG). Analyses suggested that in SH-M-TiO2 the grafted mercaptopropyl groups attributed to about 3.5% of the total weight of the matrices. For the immobilization of BG, SH-M-TiO2 or M-TiO2 powder was added with free BG solution and stirred at 4 °C for 8 h, followed by centrifugation and washing. BG was effectively immobilized on SH-M-TiO2 with an enzyme activity of 10.9 U/g, which was 92.8% of the total enzyme activity in the starting solution of free BG. BG immobilized on SH-M-TiO2 (BG-SH-M-TiO2) was more stable in pH, thermal, and storage tests than BG on M-TiO2 or free BG in solution. The Km and vmax of BG-SH-M-TiO2 were similar to values for free BG in solution. Batch hydrolysis of cellobiose-containing substrate by BG-SH-M-TiO2 was conducted. Conversion rates of cellobiose in 10 batches were consistently around 90%. Although the residual enzyme activity of BG-SH-M-TiO2 decreased gradually, only a small amount of BG activity was detected in supernatants. This indicated effective adsorption of enzyme molecules to SH-M-TiO2 matrices.Download full-size image