Enzymatic hydrolysis of cellulose is often conducted in batch processes in which hydrolytic products tend to inhibit enzyme activity. In this study, we report a method for continuous hydrolysis of carboxymethyl cellulose (CMC) by using cross-linked cellulase aggregate (XCA) trapped inside a membrane. XCA particles prepared by using a millifluidic reactor have a uniform size distribution around 350 nm. Because of their large size, XCA particles in solutions can be filtered through a polyethersulfone membrane to collect 87.1 ± 0.9% of XCA particles. The membrane with impregnated XCA can be used as a catalyst for hydrolysis of CMC in a continuous mode. When the CMC concentration is 1.0 g/l and the flow rate is 2 μl/min, 53.9% of CMC is hydrolyzed to reducing sugars. The membrane with XCA is very stable under continuously flowing solutions. After 72 h of reaction, 97.5% of XCA remains inside the membrane.
