In the present study whole cellulase mixtures were covalently immobilized on non-porous magnetic particles to enable enzyme reuse. It was shown that CellicCTec2 immobilized on magnetic particles activated with cyanuric chloride gave the highest bead activity measured by mass of reducing sugar produced per mass of particles (2.8 gkg(-1) min(-1)), when using microcrystalline cellulose as substrate. The ratio of the individual activities of endoglucanase, cellobiohydrolase and p-glucosidase changed after immobilization of CellicCTec2 and it was observed that the specific activity in units per milligram of protein (U mg(-1)) decreased upon immobilization. The amount of protein attached per mass of particles (using Cellic CTec2) to particles activated with cyanuric chloride and polyglutaraldehyde was 14.6 mg g(-1) and 11.9 mg g(-1), respectively. The effect of surfactant addition (using Tween 80, PEG 6000 orbovine serum albumin (BSA)) on hydrolysis yield was studied for free and immobilized CellicCTec2. It was observed that for both free and immobilized CellicCTec2 the hydrolysis yield was increased when Tween 80, PEG 6000 or BSA was included. Interaction between magnetic particles (containing immobilized CellicCTec2) and lignin was examined and it was demonstrated that addition of BSA completely inhibited interaction while Tween 80 and PEG 6000 had no effect on decreasing magnetic particle-lignin interaction. Hydrolysis of pretreated wheat straw biomass was performed in two consecutive cycles using the immobilized cellulases and the results confirmed the potential of hydrolyzing real lignocellulosic substrate and enabling enzyme reuse for biorefinery and biomass derived ethanol applications. (C) 2014 Elsevier Ltd. All rights reserved.