In the present investigation, a dead-end membrane module, equipped with beta-galactosidase immobilized flat-sheet ultrafiltration (UF) membrane, has been considered as a membrane bioreactor (MBR) for galacto-oligosaccharide (GOS) synthesis and separation. beta-galactosidase was immobilized on the membrane surface by the combination of a series of mechanisms, such as adsorption of polyethyleneimine (PEI) on membrane matrix, formation of PEI and beta-galactosidase aggregates, and finally cross-linking of PEI and beta-galactosidase complex on the membrane surface by glutaraldehyde. The optimum pH for PEI-enzyme aggregation was found to be 82 and the optimum pH for crosslinking of PEI-enzyme aggregates on the membrane surface was found as 7.0 when the ratio of PEI to beta-galactosidase was 1:25. Stable immobilization could be achieved over broad temperature range (4 degrees C to 65 degrees C). It was found that at optimum condition beta-galactosidase loading on the membrane surface was 85% at multilayer form. GOS was synthesized by MBR with retentate recirculation, using simulated lactose solution, as well as de-proteinated whey as a substrate. Optimum operating conditions for the GOS synthesis were found at 0.045 M initial substrate concentration, 25 mL.h(-1) feed flow rate, 45 degrees C temperature, 3.0 kgf cm(-2) trans-membrane pressure (TMP), 80 r.p.m. stirrer speed. (C) 2013 Elsevier B.V. All rights reserved.