A novel packed-bed bioreactor, operating under isothermal and non-isothermal conditions, has been constructed. The core of the apparatus consisted in a polypropylene ring filled with beta-galactosidase immobilized on beads of polyacrylic acid, grafted with dimethylaminoethyl methacrylate. Phenylendiamine and glutaraldehyde were used as spacer and coupling agent, respectively. Two lateral nylon membranes held the enzyme beads into the ring and allowed the occurrence of the process of thermodialysis when the bioreactor was operating under non-isothermal conditions. Comparison of the enzyme activity under isothermal and non-isothermal conditions has shown that in the presence of temperature gradients the rate of lactose hydrolysis was increased, with a reduction of the apparent Km value. Under non-isothermal conditions the percentage increases of enzyme activity were found to decrease with the increase of the substrate concentration. The results have been explained within the frame of reference of the process of thermodialysis. (C) 2004 Wiley Periodicals, Inc.