In this paper, production of 5'-nucleotides using a free enzyme membrane reactor was presented. The enzyme membrane reactor consists in a coupling of a membrane separation process with an enzymatic reaction. Retention of substrate (RNA) and catalyst (nuclease PI) by the ultrafiltration membranes was observed. The best enzyme membrane reactor configuration was achieved with a polyethersulfone membrane of 30 kDa molecular weight cut-off, since the substrate and biocatalyst were retained with no loss of enzyme activity. Enzyme stability and optimum temperature were investigated. Other optimized operating conditions were also obtained: working temperature 65 degrees C, flow rate 2.0 mL/min (transmembrane pressure 0.050 MPa), and reactor volume 100 mL. The results demonstrated that enzymatic production of 5'-nucleotides from RNA was efficiently conducted by employing an ultrafiltration membrane reactor. (C) 2009 Elsevier B.V. All rights reserved.