Cell extracts of Pseudomonas putida F6 expressing tyrosinase activity were immobilised in a calcium alginate matrix. Immobilised cell extracts consumed 21% more tyrosol substrate (2.5 mM) than non-immobilised cell extracts of P. putida F6. However, approximately the same level of hydroxytyrosol accumulation was observed. A number of approaches were attempted for complete removal of tyrosol substrate and maximum hydroxytyrosol accumulation. These approaches included addition of ascorbic acid, washing and re-use of immobilised cell extracts, sequential addition of fresh immobilised cell extracts to the biotransformation, and alteration of the starting concentration of tyrosol. Complete substrate removal was observed in the presence of both 1.0 and 2.5 mM tyrosol with equimolar concentrations of ascorbic acid. The sequential addition of immobilised cell extracts was required to ensure complete removal of 2.5 mM substrate. However, only 6% of the 2.5 mM substrate accumulated as product (hydroxytyrosol). The biotransformation of 1 mM tyrosol resulted in the accumulation of 0.8 mM hydroxytyrosol in a single biotransformation with immobilised cell extracts. Ninety-six percent of the hydroxytyrosol was subsequently recovered from the biotransformation reaction using an Amberlite XAD-4 (R) resin. Immobilised cell extracts retained 58% of their activity after 9 days of storage on filter paper at 4 degrees C while cell free extracts retained only 22% of their activity under the same storage conditions. (c) 2005 Elsevier Inc. All rights reserved.