FastPrep cross-linked enzyme aggregates of N-acetylneuraminate aldolase from Staphylococcus carnosus (ScNAL-FpCLEAs) were prepared in order to improve the synthesis of 2-keto-3-deoxy-D-glycerogalactononulosonic acid (KDN), an important building block for therapeutic glycolipids and a possible marker for human prostate cancer. ScNAL-FpCLEAs showed improved thermostability compared with the free enzyme, doubling its half-life at 60 C. When the effect of substrate ratio (pyruvate:D-mannose) and temperature on the yield of KDN was studied at its optimum pH (pH 7.0), 90% conversion in only 8 h was reached in the presence of 0.6 M D-mannose and 1.2 M pyruvate at 37 C. This is the highest conversion described to date for enzymatic KDN synthesis. In addition, ScNAL-FpCLEAs exhibited enhanced catalytic activity and stability and could be recycled 10 times with no loss of activity. These results suggest the biotechnological potential of using FastPrepCLEAs to obtain valuable biocatalysts. (C) 2013 Elsevier Ltd. All rights reserved.