This study focuses on well-known but commonly overlooked or unreported factors in the preparation of cross-linked enzyme nano-aggregates (nano-CLEAs). The parameters including the ionic strength of the protein solution, protein, precipitant and cross-linker concentrations, pH and addition order of the reagents were fine-tuned for nanoaggregate preparation without the need of non-protein support material, special equipment or sophisticated procedures. For this purpose, precipitation as nano-aggregates and then cross-linking while maintaining submicron size distribution were studied independently. Moreover, nano-aggregate formation from reverse micellar solutions was also investigated to improve the scope of the method to membrane-bound enzymes. Five different precipitation agents together with three different cross-linkers were investigated for immobilization of the Trametes versicolor laccase as cross-linked nano-aggregates. Although complete activity recovery was possible for micro-aggregates, the best activity results for nano-aggregates were 40.5%+/- 5.0. The K-m values of the immobilized enzymes were slightly lower than the K-m values of the free counterparts which indicate little or no mass transfer limitation due to the nano-immobilization process. However, V-max values were also lower. The activity loss and V-max reduction upon immobilization were found to mainly result from the modification of the amine groups instead of excess crosslinking. The thermal stabilities of the crosslinked laccase nano-aggregates were significantly higher (similar to 10-30 fold at 60 degrees C) compared to free laccase and the nano-CLEAs retained up to 30% of their initial activities upon 7 consequent usages. The sizes of the obtained immobilized enzyme products were found to be greatly variable depending on the cross-linker type. The smaller particles (similar to 200 nm radius) were obtained when EDAC was used as the cross-linker. The larger products (similar to 600 nm radius) were prepared when the cross-linker was dextran poly-aldehyde. The addition order of the reagents was found to be effective on particle size and thermal stability values.