Fine-tuned preparation of cross-linked laccase nanoaggregates

Abstract

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 (∼10–30 fold at 60 °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 (∼200 nm radius) were obtained when EDAC was used as the cross-linker. The larger products (∼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.

Keywords:

• Laccase
• cross-linked enzyme aggregates
• micro-emulsions
• nano-immobilization

Acknowledgements

The authors want to thank Professor Nurettin ŞAHİNER for letting us use DLS equipment.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by The Scientific and Technological Research Council of Turkey (TÜBİTAK) under grant number 215Z097.