Hyperbranched polymer-crosslinked laccase aggregates for efficient aerobic oxidation of alcohols

The crosslinked enzyme aggregate (CLEA) technique has been developed as an easy and convenient strategy for carrier-free immobilization of enzymes. However, the irregular voids of enzyme aggregates limit the controlled crosslinking process by using regular crosslinkers such as glutaraldehyde. To overcome this limitation, here we have developed a simple strategy for the preparation of hyperbranched polymer-crosslinked laccase aggregates (HPCLEAs). Hyperbranched polymers were generated in voids of laccase aggregates, and the in situ crosslinking through the formation of hyperbranched polymers provided access to the void-adaptive crosslinking process. These HPCLEAs had irregular shapes and sizes of ∼2-10 microm. 99 % of the initial activity was maintained under the optimized preparation conditions. Further incorporation of 2,2,6,6-tetramethyl-1-piperidine-N-oxyl (TEMPO) facilitated the proximity between laccase and TEMPO, resulting in efficient aerobic oxidation of alcohols. Additionally, these catalysts could be easily recovered and reused four times with a slight loss of activity. This strategy may open an avenue for the rational design and co-immobilization of enzyme and molecular catalysts used for chemoenzymatic catalysis.