Method for Accurately Assessing the Impact of Enzyme Encapsulation in a Protein Nanocage

ABSTRACT Encapsulating enzymes within protein cages is attracting increasing attention for its potential to enhance reaction efficiency, similar to mechanisms found in nature, as well as the promise for practical applications. Although various encapsulated enzymes have been developed artificially, elucidating the effects of encapsulation remains a challenge. One key difficulty lies in measuring the activity of encapsulated enzymes and free enzymes under unified reaction conditions, such as accurately determined enzyme concentrations. In this study, we developed an encapsulated enzyme using alcohol dehydrogenase from Thermoanaerobacter brockii (TbADH) as well as an artificially designed protein nanocage, TIP60, as the model enzyme and cage. TIP60 is a 60‐mer protein that easily disassembles into smaller oligomeric states above 65°C, which is a temperature lower than the melting point of TbADH (98°C). Thus, the TbADH encapsulated in TIP60 can be released by heat treatment, thereby enabling the measurement of activity by using the same sample under the same reaction conditions. Using this method, we found that cofactor accessibility to TbADH and limited molecular motion to form productive conformations may be key factors controlling their activity.