Co-Immobilization of SpyTag-Cyclized Enzymes on a γPFD-SpyCatcher Hydrogel to Address Broad Specificity

to ensure directed catalytic flux. Using this strategy, the yields of pinene and caffeoyl-CoA were enhanced by 5.8-fold (reaching 94.5 mg/L) and 2.4-fold (reaching 78.6 mg/L), respectively, compared to free enzyme systems. This work establishes an effective approach to mitigate the limitations posed by broad enzyme specificity and demonstrates its potential for applications in synthetic biology and industrial biotechnology.