LAUSR

Cascade enzymes from the same origin or different resources often differ in their pH windows of stability and activity, thus impeding one‐pot enzyme cascade reactions. Here we present a method to assemble enzyme cascades using enzyme‐polyelectrolyte as the building block, in which the polyelectrolytes not only assemble enzymatic cascades via electrostatic interactions but also create favorable microenvironments for enzymes to expand their pH windows of stability and activity. We first demonstrated the effectiveness of this method for the oxidization of p‐xylene to p‐toluic acid using chloroperoxidase (CPO) and xanthine oxidase (XO) cascade, in which CPO from Caldariomyces fumago and XO from bovine milk were covalently linked to polymethacrylic acid (PMAA) and chitosan quaternary ammonium salt, respectively. Conjugation of the enzymes with PMAA and chitosan expanded the stability and activity pH windows for CPO and XO and increased the yield of p‐toluic acid production from p‐xylene by 7.6‐fold in comparison to that obtained from their native counterparts in soluble form. We then applied this method to assemble glucose oxidase (GOx) and horseradish peroxidase (HRP) cascade. The GOx‐PMAA@HRP‐chitosan displayed a lower Km and higher apparent activity at low glucose concentration, as compared to GOx/HRP cascade in soluble form, suggesting that the assembly method boosted the outcome of the one‐pot enzyme cascade reaction.