In situ and layer-by-layer immobilization of ternary enzyme cascades based on CaCO3 microspheres

The efficiency of multi-enzyme immobilization is often influenced by the carrier and immobilization techniques employed. In this work, we proposed a ternary enzyme cascade reaction system composed of β-glucosidase (β-G), glucose oxidase (GOD) and horseradish peroxidase (HRP). CaCO 3 microspheres served as a template for in situ immobilization, while poly (allylamine hydrochloride) (PAH) and sodium poly (4-styrene sulfonate) (PSS) were utilized to create PAH-CaCO 3 and PSS-CaCO 3 templates for layer-by-layer multi-enzyme immobilization. The effects of template structure, glutaraldehyde (GA) addition, ethylenediaminetetraacetic acid (EDTA) concentration, and the structure of layer-by-layer assembly were systematically investigated. Our results indicated that an assembly of PSS-CaCO 3 --PAH--β-G--HRP--GOD presented simultaneous high enzyme loading and relative activity. The loadings of β-G, GOD and HRP were 75.2 ± 2.0%, 87.7 ± 4.4% and 85.5 ± 3.2%, respectively, while the relative activities were 81.8 ± 2.7%, 93.6 ± 4.7% and 90.6 ± 3.1%, respectively. Kinetic parameters confirmed that the reaction rate of the immobilized ternary enzymes in this assembly was close to that of the free enzymes. This work provides insights for constructing other multi-enzyme cascade reaction systems using immobilization techniques.