Site-Specific and High-Loading Immobilization of Proteins by Using Cohesin–Dockerin and CBM–Cellulose Interactions

Immobilization of enzymes enhances their properties for application in industrial processes as reusable and robust biocatalysts. Here, we developed a new immobilization method by mimicking the natural cellulosome system. A group of cohesin and carbohydrate-binding module (CBM)-containing scaffoldins were genetically engineered, and their length was controlled by cohesin number. To use green fluorescent protein (GFP) as an immobilization model, its C-terminus was fused with a dockerin domain. GFP was able to specifically bind to scaffoldin via cohesin-dockerin interaction, while the scaffoldin could attach to cellulose by CBM-cellulose interaction. Our results showed that this mild and convenient approach was able to achieve site-specific immobilization, and the maximum GFP loading capacity reached ∼0.508 μmol/g cellulose.