Cry3Aa*SpyCatcher Fusion Crystals Produced in Bacteria as Scaffolds for Multienzyme Coimmobilization.

The production of Cry3Aa enzyme fusion crystals in Bacillus thuringiensis provides a direct method to immobilize individual enzymes and thereby improve their stability and recyclability. Nevertheless, many reactions require multiple enzymes to produce a desired product; thus a general strategy was developed to extend our Cry3Aa technology to multienzyme coimmobilization. Here, we report the direct production of particles comprising a modified Cry3Aa (Cry3Aa*) fused to SpyCatcher002 (Cry3Aa*SpyCat2) for coimmobilization of model enzymes MenF, MenD, and MenH associated with the biosynthesis of menaquinone. The resultant coimmobilized particles showed improved reaction rates compared to free enzymes presumably due to the higher local enzyme substrate concentrations and enhanced enzyme coupling made possible by colocalization. Furthermore, coimmobilization of these enzymes on Cry3Aa*SpyCat2 led to increased thermal stability and recyclability of the overall multienzyme system. These characteristics together with its overall simplicity of production highlight the benefits of Cry3Aa*SpyCat2 crystals as a platform for enzyme coimmobilization.