(593n) Co-Immobilization of Multiple Cascade Enzymes On the Surface of Magnetic Nanoparticles: Easy Separation and Substrate Channeling

Cell-free synthetic pathway biotransformation (SyPaB) is in vitro assembly of numerous enzymes for implementing complicated biological reactions that microbes and chemical catalysts cannot do.  To decrease labor for the purification of numerous enzymes and facilitate easy separation of biocatalysts and products, we are developing to a new approach to co-immobilizing three cascade enzymes on the surface of magnetic nanoparticles. Three enzymes are Thermus thermophilus triose phosphate Isomerase (TtcTIM), Thermotoga maritima fructose bisphosphate aldolase (TmALD), and Thermotoga maritima fructose 1,6-bisphosphatase (TmFBP), which are key enzymes in non-reductive pentose phosphate pathway.  First, we add polysaccharides on the surface of magnetic nanoparticles. Second, the carbohydrate-binding module-containing mini-scaffoldin is bound on the surface of polysaccharides. Third, three dockerin-containing enzymes can bind with the mini-scaffoldin through the high-affinity interaction among the dockerin and cohesin modules. Such self-assembled enzyme complex can be separated easily by using magnetic force and their proximate organization resulted in drastic increases in reaction rates compared to the free enzyme mixture at the same enzyme concentration.