Step-wise immobilization of multi-enzymes by zirconium-based coordination polymer in situ self-assembly and specific absorption

The simple and quick method for co-immobilization of multiple enzymes with clear spatial distribution has presented great challenges for decades. Herein, Zr4+ and 2-methylimidazole (2MIm) coordination polymers (CPs) were used to synthetize co-immobilized nanoreactor by a simple two-step procedure in aqueous environment. The CPs was first self-assembled in situ encapsulating glucose-6-phosphate dehydrogenase (G6PD) and then utilized coordination unsaturated metal sites on the surface of CPs to selectively adsorb hexahistidine-tagged ?, ?-unsaturated ketoreductase (his-tagged KRED). The obtained multi-enzymes system (G6PD@Zr-2MIm/KRED) was employed as an enzymatic reactor involving coenzymes regeneration. G6PD@Zr-2MIm/KRED still exhibited good repeatability and storage stability. The bi-enzymatic reactor could achieve more than 95% chalcone conversion ratio after 15 min and good tolerance at high temperature and different pH, retained about 70% and 80% of its initial activity after storage for 4 days and after 4 cycles, respectively. This step-wise enzyme immobilization method is easy to operate and can be used to prepare multi-enzyme systems with clear spatial distribution of the biocatalysts and allowing the coenzymes regeneration.