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In vitro  synthetic enzymatic biosystem is considered to be the next generation of biomanufacturing platform. This biosystem contains multiple enzymes for the implementation of complicated biotransformatiom. However, the hard-to-reuse and instability of multiple enzymes limit the utilization of this biosystem in industrial process. Multi-enzyme immobilization might be a feasible alternative to address these problems. Herein, porous microspheres are used as carriers to co-immobilize multiple enzymes for producing inositol from starch. At first, all the enzymes (i.e., α-glucan phosphorylase aGP, phosphoglucose mutase PGM, inositol 1-phosphate synthase IPS, and inositol monophosphatase IMP) for converting starch to inositol were immobilized on porous microspheres individually to check the effect of immobilization, then all the enzymes are co-immobilized on porous microspheres. Compared to reaction system containing all the individual immobilized enzymes, the reaction system containing the co-immobilized enzymes exhibit ∼3.5 fold of reaction rate on producing inositol from starch. This reaction rate is comparable to that by free enzyme mixture. And the co-immobilized multi-enzyme system show higher thermal stability and recovery stability than free enzyme mixture. After 7 batches, the immobilized enzymes retain 45.6% relative yield, while the free enzyme mixture only retain 13.3% relative yield after 3 batches. Co-immobilization of multiple enzymes on porous microspheres for biomanufacturing would shed light on the application of  in vitro  synthetic enzymatic biosystem in industrial scale.

Efficient Multi-Enzymes Immobilized on Porous Microspheres for Producing Inositol From Starch