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

Abstract

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.

Keywords: cascade biocatalysis; immobilization; inositol; multi-enzymes; porous microspheres.

FIGURE 1

(A) Scheme of in vitro synthetic enzymatic pathway for inositol synthesis from starch. (B) The scheme preparation process of immobilized enzymes on porous microspheres.

FIGURE 2

Themostability of free and immobilized IPS at 70°C and 80°C.

FIGURE 3

The inositol production as a function of reaction time catalyzed by the reaction systems containing free and immobilized IPS.

FIGURE 4

Recycling stability of free and immobilized IPS.

FIGURE 5

The inositol production as a function of the reaction time catalyzed by free enzyme mixture, co-immobilized multi-enzymes, and mixture of solely immobilized enzymes.

FIGURE 6

Recycling stability of free enzyme mixture and co-immobilized multi-enzymes (A) and the thermostability of free enzyme mixture and co-immobilized multi-enzymes (B).