High-level expression of Thermobifida fusca glucose isomerase for high fructose corn syrup biosynthesis.

Glucose isomerase (GIase), an efficient enzyme in the isomerization of d-glucose to d-fructose, has been widely used in food processing. In this study, an efficient expression system for a Thermobifida fusca GIase (GIase_Tfus) in Escherichia coli was firstly designed via a two-stage feeding strategy for improving expression level. The cultivation strategy was performed at an exponential feeding rate during the pre-induction phase, followed by a gradient-decreasing feeding rate at the induction phase in a 3-L fermenter. During this process, the effect of induction conditions and the complex nitrogen supplementation in feeding solutions on GIase_Tfus production were investigated and optimized. Under the optimal conditions, the yield of GIase_Tfus reached 124.1 U/mL, which is the highest expression level of GIase by recombinant E. coli reported to date. Additionally, the obtained GIase_Tfus was performed to produce high fructose corn syrup (HFCS) with conversion approacing 55 % from glucose (45 %, w/v) to fructose. According to the molecular dynamic simulation, a number of hydrogen bonds existed in the enzyme-substrate complex could stablilize the transient states, and a appreciate reaction distance of M1 catalytic site and oxygen atom of glucose make the reaction proceed easily, thus resulting in the efficient biosynthesis of HFCS. The function of GIase_Tfus renders it a valuable catalyst for HFCS-55 (containing 55 % d-fructose) manufacturing, the most favorable industrial product of HFCS. The efficient expression of GIase_Tfus and its efficient HFCS production lays the foundation for its proming industrial application.