High-level expression of Thermobifida fusca glucose isomerase for high fructose corn syrup biosynthesis.
Journal:
Enzyme and microbial technology
PMID:
32146933
Abstract
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) 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 production were investigated and optimized. Under the optimal conditions, the yield of GIase reached 124.1 U/mL, which is the highest expression level of GIase by recombinant E. coli reported to date. Additionally, the obtained GIase 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 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 and its efficient HFCS production lays the foundation for its proming industrial application.