| High fructose corn syrup (HFCS), produced enzymatically using immobilized glucose isomerase (GI), dominates 70% of today's nutritive sweetener market. Due to poor enzyme thermostability and by-product formation, the current commercial glucose isomerization reaction can only be carried out at 60°C, producing ≤ 50 % fructose (55% fructose is more desirable). Identifying the mechanisms that cause GI to deactivate can facilitate efforts to increase GI thermostability, allowing increased reaction temperatures that could substantially improve the economics of HFCS production.; In this study, the mechanisms of thermoinactivation of GI from Streptomyces rubiginosus (in soluble and immobilized forms) were investigated, particularly the contributions of thiol oxidation of the enzyme's cysteine residue and the Maillard reaction between the enzyme and sugars in HFCS. Soluble GI (SGI) was successfully immobilized on Zeolite A and silica gel, with an activity yield between 20 and 40%. However, only GI immobilized on silica gel (LabIGI) has high enzyme retention on the support.; In batch reactors, SGI (half-life = 145 hours) was more stable than LabIGI (half-life = 27 hours) at 60°C in HFCS, while at 80°C, LabIGI (half-life = 12 hours) was more stable than SGI (half-life = 5.2 hours). Thiol oxidation caused LablIGI to deactivate at 60°C, and SGI at 80°C. The Maillard reaction between SGI and the sugars also caused SGI thermoinactivation at 60, 70 and 80°C, but had minimal effect on LabIGI. At 60 and 80°C, LabIGI had higher thermostability in continuous reactors than in batch reactors, possibility due to reduced contact with deleterious compounds in HFCS. IGI from Genencor (GenIGI) was more stable than LabIGI in continuous reactor experiments at 60 and 80°C. A first-order enzyme deactivation model fit the experimental data well.; A reversible Michaelis-Menten kinetics model was used to model the reaction at 40 to 90°C. Best-fit reaction kinetics parameter values were determined. The kinetics model that incorporated enzyme deactivation (based on kinetics parameters from the thermoinactivation study) showed good agreement with experimental data in batch and continuous isothermal and non-isothermal reactors. |
