Biochemical, biophysical and biotechnological studies of class II xylose isomerases from hyperthermophilic Thermotoga species

Xylose isomerase (XI) (D-xylose ketol isomerase, EC 5.3.1.5) is used to convert D-glucose to D-fructose in the production of high fructose corn syrup (HFCS). Here, the biochemical and biophysical properties of xylose isomerases from hyperthermophilic Thermotoga species are examined with regard to their potential for HFCS production at elevated temperatures.; The effects of divalent metal cations on structural thermostability and inactivation kinetics of class II XIs from two mesophilic, one thermophilic, and one hyperthermophilic bacteria were examined. The three less thermophilic XIs were stabilized in the presence of Co2+ and Mn2+ (and Mg2+ to a lesser extent), while the melting temperature of TNXI (Tm∼100°C) showed little significant variation. TNXI's kinetic inactivation was non-first order for all metal cases, and was modeled as a two-step sequential process. Unlike other class II enzymes examined, metals are required for TNXI activity but are not essential for structural thermostability.; To determine if xylose isomerases from Thermotoga maritima (TMXI) and Thermotoga neapolitana (TNXI) could be utilized in HFCS production, the enzymes were compared with a commercial class I enzyme from Streptomyces murinus (SMXI) (Sweetzyme T(TM)). While the soluble enzymes exhibited bi-phasic inactivation, the immobilized enzymes were characterized by a first order decay rate. A simple mathematical model was developed which utilizes the soluble enzyme kinetic data and immobilized inactivation rates to calculate productivities as a basis to compare enzymes under different process conditions.; The extended N-terminus of class II XIs makes them attractive targets for attaching a carbohydrate-binding domain (CBD) for immobilization. Modifying the length of the N-terminal amino acid insert demonstrated that approximately half of the insert (to about residue 19) could be deleted while retaining activity; removing larger sections or the entire N-terminus caused the enzyme to misfold. A fusion protein (TNXI-CBD) with a thermostable CBD cloned from a hyperthermophilic chitinase (Pyrococcus furiosus 1233) attached to TNXI's N-terminus was created. The ability of the fusion protein to immobilize the enzyme to chitin beads was examined.