N-terminal Fusion of a Hyperthermophilic Chitin-Binding Domain to Xylose Isomerase from Thermotoga neapolitana Enhances Kinetics and Thermostability of Both Free and Immobilized Enzymes

摘要

Immobilization of a thermostable D-xylose isomerase (EC 5.3.1.5) from Thermotoga neapolitana 5068 (TNXI) on chitin beads was accomplished via a N-terminal fusion with a chi-tin-binding domain (CBD) from a hyperthermophilic chitinase produced by Pyrococcus furiosus (PF1233) to create a fusion protein (CBD-TNXI). The turnover numbers for glucose to fructose conversion for both unbound and immobilized CBD-TNXI were greater than the wild-type enzyme: k_(cat) (min~(-1)) was ～1,000, 3,800, and 5,800 at 80°C compared to 1,140, 10,350, and 7,000 at 90°C, for the wild-type, unbound, and immobilized enzymes, respectively. These k_(cat) values for the glucose to fructose isomerization measured are the highest reported to date for any XI at any temperature. Enzyme kinetic inactivation at 100°C, as determined from a bi-phasic inactivation model, showed that the CBD-TNXI bound to chitin had a half-life approximately three times longer than the soluble wild-type TNXI (19.9 hours vs. 6.8 hours, respectively). Surprisingly, the unbound soluble CBD-TNXI had a significantly longer half-life (56.5 hours) than the immobilized enzyme. Molecular modeling results suggest that the N-terminal fusion impacted subunit interactions, thereby contributing to the enhanced thermostability of both the unbound and immobilized CBD-TNXI. These interactions likely also played a role in modifying active site structure, thereby diminishing substrate-binding affinities and generating higher turnover rates in the unbound fusion protein.