title: 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.
abstract: 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 chitin-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 approximately 1,000, 3,800, and 5,800 at 80 degrees C compared to 1,140, 10,350, and 7,000 at 90 degrees 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 degrees 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.
authors: Dept. of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695-7905, USA.
authors: James M
authors: JM
authors: Harris
authors: Kevin L
authors: KL
authors: Epting
authors: Robert M
authors: RM
authors: Kelly
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000235:genetics / Q000378:metabolism*; D002686:Chitin / Q000378:metabolism*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D046949:Thermotoga neapolitana / Q000201:enzymology*
chemical_list: D004800:Enzymes, Immobilized; D011994:Recombinant Proteins; D002686:Chitin; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase