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: kcat (min−1) was approximately 1000, 3800, and 5800 at 80°C compared to 1140, 10350, and 7000 at 90°C, for the wild-type, unbound, and immobilized enzymes, respectively. These kcat 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.

Immobilization of β-Galactosidases from Lactobacillus on Chitin Using a Chitin-Binding Domain

“…The patterns of chitin bead samples, with only two bands corresponding to the two subunits of immobilized LacLM (LacL and LacM-ChBD in lane 3 and ChBD-LacL and LacM in lane 6, Figure 2B) or the single strong band of LacZ-ChBD (Figure 2C, lane 6) with only a minor very faint band indicating an impurity, indicate the high specific affinity of ChBD to chitin 25. This observation is in accordance with many other studies using ChBD for enzyme immobilization 28,29,39. LacLM from L. reuteri L103 is a heterodimer consisting of two subunits.…”

Common Causes of Glucose Oxidase Instability in In Vivo Biosensing: A Brief Review

“…77 Another synthetic approach is to engineer recombinant versions of the enzyme at the genetic level to include components or chimeras that lead to stabilization. Engineered protein tags that may improve stability include the chitin binding domain from Pyrococcus furiousus 78 and elastin-like polypeptides (ELPs). 79 We attached the chitin-binding domain (CBD) to the N-terminus of a xylose isomerase from Thermotoga neapolitana and immobilized the fusion enzyme to chitin beads.…”

Extreme thermophiles: moving beyond single-enzyme biocatalysis

“…For example, entrapment in a porous gel as a means of immobilization, commonly employed for mesophilic enzymes, is problematic because of the thermolability of the matrix. Alternatively, carbohydrate binding domains from extreme thermophiles can be employed in fusions with extremely thermophilic enzymes for immobilization, as was demonstrated with the chitin-binding domain from a P. furiosus chitinase and the xylose isomerase from Thermotoga neapolitana [79]. …”