(664b) Stabilization of Xylanase By Its Random Insertional Fusion to a Thermophilic Host Protein Domain | AIChE

(664b) Stabilization of Xylanase By Its Random Insertional Fusion to a Thermophilic Host Protein Domain


Protein stabilization has been of continuous interest to researchers and is an intense field of study with various applications in pharmaceutical sectors and energy industries. In particular, improved thermal stability of an enzyme enables its application at high temperature, offering many advantages such as low microbial growth, high substrate solubility and an enhanced reaction rate. Our previous study has demonstrated that a target enzyme domain (guest) may be stabilized by inserting into a stable scaffold (host) protein domain. It was also found that stability conferred was a direct influence of the host protein as well as the point of insertion. In the present study, to systematically explore all potential insertion sites of a host protein for guest enzyme stabilization, we facilitated a combinatorial approach. To this end, we engineered a MuST transposon in order to construct insertional fusion libraries, where xylanase from Bacillus circulans (BCX) as a guest domain protein was randomly inserted into maltose binding protein from a hyperthermophilic archaeon Pyrococcus furiosus (PfMBP) as a host domain protein. The random insertion libraries were screened based on xylanase activity and stability to identify enzymatically active and thermally stable PfMBP-BCX insertional fusion complexes. Our results indicate that thermal unfolding of the BCX domain inserted into PfMBP was reversible in contrast to irreversible thermal unfolding of the wild-type BCX. We also found that while thermodynamic stability of the BCX domain within the selected PfMBP-BCX fusion proteins remained similar, its kinetic stability during prolonged thermal denaturation was substantially higher when compared to the wild-type BCX and another control, an end-end fusion complex of PfMBP-BCX. In summary, our results demonstrate that combinatorial insertional fusion can serve as an important molecular platform for protein stabilization.