Investigation on the underlying mechanism: How fusion xylanase-ELPs self-assembles into insoluble active aggregates

We have successfully obtained a recombinant xylanase by fusing with elastin-like polypeptides (ELPs), the xylanase SoxB underwent a sharp irreversible phase transition, and self-assembled into an insoluble but more catalytically operative particle. This was analogous to the immobilized xylanase to a large extent and aroused our interest to gain new insights into the determinant factor that may cause this phenomenon. We herein listed several candidate factors including the length of ELPs, linker sequence, buffer properties, and the target protein, subsequently we evaluated their contributions to the formation of the active aggregates. The results suggested that SoxB was fused with ELPs as desired protein partners, neither ELPs length nor the linker type made crucial contribution to the formation of active aggregates. However, when Na₂CO₃ was chosen as the salt to trigger the phase transition, the catalytic activities detected in aggregates accounted for more than 87.7% of total activity, whereas above 83.8% of the activity remained in supernatant when using Na₂SO₄. Then we introduced an alkali-tolerant xylanase termed as Xyl and compared it with SoxB, and found that the activity ratio in insoluble particle dropped to 15.3% in Na₂CO₃ and 19.3% in Na₂SO₄ respectively, only a few insoluble aggregates observed during the purification. Therefore, we speculated the property of xylanase partner fused to ELPs should be the predominant factor to form the catalytically active insoluble aggregates, and this provides a promising not yet reported perspective for industrial application of “immobilized” SoxB.