The Design, Preparation And Characteristic Of Self-assembled ELPs-xylanase Aggregates

In the preceding work of our project group,using elastin like peptides(ELP [KV8F-20]) as purification tag, purified the xylanase through inverse thermal cycling(ITC). While we found that a large part of ELPs-xylanase fusion proteins form insoluble aggregates in the course of ITC. Meanwhile, the aggregates had a high xylanase activity, similar to the xylanase was immobilized on the ELPs aggregates. This phenomenon was first reported by our group, whose mechanism of formation has not yet been investigated.The thesis will discuss the mechanism of this phenomenon from multiple angles.Firstly, the clone and expression vector was constructed. Then evaluated the characterizations of the fusion protein which different lengths ELPs fusion with the xylanase Sox. The result showed that the optimum temperature of all the fusion protein were about 70?, compared with xylanase without fusion with ELPs has increased about 20?. It helped xylanase endure high temperature conditions which will broadened their application in industry. Xylanase terminus with the same or larger size of the ELPs, can improve the activity at low temperatures and reduce the loss of activity under high temperature. The optimum p H of fusion protein was 7.0, while shorter ELPs made xylanase adapt basic conditions, longer ELPs make it adapt acidic conditions. After fusion with ELPs, xylanase retain more activity under basic conditions. Longer ELPs also helps improving the reusability of enzyme. In summary, the xylanase immobilized on ELPs helped improving the stability of the enzyme.Secondly, investigated the causes of insoluble aggregates xylanase-ELPs. The possible relevant factors including the length of ELPs, the property or the length of linker, the p H of buffer, salt and the target protein. The results showed that the length of ELPs and the linker were not the key factor, caused the forming insoluble aggregates. It was easy to make the xylanase Sox exposed in alkaline solution lose activity. Meanwhile denatured protein wrapped the ELPs, then made ELPs isolated with the aqueous solution, so it can not be restored to soluble state. The portion of the xylanase inside the circle ELPs made were still retain activity. However xylanase Xyl fusion with with ELPs did not form insoluble aggregates not matter using sodium carbonate or sodium sulfate in ITC. Compared with the properties of this two xylanase, it found that the p H tolerance and the surface charge distribution were quite different. So we think the main factor related to the properties of the target protein.