RBS + Circular Xylanase
Xylanases are enzymes that degrad Xylan, a major component of wood. This is the circular counterpart of BBa_K1362020. A circular Xylanase should be resistent to exo-peptidases and could be more heat stable because of its reduced conformational space. This could be interesting for industrial applications, as they are often performed at higher temperatures and harsher environments.
Usage and Biology
The first interesting question for the circular xylanase is did the circulrization was actually successful. We therefore made a SDS Polyacrylamide Gel stained with Coomassie Billiant Blue. It shows a visible sift between the linear and the circular construct as the circular is running a little bit faster due to increased electrophoretic mobility (Fig. 1)
To characterize our part in respect to its thermal stability we conducted a Heatshock Assay. Firstly the Xylanases were incubated at 37 °C, 50 °C, 56 °C, 60 °C and 66 °C. After 30 minutes substrate (EnzChek® Ultra Xylanase Assay Kit) was added and the fluorescence was measured for 150 minutes in a platereader at 37 °C. The results indicate a reduction of activity at temperatures higher than 50 °C and complete loss of function for temperatures higher than 60 °C.
In order to test the results with a biological replicate and to find out how a shorter heatshock influences the protein, we expressed the construct again and run another set of heatshock for 5 minutes at 63 °C. The data actually shows that with heatshock of 63 °C for the circular xylanase not all activity is lost but there is still some remaining activity. In contrast the assay confirms the initial data that the linear xylanase is not able to survive temperatures higher than 60 °C.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 153
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000Illegal BsaI.rc site found at 557
Illegal SapI.rc site found at 611