Part:BBa_K3990011
T7+Laccase CotA [H497D;L386E] Expressing Construct
We will express the enzyme LaccaseCotA in BL21(DE3) E.coli to decompose the ethylene vinyl acetate copolymer(EVA).
Team: SMS_Shenzhen 2021
In order to determine the optimal temperature for modified Laccase CotA, we conducted a temperature gradient experiment. We found that 60℃ is the optimal temperature of the original Laccase CotA, which is close to the normal temperature of the ground in semitropic and tropic zones, suggesting that Laccase CotA can degrade gum base that stuck to the ground effectively.
Fig.1 Temperature gradient for CotA
We then utilized two approaches to improve the enzyme activity of Laccase CotA. One is rational design serving to modify the substrate affinity between Laccase CotA and ABTS, and the other one is directed evolution with the purpose of improving the thermal stability of Laccase CotA at ground temperature.
We carried out homology modeling to identify the probable 3D-structure of laccase CotA, molecular docking to predict the binding sites on laccase CotA, and multiple sequence alignment to verify the validity of binding sites. Then, recommended mutations, which can improve substrate affinity, were compared by molecular docking software. Laccase CotA with mutation of HIS496>ASP, LEU386>GLU, and LEU386>GLU& HIS496>ASP, which show the highest mutation energy, was determined via rational design.
We also made a plan of conducting directed evolution experiments. Randomly mutated sequences coding Laccase CotA would be yielded from Error Prone-PCR(EP-PCR). Then, single colonies would be cultured to express mutated Laccase CotA. We implemented library screening for beneficial variations by testing activity of crude enzymes. Finally, two beneficial variations were obtained, CotA-2-12 and CotA-2-8.
We then characterized those mutations separately. As for the rational design mutations, CotA-386 shows approximately none activity. The enzymatic activities of CotA-497 and CotA-386-497 increased compared to the original one. Surprisingly, enzymatic activities for mutation from directed evolution, CotA-2-12 and CotA-2-8, shows a higher increase.
Fig.2 Enzymatic Activities of original and mutaional CotA
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 917
Illegal AgeI site found at 1224
Illegal AgeI site found at 1380 - 1000COMPATIBLE WITH RFC[1000]
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