Part:BBa_K3656307:Experience

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Applications of BBa_K3656307

By using the software Autodock for molecular docking, we studied the docking conformation of the substrate at the catalytic site, and analyzed the interaction between the residue at the catalytic site and the substrate. Autodock and PyMOL were used to further investigate the effects of secondary and tertiary structures of catalytic sites on catalytic processes. After research and discussion, we set up the mutation site and the alternative residue scheme, carried on the molecular docking of the recombinant enzyme, and then compared the enzyme-substrate docking conformation before and after the reorganization. Finally, suitable mutation sites and alternative residues were selected to simulate the mutation and the catalytic performance was improved theoretically.

Based on the simulation results, we finally determine appropriate mutations are respectively: 122: P and K (proline mutation of lysine) 122, 123, P and K (proline mutation of lysine) 123, 128, P and K (proline mutation of lysine) 128, 132: L - > K (leucine mutation of lysine) 132, 134: L - > K (134th leucine mutation of lysine), and 137 - bit: P→K (leucine at 137 was mutated to lysine), and the mutated OT3-CA (OT3-ca-Mu, BBa_K3656307, Fig.1) was finally obtained, which enhanced its catalytic activity under high temperature conditions.

Fig. 1 Structure of Mutant OT3-CA

！Name ！ OT3-CA-WT ！ OT3-CA-MU |-

BBa_K3656305 | BBa_K3656309 |-

-5.08 | -5.37 |-

-1.27 | -1.34 |-

187.83 | 116.58 |-

uM | uM |-

-5.68 | -5.96 |-

-1.76 | -1.92 |-

-3.92 | -4.04 |-

0.05 | 0.02 |-

0.6 | 0.6 |-

0.05 | 0.02 |-

Table 1. Using Auto Dock software to analyze the docking results of OT3-CA-WT and OT3-CA-MU with carbonic acid respectively.*

Secondly, we also use SnapGene design software to simulate and construct OT3-CA-MU-His recombinant vector (Fig. 2 with pET-28a (+) as the carrier.

Fig. 2 Map of OT3-CA-MU-His recombinant vector

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