Team:AHUT-ZJU-China/Proof Of Concept
AHUT-ZJU-China
Proof Of Concept
CO2 is the most important greenhouse gas which can cause greenhouse effect and so on which has serious impact on ecological environment of earth. Over the past 20 years 75% of atmospheric emissions have been generated from combustion and use of fossil fuels such as exhaust emissions from automobiles waste coal plants emissions coal production etc. Every country in the world seeks to reduce emissions by reducing emissions thereby reducing damage to humans caused by excessive levels of greenhouse gases and greenhouse gases thereby meeting Kyoto Protocol requirements.
In order to help countries reduce environmental levels of Carbonic anhydrase, we studied human Carbonic anhydrase 2 (CA2) during 2018 and 2019, and mutated them appropriately to improve their activity and heat resistance. This year Carbonic anhydrase (OT3-CA-WT) is extracted from Pyrococcus horikoshii (thermophilic bacteria). Due to its high heat resistance and low activity, we decide to improve its activity under the premise of maintaining its heat resistance, thus constructing Carbonic anhydrase (OT3-CA-MU) with high activity and heat resistance. Molecular simulations and experimental verification were used to detect whether their activity and heat resistance were improved.
We use PyMOL and AutoDock molecular simulation software to investigate and study active sites and sites of Zn2 + catalytic sites in OT3-CA-WT and analyze amino acid sites and spatial structures affecting activity and heat resistance of OT3-CA-WT. We read a lot about literature and tried to change amino acids at important sites in OT3-CA-WT. Meanwhile we carried out lots of simulation mutations and molecular docking of recombinant enzymes compared with enzyme-substrate docking conformation before and after recombination. Finally we choose suitable mutation sites and substitute residues and study the optimal mutation scheme for improving its catalytic performance.
Fig. 1 Three dimensional structure of OT3-CA-WT
Fig. 2 Structure of OT3-CA-WT
We constructed OT3-CA-WT-pET28a (+) and OT3-CA-MU-pET28a (+) vectors along with members of ZJU-China, expressed in engineered bacteria from E.coli TB1 and extracted purified of OT3-CA-WT and OT3-CA-MU. We detected and identified enzyme activity and heat resistance by using Western bolt technology and SDS-PAGE and compared with experimental data obtained from 2018 and 2019.
Fig. 3 Map of OT3-CA-WT recombinant vector
Fig. 4 Map of OT3-CA-MU recombinant vector
Through analyzing and identifying simulation results and experimental data we found that the activity expressed by mutant Auto Dock was much higher than that of CA2 and compared with simulated activity of OT3-CA-MU. We have already had relatively complete experimental methods and design flow for OT3-CA-MU. We will continue to study OT3-CA-WT and OT3-CA-MU in future and try to combine our research results with practical applications including automobile exhaust emissions industrial production exhaust emissions and coal chemical industry resulting in excessive emissions of industrial fields design. We will contact more sponsors to create more commercial or public goods that have greater impact on capture collection.