Design Thermally Stable Carbonic Anhydrase Using Molecular Simulation

Human carbonic anhydrase II（hCAII）is one of the fastest enzymes for catalyzing the hydration rate of carbon dioxide in nature.However,due to its poor thermal stability and easy to inactivation,it is a serious obstacle to large-scale industrial applications.This paper will use molecular simulation methods to investigate thermal stability and catalytic activity of hCAII,and further design mutant CA by replacing the unstable amino acid without affecting the catalytic activity.Then the thermal stability,acid and alkali resistance and anion resistance of the mutant CA were studied.The enzyme activity of the mutant CA under different conditions was explored.A new type of CA with high stability,comparable catalytic activity was obtained,which can be used for industrial applications.This research mainly includes the following contents:（1）In vitro evolution of CA:Human carbonic anhydrase was used as a research object,and its thermal stability at different temperatures was determined by molecular simulation to determine the mutation region.Further analysis of the effects of amino acid sequence and environmental factors on the secondary structure,biological conformation and properties of the enzyme,and the modified residues such as 65Ala,198Leu,203Leu,204Leu were determined.The mutant carbonic anhydrase L204K（L204K-hCAII）was identified as a candidate mutant enzyme by comparing the parameters of radius of gyration,cavity structure and root mean square deviation（RMSD）of the carbonic anhydrase after the mutation at different temperatures.（2）Expression and purification of recombinant enzyme:construct the target gene of mutant carbonic anhydrase L204K,design primers,clone the target gene L204K-hCAII,establish the vector containing the target gene pET30a,and introduce it into E.coli BL21 for expression.The target protein L204K-hCAII was obtained after the separation and purification.（3）Enzyme activity of recombinant carbonic anhydrase:The esterase activity and CO₂ hydration activity of the carbonic anhydrase L204K-hCAII after mutation were detected by p-NPA method and electrode method,respectively.The results showed that the esterase activity of mutant L204K-hCAII is 8 times higher than that of wild type,and 70%esterase activity is preserved at 95℃while wild type hCAII retained only 10%activity at 55℃.The hydration rate of L204K-hCAII is increased by about 20%compared with the wild type.100%hydration rate is maintained at 45℃,50%at 55℃while the wild type hCA II is almost completely inactivated at 45℃.The thermal stability and catalytic activity are improved after replace L204 with K.In addition,L204K-hCAII has good anion tolerance.