Increased Reaction Efficiency of Enzyme Complexes Based on Irreversible Covalent Bonding Systems

Abstract

Carbonic anhydrase (CA) and cyanase can be a solution to the reduction of CO2 and cyanide that adversely affect the environment. However, the high temperature inactivation of enzymes and their dependence on bicarbonate limits their industrial applications. Therefore, this problem was overcome by changing the structure of the enzyme using the Catcher/Tag system with irreversible covalent bonding and combining them with each other. Cyclization and polymer formation of hmCA recovered its activity even at a high temperature of 70 °C or higher compared with the wild type, and polymeric hmCA showed a result that was more than 4 times different from that of the control in CaCO3 production through enzymatic reaction. This suggests Thatcher the binding of enzymes by the Catcher/Tag system improved thermal and structural stability and could be a new strategy to overcome the existing limitations. The subsequent binding of cyanase and hmCA increased cyanide degradation compared to when cyanase alone was used, but the degradation reaction efficiency was lowered compared to the condition in which the two enzymes were not linked. The cause of these results is that the formation of the complex in a linear form in the binding through the peptide module brought a limitation in distance, resulting in decreased efficiency. To improve this, the complex was engineered to form a circular structure. As a result of measuring the reaction efficiency of the enzyme, it showed more than two times higher activity than the condition in which cyanase alone was used, and it was confirmed that the reaction efficiency was improved compared to the linear complex formed by mixing Catcher, cyanase, and hmCA. In conclusion, an enzyme complex with reduced HCO- dependence and improved reaction efficiency was prepared in the presence of the same concentrations of oxidized cyanide and HCO- than that in the presence of cyanase alone.