Charge controlled immobilization of chloroperoxidase on both inner/outer wall of NHT: Improved stability and catalytic performance in the degradation of pesticide

Halloysite nanotubes (HNT) is a kind of kaolin clay consisting of one alumina octahedron sheet and one silica tetrahedron sheet formed by rolling flat sheets into a hollow tubular structure. It is an ideal carrier for immobilization of enzyme due to its large surface area, biocompatibility, and chemical and mechanical stability besides abundance and low-cost. In order to make the most of this carrier for enzyme immobilization, in this work, two strategies were proposed for entrapping and embedding chloroperoxidase (CPO) on both inner/outer wall of HNT rather than in the hollow space alone by pH modulated electrostatic adsorption. Besides the enhanced loading amount, the thermal stability and tolerance to organic solvents of immobilized CPO (I-CPO) was greatly improved compared to the free enzyme. The free CPO can retain only 11.66% activity after 1 h incubation at 80 °C, while the I-CPO remained 87.63% activity at the same condition. Even after 1.5 h incubation at 90 °C, when the free CPO lost all its activity, the I-CPO can still remain 40.3% activity; Moreover, in the presence of organic solvent (ethyl acetate, acetonitrile, methanol, and DMF) with volume fraction of 10%, almost no loss of activity of I-CPO was observed, but free CPO can only remain 41.6%, 38.2%, and 23.5% of its initial activity in ethyl acetate, acetonitrile, methanol-water mixed system respectively, and even inactivated completely in DMF in the same condition. Furthermore, the enzymatic kinetic parameters (Km, and kcat/Km) suggested the affinity and specificity of I-CPO to the substrate was improved.