Covalent immobilization of Proteinase K and Catalase onto single-walled carbon nanotubes: secondary structure and thermal stability

Stabilization of enzymes against extreme conditions etc. high temperature is one of the most complicated challenges in protein science. Amino acids substitution, chemical modification and protein immobilization on various support materials are the main classical strategies for enzyme stabilization. Carbon nanotube (CNT) as a nano-scale material with dedicated attributes, like enormous rigidity, stability and resistance to bio- and chemical degradation, could be used as a good candidate for enzyme immobilization. In the current study, catalase and proteinase K were covalently immobilized onto single-walled carbon nanotube (SWCNT). Enzyme functionalization of carboxylated SWCNTs was performed using carbodiimide chemistry according to Jiang method. Briefly, N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) was used for coupling of N-hydroxysuccinamide (NHS) to the carboxylic groups on the oxidized nanotubes. The resulting stable active esters on CNTs were reacted with the amine groups on the catalase and proteinase K. Secondary structures of CNT-conjugated forms of enzymes were assessed byCD spectrophotometry. Catalase activity was measured using H2O2 as substrate and proteinase K activity was determined via the hydrolysis of casein. Thermal stabilization was followed by fluorescence spectrophotometry at different temperatures. Catalase and proteinase K in conjugation with SWCNT remain kinetically active. Conjugation to SWCNTs could induce extensive changes in the secondary structure of catalase. In contrast, the secondary structure of proteinase k in the conjugated form remains unchanged in comparison to unconjugated form. Immobilization of proteinase K on SWCNTs could stable its structure against high temperature but conjugation of catalase to SWCNTs had no obvious effect on its thermostability. It seems that immobilization of proteins on CNTs to obtain thermostable biocatalysts could not be used as a general route for any enzyme.