The influence of carbon nanotubes on enzyme activity and structure: investigation ofdifferent immobilization procedures through enzyme kinetics and circular dichroismstudies

Abstract

In the last decade, many environmental organizations have devoted their efforts toidentifying renewable biosystems, which could provide sustainable fuels and thus enhanceenergy security. Amidst the myriad of possibilities, some biofuels make use ofdifferent types of waste biomasses, and enzymes are often employed to hydrolyzethese biomasses and produce sugars that will be subsequently converted intoethanol. In this project, we aimed to bridge nanotechnology and biofuel production:here we report on the activity and structure of the enzyme amyloglucosidase(AMG), physically adsorbed or covalently immobilized onto single-walled carbonnanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs). In fact, carbonnanotubes (CNTs) present several properties that render them ideal supportsystems, without the diffusion limitations displayed by porous material and with theadvantage of being further functionalizable at their surface. Chemical ligationwas achieved both on oxidized nanotubes (via carbodiimide chemistry), as wellas on amino-functionalized nanotubes (via periodate-oxidized AMG). Resultsshowed that AMG retained a certain percentage of its specific activity for allenzyme–carbon nanotubes complexes prepared, with the physically adsorbedsamples displaying better catalytic efficiency than the covalently immobilizedsamples. Analysis of the enzyme’s structure through circular dichroism (CD)spectroscopy revealed significant structural changes in all samples, the degree of changebeing consistent with the activity profiles. This study proves that AMG interactsdifferently with carbon nanotubes depending on the method employed. Due to thehigher activity reported by the enzyme physically adsorbed onto CNTs, thesesamples demonstrated a vast potential for further development. At the same time,the possibility of inducing magnetic properties into CNTs offers the opportunityto easily separate them from the original solution. Hence, substances to whichthey have been attached can be separated from a reaction medium, or directedby an external magnetic field to achieve efficient biofuel production. This pavesthe way for future design of efficient CNT–enzyme nanostructure bioreactors.