Fabrication of a Cellulolytic Enzyme Cocktail Immobilised on Magnetic Multi-walled Carbon Nanotubes: Catalytic Kinetics and Reusability

An enzyme-immobilised magnetised multi-walled carbon nanotube composite (E-mMWCNT) was fabricated by acid oxidation of pristine MWCNT, Fe₃O₄ deposition, AEAPTMS silanisation, glutaraldehyde activation, and covalent immobilisation of the NS22257 enzyme cocktail (Novozymes A/S).Raman, FTIR, FESEM and VSM analyses supported stepwise assembly.Saturation magnetisation increased from 6.95 emu g⁻¹ for pristine MWCNT to 46.7 emu g⁻¹ for E-mMWCNT, with a remanence of 2.02 emu g⁻¹ and coercivity of 2.07 G, indicating soft-magnetic behaviour suitable for rapid magnetic separation.The immobilised system performed best at pH 4.0, 55 °C and 200 rpm using carboxymethyl cellulose (CMC), a soluble model substrate for endoglucanase activity.The immobilisation yield was 80 %, the activity recovery was 39 %, and the activity yield was 48 %, giving a preparation of 1000 U g⁻¹. E-mMWCNT showed saturation kinetics, with apparent Michaelis-Menten parameters of K m = 17.2 g L⁻¹ and V max = 0.36 g L⁻¹ min⁻¹, against 12.9 g L⁻¹ and 2.22 g L⁻¹ min⁻¹, respectively, for the free enzyme, corresponding to an 8.2-fold increase in K m /V max attributed to external and internal transport limitations.A resistances-in-series treatment of the double-reciprocal plots separated intrinsic kinetics from external transport limitation, giving an effective liquid-solid volumetric mass-transfer coefficient of 0.016 min⁻¹.Over twelve consecutive reaction cycles with magnetic recovery, the preparation retained 91 % of its first-cycle yield at the fifth cycle and 55 % at the eighth cycle, giving 8.0 first-cycle batch equivalents from a single enzyme charge.The trade-off is therefore between single-pass rate and cumulative output per enzyme charge, and on this evidence, the latter favoured immobilisation.