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

C. W. Low, M. S. Mohamad Annuar and S. Ibrahim

Abstract
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 Km = 17.2 g L⁻¹ and Vmax = 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 Km/Vmax attributed to external and internal trans port 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.

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Keywords
enzyme, immobilization, multi-walled carbon nanotubes, magnetic, nanobiocatalyst