Preparation of magnetic xylanase cross-linked aggregates (m-CLXAs) for the hydrolysis of arabinoxylan by mechano-enzymology

The development of a biocatalyst consisting of immobilised xylanases as cross-linked enzyme aggregates (CLEAs) on magnetic nanoparticles (MNPs) as support was undertaken. MNPs were prepared by a coprecipitation reaction of Fe^{3 +} and Fe²⁺, followed by surface modification with (3-aminopropyl)-trimethoxysilane using a polycondensation reaction. Magnetic CLEAs were prepared via glutaraldehyde cross-linking after precipitation with acetone, and the physicochemical characterisation of the immobilised enzyme was performed at all stages using several techniques (FTIR, PXRD, TGA, and DLS). A one-factor-at-a-time approach (OFAT) was used to investigate the impact of temperature, time, enzyme: MNP ratio, precipitant, and cross-linking agent to determine their effect on the enzyme's recovered activity. The results demonstrated that all parameters impacted the immobilisation differently, with the optimised conditions determined as 4°C, 12 h, 10 mg/g, 60 % (v/v) acetone, and 200 mM glutaraldehyde, respectively. The immobilisation did not affect the pH and temperature optima of the enzyme, which were 6.0 and 50-70°C, respectively, for both the free and immobilised enzymes. In contrast, the immobilised enzyme could be reused more than ten times to hydrolyse wheat arabinoxylan, without losing 50 % of its initial activity. Values for V_max and K_m only decreased slightly compared to those obtained for the free enzyme. The thermal inactivation parameters showed that the immobilisation procedure did not adversely affect the enzyme's catalytic properties after immobilisation. Finally, we assessed the immobilised enzyme for its ability to catalyse reactions under mechanochemical conditions (grinding and ageing) and found that the free and immobilised enzymes were active during solvent-free and liquid-assisted grinding (LAG).