In this work, porous biochar was obtained from sugarcane bagasse by alkali activation and pyrolysis and then magnetized with γ-Fe2O3 by calcination. After functionalization with chitosan and activation with glutaraldehyde, the as-prepared chitosan/magnetic porous biochar served as a support to immobilize cellulase by covalent bonds. The immobilization amount of cellulase was 80.5 mg cellulase/g support at pH 5 and 25 °C for 12 h of immobilization. To determine the enzymatic properties, 1% carboxymethyl cellulose sodium (CMC) (dissolved in 0.1 M buffer) was considered as a substrate for hydrolysis at different pH values (3–7) and temperatures (30–70 °C) for 30 min. The results showed that the optimum pH and temperature of the free and immobilized cellulase did not change, which were pH 4 and 60 °C, respectively. The immobilized cellulase had a relatively high activity recovery of 73.0%. However, it also exhibited a higher Michaelis–Menten constant (Km) value and a slower maximum reaction velocity (Vmax) value compared to the free enzyme. In the reusability assay, the immobilized cellulase showed initial glucose productivity of 330.9 mg glucose/g CMC and remained at 86.0% after 10 uses. In conclusion, the chitosan/magnetic porous biochar has great potential applications as a support for enzyme immobilization.
Two magnetic supports with different morphologies and particle sizes were designed and prepared for cellulase immobilization based on chitosan and industrial by-product magnetic coal fly ash (MCFA). One was prepared by coating chitosan onto spherical MCFA particles to form non-porous MCFA@chitosan gel microcomposites (Support I) with a size of several micrometers, and the other was prepared using the suspension method to form porous MCFA/chitosan gel beads (Support II) with a size of several hundred micrometers. Cellulase was covalent binding to the support by glutaraldehyde activation method. The morphology, structure and magnetic property of immobilized cellulase were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy and a vibrating-sample magnetometer. The cellulase loading on Support I was 85.8 mg/g with a relatlvely high activity recovery of 76.6%, but the immobilized cellulase exhibited low thermal stability. The cellulase loading on Support II was 76.8 mg/g with a relative low activity recovery of 51.9%, but the immobilized cellulase showed high thermal stability. Cellulase immobilized on Support I had a glucose productivity of 219.8 mg glucose/g CMC and remained 69.9% of the original after 10 cycles; whereas the glucose productivity was 246.4 mg glucose/g CMC and kept 75.5% of its initial value after 10 repeated uses for Support II immobilized cellulase. The results indicate that the two supports can be used as cheap and effective supports to immobilize enzymes.
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