Abstract
Enzyme immobilization on different supports has emerged as an efficient and cost-effective tool to improve their stability and reuse capacity. This work aimed to produce a stable immobilized multienzymatic system of xylanase and filter paper-ase (FPase) onto magnetic chitosan using genipin as a cross-linking agent and to evaluate its biochemical properties and reuse capacity. A mixture of chitosan magnetic nanoparticles, xylanase, and FPase was covalently bonded using genipin. Immobilization yield and efficiency were quantified. The activity of free and immobilized enzymes was quantified at different values of pH, temperature, substrate concentration (Km and Vmax), and reuse cycles. The immobilization yield, immobilization efficiency, and activity recovery were 145.3% ± 3.06%, 14.8% ± 0.81%, and 21.5% ± 0.72%, respectively, measured as the total hydrolytic activity. Immobilization confers resistance to acidic/basic conditions and thermal stability compared to the free form. Immobilization improved 3.5-fold and 78-fold the catalytic efficiency (Kcat/Km) of the xylanase and filter paper-ase activities, while immobilized xylanase and FPase could be reused for 34 min and 43 min, respectively. Cross-linking significantly improved the biochemical properties of immobilized enzymes, combined with their simplicity of reuse due to the paramagnetic property of the support. Multienzyme immobilization technology is an important issue for industrial applications.
Highlights
Among the numerous compounds employed to formulate polymeric nanoparticles, chitosan has been used due to its biodegradability, biocompatibility, and non-toxicity [1]
The magnetic chitosan composite was synthesized in a single step, and was covalently bonded to two enzymes using a natural and low-toxicity cross-linking agent
The structural characterization of magnetic nanoparticles cross-linked to two hydrolytic enzymes has been previously described [22]
Summary
Among the numerous compounds employed to formulate polymeric nanoparticles, chitosan has been used due to its biodegradability, biocompatibility, and non-toxicity [1]. Genipin is commonly used in pharmaceutical, food, and health industries due to its unique biocompatibility and low cytotoxicity relative to conventional cross-linkers such as glutaraldehyde and epoxy compounds (5000–10,000 fold) [2,3]. Since genipin can cross-link amino-group-containing molecules, it has been used as a support activator for enzyme immobilization [4,5]. Enzymes show great potential for scientific and industrial processes because of their highly efficient catalytic mechanisms and substrate selectivity [6]. Free enzymes are generally unstable, practically non-reusable and many are denatured at relatively low temperatures. Enzyme immobilization on different supports has emerged as an efficient and cost-effective tool to improve their stability and reuse capacity [7]. Enzyme immobilization may alter their activity, specificity, or selectivity, which could be a tool to improve
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