Enzyme Storage and Recycling: Nanoassemblies of α-Amylase and Xylanase Immobilized on Biomimetic Magnetic Nanoparticles

Abstract

Immobilization of enzymes has been extensively required in a wide variety of industrial applications as a way to ensure functionality and the potential of enzyme recycling after use. In particular, enzyme immobilization on magnetic nanoparticles (MNPs) could offer reusability by means of magnetic recovery and concentration, along with increased stability and robust activity of the enzyme under different physicochemical conditions. In the present work, microbial α-amylase (AmyKS) and xylanase (XAn11) were both immobilized on different types of MNPs [MamC-mediated biomimetic MNPs (BMNPs) and inorganic MNPs] by using two different strategies (electrostatic interaction and covalent bond). AmyKS immobilization was successful using electrostatic interaction with BMNPs. Instead, the best strategy to immobilize XAn11 was using MNPs through the hetero-crosslinker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N -hydroxysuccinimide (NHS). The immobilization protocols were optimized by varying glutaraldehyde (GA) concentration, enzyme quantity, and reaction time. Under optimal conditions, 92% of AmyKS and 87% of XAn11 were immobilized on BMNPs and MNPs–E/N, respectively (here referred as AmyKS–BMNPs and XAn11–MNPs nanoassemblies). The results show that the immobilization of the enzymes did not extensively alter their functionality and increased enzyme stability compared to that of the free enzyme upon storage at 4 and 20 °C. Interestingly, the immobilized amylase and xylanase were reused for 15 and 8 cycles, respectively, without significant loss of activity upon magnetic recovery of the nanoassemblies. The results suggest the great potential of these nanoassemblies in bioindustry applications.

Programming an Orthogonal Self-Assembling Protein Cascade Based on Reactive Peptide–Protein Pairs for In Vitro Enzymatic Trehalose Production

“…This on-demand assembly of protein scaffolds can also be adapted for other enzyme cascade systems, as the fusion of short peptides and post-translational ligation does not affect the expression and correct folding of proteins. In the future, we still need to explore the possibility of combining EutM scaffolds with magnetic nanoparticles, 53 silica nanoparticles, 41 and other materials to construct hybrid biomaterials with magnetic and mechanical properties. 18 In order to expand the application prospect of this modular and self-assembling EutM scaffold platform, the cascade system in this paper will be optimized from four directions: (i) improvement of the stability of the scaffold; (ii) achievement of one-step purification of immobilized enzymes; (iii) integration of functional and structural modules; and (iv) making the cascade system repeatable.…”

Biomimetic Nanomaterials: Diversity, Technology, and Biomedical Applications

“…When implementing this method, the biomineralization of Fe 3 O 4 is carried out in an oxygen-free aqueous solution containing the recombinant magnetosome protein, usually for 30 days. Such magnetic NPs can be used for photothermia or magnetic hyperthermia, chemotherapy, and enzyme immobilization [ 32 , 48 , 85 , 86 , 87 ]. It was experimentally established in [ 32 ] that, when two magnetosome proteins (MamC and Mms6) were used for biosynthesis at once, it was possible to obtain magnetic NPs that were close in shape and size to magnetosome crystals [ 88 ].…”

“…Methods of the second group, from our point of view, were more promising due to the ability to create a material structure similar to natural analogues at the level of individual atoms. They included the biosynthesis of magnetic NPs from oxygen-free solutions containing recombinant MamC in anaerobic conditions [ 32 , 48 , 85 , 86 , 87 ], magnetite biomineralization using PEGylated human ferritin NPs [ 52 ], and the silica encapsulation or biotinylation of isolated bacterial magnetosomes [ 44 , 89 ]. Thus, the methods of the second group made it possible to achieve a combination of the advantages of biogenic magnetic NPs (magnetosomes) and their synthetic counterparts, minimizing the disadvantages of both.…”

Site-Specific and Covalent Immobilization of Lipase on Natural Polyphenol-Modified Magnetic Nanoparticles for Effective Biodiesel Production

“…To date, biomimetic green chemistry provides an environmentally friendly strategy to fabricate functional nanocarriers for efficient enzyme immobilization. , Thus, using non-toxic natural polyphenol as a “secondary functional platform” for site-specific immobilization of enzymes is appealing to overcome such disadvantages. Natural polyphenols can oxidize and self-polymerize on various substrate surfaces. − Among various substrates, magnetic nanoparticles have become excellent carriers for enzyme immobilization due to their biocompatibility, superparamagnetism, high specific surface area, and outstanding mechanical strength. − The formed polyphenol coating with abundant quinone groups could conjugate with amine groups in residues such as lysine through a Schiff base reaction . As a result, it allows enzymes to be covalently attached to the polyphenol-functionalized surface in a well-defined manner.…”