Preparation of Streptavidin-Coated Magnetic Nanoparticles for Specific Immobilization of Enzymes with High Activity and Enhanced Stability

Abstract

In this study, streptavidin-functionalized magnetic nanoparticles were developed as a support for enzyme immobilization based on the specific recognition between biotin and streptavidin. The enzymeimmobilizing ability was evaluated using pullulanase as a model enzyme. Magnetic poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) nanoparticles were prepared and functionalized with streptavidin using cyanuric chloride. The immobilized enzymes were characterized using UV−vis spectra, Fourier-transform infrared spectroscopy, and transmission electron microscopy. The immobilized pullulanase retained high levels of activity (85.3%) and exhibited significantly improved pH and thermal stability as compared to the free enzyme. At pH 5.5, the relative activity of the immobilized enzyme (75.2%) was significantly greater than that of the free enzyme (15.8%; p < 0.01). After incubation for 360 min at 60 °C, the residual activity of the free enzyme was only 21.5%, while the immobilized enzyme retained more than 70.6% of its residual activity. Moreover, the immobilized pullulanase also exhibited excellent recyclability, retaining more than 74.2% of its initial activity after 8 consecutive reuses. These results indicated that streptavidin-coated magnetic nanoparticles have great potential for use as a support for the immobilization of the various enzymes required for continuous biotechnological applications.

Magnetic Nanoplatforms for Covalent Protein Immobilization Based on Spy Chemistry

“…Nevertheless, site-selective protein conjugations are challenging since there are many amine groups from lysine units in one protein chain and most of the cysteine thiol groups in natural proteins are in disulfide forms. As a result, the protein tertiary structure and biofunction are often compromised and even lost after immobilization by these random nonspecific reactions. , To solve these problems, highly selective natural ligand–receptor complexes were used for protein immobilization, represented by the popularly used streptavidin–biotin complex with dissociation constant K d ∼ 10 –15 M –1 for protein immobilization onto nanoparticles − and flat solid surfaces. , However, to bind any biotinylated protein, sizable streptavidin (53 kDa) has to be immobilized to the material surface, which limits the density of immobilized proteins …”

Engineering Mussel-Inspired Coating on Membranes for Green Enzyme Immobilization and Hyperstable Reuse

“…As shown in Figure C, after 7 h of incubation at 60 °C, the TA/AEAPTES-E and TA/APTES-E preserved 65% and 52% of their initial activity, which were 3.1-fold and 2.5-fold of the free enzyme (only 21%), respectively. The better thermal tolerance of the TA/AEAPTES-E might benefit from the stronger conformation of enzyme after covalent immobilization …”

“…The better thermal tolerance of the TA/AEAPTES-E might benefit from the stronger conformation of enzyme after covalent immobilization. 31 3.2.2. pH Tolerance and Mechanism Exploration. Since the pH tolerance plays an important role in enzyme commercial application, the pH tolerance of free enzyme, TA/APTES-E, and TA/AEAPTES-E was studied in the pH range 1.5−12 for 7 h (Figure 4 and Figure S5).…”

Aptamer-Assisted Protein Orientation on Silver Magnetic Nanoparticles: Application to Sensitive Leukocyte Cell-Derived Chemotaxin 2 Surface Plasmon Resonance Sensors

“…Usually, proteins have been bound to nanoparticles either in random or in oriented conformations. Random binding refers to conjugation approaches that partly render the active site of the protein inaccessible or not functional such as chemical coupling via cysteine, histidine, or lysine. , In contrast, the most common strategy utilized so far for oriented or site-specific protein immobilization is using biotinylated protein combined with streptavidin immobilized on the nanoparticle surface. , While this strategy is efficient, it still has quite a few disadvantages, such as the increased size of nanoparticles by the addition of streptavidin and the difficulty to accurately control the orientation and directionality of protein owing to tetravalent binding of biotin to streptavidin . In short, since the protein immobilization strategy greatly affects the activity of the protein on the surface of the biosensor, improper surface modification methods will affect the performance of the biosensor.…”