Facile Oriented Immobilization and Purification of His-Tagged Organophosphohydrolase on Viruslike Mesoporous Silica Nanoparticles for Organophosphate Bioremediation

Abstract

In this study, nickel-nitrilotriacetic acid-modified viruslike mesoporous silica nanoparticles (Ni-NTA-VMSN) were fabricated by a facile method for the first time. The Ni2+ ions on the surface of Ni-NTA-VMSN provided abundant anchoring sites for directly selective oriented immobilization of His-tagged proteins from crude enzymes. By incubation of the Ni-NTA-VMSN in the His-tagged organophosphohydrolase (OpdA)-contained cell lysates, a nanobiocatalyst, OpdA@Ni-NTA-VMSN, could be constructed. At the optimal immobilization conditions, the specific activity of OpdA@Ni-NTA-VMSN was 353.0 U/gprotein, which was slightly higher than that of free OpdA in cell lysates. The substrate’s affinity capacity of OpdA became higher after oriented immobilization, and the OpdA@Ni-NTA-VMSN exhibited excellent catalytic activity and stability. In addition, when OpdA@Ni-NTA-VMSN was applied in a plug-flow reactor, the degradation percent of methyl parathion could be maintained at more than 90% with the injection of 20 mL of reaction mixture. Thus, the Ni-NTA-VMSN will be a promising material for purifying and immobilizing His-tagged enzymes. The proposed OpdA@Ni-NTA-VMSN will be an excellent nanobiocatalyst for biodegradation of organophosphorus pesticides.

Parameters necessary to define an immobilized enzyme preparation

“…So this will underestimate the actual immobilization yield since other proteins will not be immobilized. For instance, this occurs with immobilization of lipases on hydrophobic supports at low ionic strength (88), the tagged proteins and affinity domains (89,90,91,92,93,94,95), but this occurs mostly with covalent or physical immobilization protocols even when it is difficult to predict beforehand. Almost no immobilization protocol can immobilize 100% of the proteins contained in a crude protein extract.…”

Immobilization of Phenylalanine Ammonia-lyase via EDTA Based Metal Chelate Complexes – Optimization and Prospects

“…In the previous IMAC applications, mainly iminodiacetic acid or nitrilotriacetic acid-based functions were applied as metal-complexing agent. Iminodiacetic acid is linked to the support via epoxy groups, and nitrilotriacetic acid is usually reacted in a C-C coupling reaction [35] at the alpha carbon or in a reaction with a lysine derivative (N α ,N αbis(carboxymethyl)-l-lysine) [32,40,41,43]. The disadvantage of the epoxy functionalized surface is that the remaining epoxy groups must be blocked to avoid subsequent covalent enzyme immobilization.…”

“…Applying this technique, immobilized biocatalyst can be produced in a one-step operation that involves the purification and the immobilization of the enzyme as well [25,[32][33][34][35]. The most used metal complexing agents in this IMAC based selective enzyme immobilization methods are the iminodiacetic acid [34][35][36][37][38] and nitrilotriacetic acid [32,[39][40][41][42][43].…”

Engineering of a Novel, Magnetic, Bi-Functional, Enzymatic Nanobiocatalyst for the Highly Efficient Synthesis of Enantiopure (R)-3-quinuclidinol

“…Notably, negligible losses of enzyme (only 0.02%) were released after each cycle [21]. Similarly, porous silica nanoparticles containing Ni-NTA groups on the surfaces have been reported to immobilize organophosphohydrolase for biodegradation of organophosphorus pesticides [24], or DNase, coagulase, and α-amylase against Gram-positive and -negative bacteria [25]. More importantly, the immobilization based on Ni-NTA-functionalized magnetic or porous silica nanoparticles can overcome some limitations of IMAC such as poor mechanical stability, limited surface area of agarose bead accessible for binding, etc.…”