Covalent Immobilization of Bacillus licheniformis γ-Glutamyl Transpeptidase on Aldehyde-Functionalized Magnetic Nanoparticles.

Yi-Yu Chen,Ming-Gen Tsai,Tzu-Fan Wang,Meng-Chun Chi,Long-Liu Lin

doi:10.3390/ijms14034613

Abstract

This work presents the synthesis and use of surface-modified iron oxide nanoparticles for the covalent immobilization of Bacillus licheniformis γ-glutamyl transpeptidase (BlGGT). Magnetic nanoparticles were prepared by an alkaline solution of divalent and trivalent iron ions, and they were subsequently treated with 3-aminopropyltriethoxysilane (APES) to obtain the aminosilane-coated nanoparticles. The functional group on the particle surface and the amino group of BlGGT was then cross-linked using glutaraldehyde as the coupling reagent. The loading capacity of the prepared nanoparticles for BlGGT was 34.2 mg/g support, corresponding to 52.4% recovery of the initial activity. Monographs of transmission electron microscopy revealed that the synthesized nanoparticles had a mean diameter of 15.1 ± 3.7 nm, and the covalent cross-linking of the enzyme did not significantly change their particle size. Fourier transform infrared spectroscopy confirmed the immobilization of BlGGT on the magnetic nanoparticles. The chemical and kinetic behaviors of immobilized BlGGT are mostly consistent with those of the free enzyme. The immobilized enzyme could be recycled ten times with 36.2% retention of the initial activity and had a comparable stability respective to free enzyme during the storage period of 30 days. Collectively, the straightforward synthesis of aldehyde-functionalized nanoparticles and the efficiency of enzyme immobilization offer wide perspectives for the practical use of surface-bound BlGGT.

Highlights

Enzymes are versatile biocatalysts that are useful for biotechnological applications, including food industries and pharmaceuticals [1,2]
The possible explanation for this phenomenon is that the multiple layers of enzyme molecules on the surface of magnetic nanoparticles are formed at high enzyme loading, which might block the active sites of the enzyme and cause the diffusion limitation of the substrate
Compared with the spectrum of naked Fe3O4, the characteristic bands appear at 1542.95 and 1645.17 cm−1 in Fe3O4/APES/BlGGT and can be assigned to the NH and NH2 bending vibration, respectively. These results indicate that the BlGGT molecules are conjugated with the APES-modified magnetic nanoparticles successfully

Summary

Introduction

Enzymes are versatile biocatalysts that are useful for biotechnological applications, including food industries and pharmaceuticals [1,2]. For industrial applications, immobilized enzymes are currently the object of considerable interest, because of the advantage benefits over the soluble form of enzymes or alternative technologies [6]. The ability to retain or recover enzymes allows biocatalyst separation from the product, thereby permitting continuous processes, and prevents carry-through of protein or activity to subsequent process steps [7]. The denaturation operation of immobilized enzymes can even be prevented [8,9,10]. A number of interesting findings indicate that enzyme immobilization has entered an exciting new phase [13]

Methods

Results

Conclusion