Magnetic Cross-Linked Enzyme Aggregates of a Transpeptidase-Specialized Variant (N450D) of Bacillus licheniformis γ-Glutamyl Transpeptidase: An Efficient and Stable Biocatalyst for l-Theanine Synthesis

Abstract

γ-Glutamyl transpeptidase (GGT) catalyzes the transfer of glutathione’s γ-glutamyl group and related γ-glutamyl amides to water, amino acids or peptides, and utilizes a conserved Thr residue to process its own polypeptide chain into a large and a small subunit that then assemble to produce a catalytically competent enzyme. In this study, the magnetic cross-linked enzyme aggregates (mCLEAs) of a transpeptidase-specialized variant (N450D) of Bacillus licheniformis GGT were successfully prepared with optimized process parameters viz.1.25:1 (v/v) of isopropanol to N450D (0.3 mg/mL) ratio/0.02:1 (w/w) of enzyme to 3-aminopropyl triethoxysilane (APTES)-coated magnetic nanoparticle ratio/20 mM of glutaraldehyde. The prepared magnetic nanoparticles and immobilized enzyme (N450D-mCLEAs) were characterized by X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy, field-emission scanning electron microscope integrated with energy dispersive X-ray spectroscopy (FESEM/EDS), and superparamagnetic analysis. As compared with free enzyme, N450D-mCLEAs displayed significantly higher heat resistance at temperatures of 55 and 60 °C, and had a greater stability over a storage period of one month. The immobilized enzyme could also be reused for 10 consecutive biocatalytic cycles with no significant reduction in the percent yield of l-theanine. Conclusively, this immobilization strategy surely provides a meaningful glance of developing N450D-mediated biocatalysis for the production of physiologically important γ-glutamyl compounds.

Streamlining Design, Engineering, and Applications of Enzymes for Sustainable Biocatalysis

“…Magnetic cross-linked enzyme aggregates (mCLEAs) of γglutamyl transpeptidase incorporating APTES-coated magnetic nanoparticles were used in the synthesis of L-theanine from Lglutamine and ethylamine and then recovered magnetically for another nine reactions without activity loss. 178 Other magneticbased immobilized biocatalysts have been demonstrated. Zhou et al (2015) used magnetic beads with cross-linked polyethylenimine and epoxide functional groups (MagReSyn) to bind pyrimidine nucleoside phosphorylase and purine nucleoside phosphorylase with high enzyme loading (up to 0.4 and 1.3 g per gram of dry beads respectively), for the synthesis of nonnatural nucleosides as precursors for anti-leukemia drugs such as Cladribine, Clofarabine, and Fludarabine.…”

“…Recent developments of magnetic CLEAs is a step forward. Magnetic cross-linked enzyme aggregates (mCLEAs) of γ-glutamyl transpeptidase incorporating APTES-coated magnetic nanoparticles were used in the synthesis of l -theanine from l -glutamine and ethylamine and then recovered magnetically for another nine reactions without activity loss . Other magnetic-based immobilized biocatalysts have been demonstrated.…”

Current Status and Future Perspectives of Supports and Protocols for Enzyme Immobilization

“…Covalent bonding is the most used method, which requires chemical reactants to create functional groups that will bond with the enzyme [6,69,[83][84][85][86][87]. This strategy can offer significant operational stability and reusability to the biocatalyst, but it may cause conformational changes in the enzymes during the immobilization procedure, reducing or even extinguishing enzyme activity [6,74,88]. Using the principles briefly presented in this paragraph, the following subsections present the trending protocols for lipase immobilization.…”

(Magnetic) Cross-Linked Enzyme Aggregates of Cellulase from T. reesei: A Stable and Efficient Biocatalyst

“…Nevertheless, when enzymes are exposed to specific conditions, changes in conformation may occur, which causes the loss of some specific enzyme features. Such drawbacks can be overcome by the process of immobilization, since immobilization enhances the operational stability [ 11 ]. To accomplish a suitable immobilization protocol, limitations such as activity and stability under certain conditions, selectivity and specificity using substrates, as well as enzyme purity must be addressed.…”