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

gamma-Glutamyl transpeptidase (GGT) catalyzes the transfer of glutathione's gamma-glutamyl group and related gamma-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 degrees 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 gamma-glutamyl compounds.

Automated summary

In this study, magnetic cross-linked enzyme aggregates (mCLEAs) of a transpeptidase-specialized variant (N450D) of Bacillus licheniformis GGT were successfully prepared using optimized process parameters. The immobilized enzyme exhibited enhanced thermal and storage stability compared to the free enzyme, and could be reused for multiple cycles without significant reduction in product yield, showing potential for biocatalysis in the production of physiologically important gamma-glutamyl compounds.