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.