Abstract
γ-Glutamyltransferase (GGT) can catalyze the transfer of a γ-glutamyl group to amino acids or peptides. Bacterial GGTs have a wide substrate specificity, which means that a variety of γ-glutamyl amino acid derivatives or peptides can be synthesized under GGT catalysis. In this work, in order to enhance the expression and stability of GGT, a recombinant plasmid harboring Bacillus amyloliquefaciens ggt gene with a Usp45 signal peptide gene fused upstream was constructed and transferred into Lactococcus lactis NZ9000 to express BaGGT. After on-column hydrolysis and purification by immobilized metal affinity chromatography, 90.2 ± 0.7 mg/L of BaGGT-6His with an enzymatic activity of 57.3 ± 0.9 U/mg was purified. Subsequently, BaGGT-6His was immobilized onto magnetic mesoporous nanoparticles (Fe3O4NPs) by covalent binding. The immobilized BaGGT-6His largely retained the activity and, importantly, could be recovered and reused. After optimization of the production and immobilization conditions, the biotechnologically relevant product l-theanine was synthesized with free and immobilized BaGGT-6His.
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