Biosynthesis of l -theanine via one-step purification and immobilization enzyme system

• Published in: Journal of applied microbiology Vol. 136; no. 3
• Main Authors: Fan, Chao, Qi, Jiakun, Zhang, Chunzhi
• Format: Journal Article
• Language: English
• Published: England 03.03.2025
• Subjects: Amide Synthases - genetics; Amide Synthases - metabolism; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biocatalysis; Enzymes, Immobilized - genetics; Enzymes, Immobilized - metabolism; Escherichia coli - genetics; Glutamates - biosynthesis; Listeria monocytogenes - enzymology; Listeria monocytogenes - genetics; Phosphotransferases (Phosphate Group Acceptor) - genetics; Phosphotransferases (Phosphate Group Acceptor) - metabolism; anchoring tags; GEM particles; one-step purification and immobilization (OSPI); l-theanine production
• ISSN: 1365-2672; 1365-2672
• DOI: 10.1093/jambio/lxaf053

l-theanine, a non-protein amino acid derived from green tea, was synthesized by a relatively substantial amount of γ-glutamylmethylamide synthetase (GMAS) and polyphosphate kinase (PPK) without efficient recycling. This study establishes a cost-efficient, industrially scalable, and continuous biocatalytic platform for sustainable l-theanine production. A functional catalyst system was engineered by fusing GMAS and PPK with the cell wall-binding domain derived from the Listeria monocytogenes p60 protein (Lm-p60). The enzyme complex was immobilized onto Gram-positive enhancer matrix particles, enabling facile separation and reuse over catalytic cycles. The enzymes were reusable and could be applied for six cycles with an l-theanine yield achieving 86%-93%. The reusable catalyst demonstrates operational sustainability over multiple cycles, offering cost savings and continuous utility.