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This distribution suggests a different ecological role of the enzyme in homofermentative and heterofermentative lactobacilli. In conclusion, three Gcls exhibited similar biochemical properties but differed with respect to their substrate specificity and thus the synthesis of kokumi-active γ-glutamyl dipeptides. KEY POINTS • Strains of Limosilactobacillus reuteri encode for up to 3 glutamyl cysteine ligases. • Gcl1, Gcl2, and Gcl3 of Lm. reuteri differ in their substrate specificity. • Gcl1 and Gcl3 produce kokumi-active dipeptides.Nitro