L-theanine, an amino acid known for its favourable taste and linked with health benefits, can be prepared by enzymatic synthesis using γ-glutamyltranspeptidase (GGT; E.C 2.3.2.2). In the present study, a novel GGT from Bacillus pumilus ML413 was expressed in Bacillus subtlis 168 and exhibited high stability at low temperature (40 °C) and alkaline pH 10, compared to the other GGTs. To enhance GGT production, firstly, PrsA lipoproteins was overexpressed in the host which resulted in the extracellular GGT activity doubled. Subsequently, a suitable poly (A/T) tail (TTTAAA) was selected and added to the 3'-terminal of ggt gene, which increased the mRNA stability of ggt gene by 58% and the activity of GGT by 60%. Finally, under optimized fed batch system, L-theanine yield was 53 g l within 16 h. In this study, we demonstrate a convenient strategy of increasing theanine yield using GGT overexpressing in a safe host B. subtilis 168.
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