Molecular modification of glutathione bifunctional synthase and its application
Glutathione(GSH)is the most abundant non-protein thiol within cells.It has strong electron donating ability and participates in redox reactions in organisms,which endows it with multiple physiological functions and it is widely used in food,pharmaceutical and cosmetics industries.Glutathione bifunctional synthase is the key enzyme for catalyzing the production of GSH and improvement of its catalytic efficiency is of significance to GSH biosynthesis.In this study,the glutathione bifunctional synthasefrom Streptococcus thermophilus(St-GshF)was chosen for study object.A high-throughput screening method based on the fluorescence colorimetric effect generated by the reaction between GSH and ortho benzaldehyde was established.Using semi-rational design,St-GshF was engineered and the mutant St-GshF(S27Q/G510P)was obtained.Its activity was 1.75 times higher than the wild type.The polyphosphate kinase was coupled to construct the efficient glutathione biosynthesis system and after optimization of the reaction conditions,17.36 g/L glutathione was obtained with a yield of 94.22％,laying an important foundation for the large-scale production of glutathione.
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