Journal
MOLECULAR BIOTECHNOLOGY
Volume 65, Issue 10, Pages 1644-1652Publisher
SPRINGERNATURE
DOI: 10.1007/s12033-023-00682-6
Keywords
Enzyme engineering; Glutathione peroxidase; Selenoprotein; Selenocysteine
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This study successfully incorporated selenocysteine (Sec) into the cysteine peroxidase (Cys-GPx) from Lactococcus lactis, resulting in improved enzyme activity and stability for industrial applications.
Glutathione peroxidase (GPx) is an important antioxidant enzyme. Selenocysteine (Sec)-containing GPxs (Sec-GPxs) are usually superior to their conventional cysteine-containing counterparts (Cys-GPxs), which make up the majority of the natural GPxs but display unsuitable activity and stability for industrial applications. This study first heterologously expressed and characterized a Cys-GPx from Lactococcus lactis (LlGPx), systematically exchanged all the three Cys to Sec and introduced an extra Sec. The results showed that the insertion of Sec at the active site could effectively increase the enzyme activity and confer a lower optimal pH value on the mutants. The double mutant C36U/L157U increased by 2.65 times (5.12 U/mg). The thermal stability of the C81U mutant was significantly improved. These results suggest that site-directed Sec incorporation can effectively improve the enzymatic properties of LlGPx, which may be also used for the protein engineering of other industrial enzymes containing catalytic or other functional cysteine residues.
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