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Milligram-scale sulfation of silybin B was optimized employing resting E. coli cells producing AstIV, thus avoiding the use of expensive 3'-phosphoadenosine-5'-phosphate cofactor and laborious enzyme purification.
Using PROSS 42, a mutant enzyme design strategy that integrates force-field-based Rosetta modeling with phylogenetic sequence information, we engineered ASTIV(M), which had 52 mutations compared with the wild-type (WT) enzyme.
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Aryl sulfotransferase IV (AstIV) from rat liver was overexpressed in Escherichia coli and purified to homogeneity. Using the produced mammalian liver enzyme, sulfation—the Phase II conjugation reaction—of optically pure silybin diastereoisomers (silybin A and B) was tested.
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