Engineering of cyclodextrin glycosyltransferase from Paenibacillus macerans for enhanced product specificity of long-chain glycosylated sophoricosides

Abstract

Poor water solubility limits the application of genistein in food and pharmaceutical fields. Glycosylation can greatly improve the water solubility of genistein by attaching long chain sugars. Here, glycosylation of sophoricoside (genistein monoglucoside) by cyclodextrin glycosyltransferase from Paenibacillus macerans (PmCGTase) was investigated. Based on molecular docking and hydrolytic activity analysis, saturation mutagenesis was conducted at residues Y195 and S77 of PmCGTase. Variants S77N, Y195S, Y195I, S77N/Y195S, and S77N/Y195I showed enhanced product specificity toward long-chain glycosylated sophoricosides (LCGS). In particular, S77N/Y195I displayed 119% increased LCGS product specificity and 20.6% higher sophoricoside conversion than the wild type. Increased comprehensive activities of Y195S and Y195I contributed to enhanced conversion, and decreased hydrolytic activity of S77N promoted LCGS accumulation. Kinetics results revealed improved catalytic efficiency and substrate affinity of variants toward both the glycosyl donor and acceptor. Furthermore, MD simulation elucidates that the decreased substrate binding energy of variants could lead to improved kinetics, and increased hydrogen binding between residues at -6/-7 subsites and the glycosyl donor is primarily responsible for enhanced LCGS specificity. An “Increased Income-Reduced Expenditure” mechanism was proposed, explaining that the enhanced LCGS product specificity of PmCGTase is closely related to the increased hydrogen bond interactions at -6/-7 subsites and decreased hydrolytic activity.