Purification and characterization of limonin D‐ring lactone hydrolase from sweet orange (Citrus sinensis (L.) Osbeck) seeds

Abstract BACKGROUND Citrus products often suffer from delayed bitterness, which is generated from the conversion of non‐bitter precursors (limonoate A‐ring lactone, LARL) to limonin under the catalysis of limonin D‐ring lactone hydrolase (LDLH). In this study, LDLH was isolated and purified from sweet orange seeds, and a rapid and accurate high‐performance liquid chromatography method to quantify LARL was developed and applied to analyze the activity and enzymatic properties of purified LDLH. RESULTS Purified LDLH (25.22 U mg −1 ) showed bands of 245 kDa and 17.5 kDa molecular weights in native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate PAGE analysis respectively. After a 24 h incubation under strongly acidic (pH 3) or strongly alkaline (pH 9) conditions, LDLH still retained approximately 100% activity. Moreover, LDLH activity was not impaired by thermal treatment at 50 °C for 120 min. Enzyme inhibition assays showed that LDLH was inactivated only after ethylenediaminetetraacetic acid treatment, and other enzyme inhibitors showed no significant effect on its activity. In addition, the LDLH activity of calcium ion (Ca 2+ ) intervention was 108% of that in the blank group, and that of zinc ion (Zn 2+ ) intervention was 71%. CONCLUSION LDLH purified in this study was a multimer containing 17.5 kDa monomer with a wide pH tolerance range (pH 3–9) and excellent thermal stability. Moreover, LDLH might be a metallopeptidase, and its activity was stimulated by Ca 2+ and significantly inhibited by Zn 2+ . These findings improve our understanding of LDLH and provide some important implications for reducing the bitterness in citrus products in the future. © 2024 Society of Chemical Industry.