Purifying and Studying a Key Enzyme from Sweet Orange Seeds

Jim Crocker
8th June, 2024

Purifying and Studying a Key Enzyme from Sweet Orange Seeds

Image Source: Natural Science News, 2024

Key Findings

  • Researchers at Huazhong Agricultural University isolated and purified an enzyme called LDLH from sweet orange seeds
  • LDLH is highly stable, maintaining nearly 100% activity in both acidic and alkaline conditions and after thermal treatment
  • The enzyme's activity can be enhanced or reduced by specific ions, suggesting potential methods to control citrus bitterness
Citrus products, such as orange juice, often experience delayed bitterness, which significantly impacts their sensory quality and consumer acceptability. This bitterness arises from the conversion of non-bitter precursors like limonoate A-ring lactone (LARL) to limonin, catalyzed by the enzyme limonin D-ring lactone hydrolase (LDLH). A recent study conducted by researchers at Huazhong Agricultural University[1] has isolated and purified LDLH from sweet orange seeds, providing new insights into its properties and potential applications in reducing citrus bitterness. The research team successfully purified LDLH, achieving an enzyme activity of 25.22 U mg-1. Through native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate PAGE analysis, they identified bands corresponding to molecular weights of 245 kDa and 17.5 kDa, respectively. These findings indicate that LDLH is a multimer containing 17.5 kDa monomers. The enzyme demonstrated remarkable stability, retaining nearly 100% activity after 24 hours of incubation in strongly acidic (pH 3) or alkaline (pH 9) conditions. Furthermore, LDLH maintained its activity even after thermal treatment at 50°C for 120 minutes. Enzyme inhibition assays revealed that LDLH was inactivated only by ethylenediaminetetraacetic acid (EDTA), suggesting that it might be a metallopeptidase. This hypothesis was further supported by the observation that calcium ions (Ca2+) enhanced LDLH activity to 108% of the control group, while zinc ions (Zn2+) reduced it to 71%. These findings highlight the enzyme's potential role in reducing bitterness in citrus products by manipulating its activity through specific ions. This study builds on previous research into the factors affecting the sensory quality of citrus products. For instance, it has been established that the bitterness in citrus fruits is primarily due to compounds like naringin and limonin[2]. Additionally, the detection and reduction of limonin in citrus juices have been explored using various methods, such as the novel interdigitated capacitive sensor[3]. The current study's purification and characterization of LDLH provide a deeper understanding of the enzymatic processes involved in limonin formation, offering a new avenue for addressing citrus bitterness. Moreover, the study's findings have practical implications for the citrus industry. By leveraging the stability and ion-dependent activity of LDLH, it may be possible to develop targeted interventions to reduce limonin content in citrus products, thereby enhancing their taste and consumer appeal. This could involve optimizing storage conditions or incorporating specific ions to modulate enzyme activity. In summary, the isolation and characterization of LDLH from sweet orange seeds by Huazhong Agricultural University researchers represent a significant advancement in our understanding of citrus bitterness. The enzyme's stability across a wide pH range and its response to various ions provide valuable insights for developing strategies to improve the sensory quality of citrus products. These findings, in conjunction with earlier studies on citrus bitterness and debittering methods[2][3], pave the way for innovative solutions to enhance the flavor and acceptability of citrus juices.

FruitsBiochemPlant Science

References

Main Study

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

Published 7th June, 2024

https://doi.org/10.1002/jsfa.13650

Related Studies

2) Citrus Taste Modification Potentials by Genetic Engineering.

https://doi.org/10.3390/ijms20246194

3) A novel electrochemical interdigitated electrodes sensor for limonin quantification and reduction in citrus limetta juice.

https://doi.org/10.1016/j.foodchem.2022.132248


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