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<p>This study presents the first immobilization of a true triacylglycerol (TAG) plant lipase, unambiguously confirmed by pH-stat analysis, from a Tunisian almond (<i>Prunus amygdalus</i> Mill., cultivar ‘Achaak’), representing a local and previously underexplored cultivar. The aim was to develop an immobilization-based strategy for the purification, stabilization, and enhancement of catalytic performance of this plant-derived TAG lipase. Although plant lipases, particularly those from oilseeds, play a central role in storage oil mobilization during seed germination, they remain far less explored for biocatalytic applications than their microbial counterparts. Purification was achieved by selective adsorption on hydrophobic supports, with Octyl Sepharose showing the highest immobilization yield (80%) and inducing marked hyperactivation, corresponding to an approximately 197-fold increase in apparent activity compared with the crude extract. The purified enzyme exhibited a high specific activity (461.25 IU·mg⁻¹), an estimated molecular weight of approximately 45&#xa0;kDa, and optimal stability at pH 8 and 40&#xa0;°C, together with high tolerance toward surfactants and enhanced operational robustness. Compared with previously reported plant-derived lipases, immobilized LipA displayed competitive catalytic efficiency and stability under mild alkaline conditions. Homology modelling revealed a canonical α/β-hydrolase fold, harbouring a conserved catalytic triad (Ser180, His383, Glu359) and hydrophobic regions surrounding the active site, consistent with an interfacial activation mechanism. Structural comparison with well-characterized lipases, including peach and pancreatic lipases, revealed similarities, supporting the classification of this enzyme as a true triacylglycerol lipase. Structural validation analyses confirmed acceptable stereochemical quality, while physicochemical profiling suggested favourable thermal stability, moderate hydrophilicity, and a slightly alkaline behaviour. Altogether, these findings provide the first structural framework for an almond TAG lipase and highlight immobilization as an effective strategy to stabilize and induce hyperactivation of plant-derived lipases, thereby supporting their potential as eco-friendly biocatalysts for lipid modification, biodiesel production, and other low-carbon biotechnological applications.</p>

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Structural and biochemical characterization of triacylglycerol lipase from almond (Prunus amygdalus Mill.) seeds: immobilization, homology modelling, and sustainable biocatalytic insights

  • 期刊论文

  • 发表日期：

    2026年5月20日

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  • Refka Dhouibi,
  • Najeh Krayem,
  • Adel Sayari,
  • Sellema Bahri

摘要

This study presents the first immobilization of a true triacylglycerol (TAG) plant lipase, unambiguously confirmed by pH-stat analysis, from a Tunisian almond (Prunus amygdalus Mill., cultivar ‘Achaak’), representing a local and previously underexplored cultivar. The aim was to develop an immobilization-based strategy for the purification, stabilization, and enhancement of catalytic performance of this plant-derived TAG lipase. Although plant lipases, particularly those from oilseeds, play a central role in storage oil mobilization during seed germination, they remain far less explored for biocatalytic applications than their microbial counterparts. Purification was achieved by selective adsorption on hydrophobic supports, with Octyl Sepharose showing the highest immobilization yield (80%) and inducing marked hyperactivation, corresponding to an approximately 197-fold increase in apparent activity compared with the crude extract. The purified enzyme exhibited a high specific activity (461.25 IU·mg⁻¹), an estimated molecular weight of approximately 45 kDa, and optimal stability at pH 8 and 40 °C, together with high tolerance toward surfactants and enhanced operational robustness. Compared with previously reported plant-derived lipases, immobilized LipA displayed competitive catalytic efficiency and stability under mild alkaline conditions. Homology modelling revealed a canonical α/β-hydrolase fold, harbouring a conserved catalytic triad (Ser180, His383, Glu359) and hydrophobic regions surrounding the active site, consistent with an interfacial activation mechanism. Structural comparison with well-characterized lipases, including peach and pancreatic lipases, revealed similarities, supporting the classification of this enzyme as a true triacylglycerol lipase. Structural validation analyses confirmed acceptable stereochemical quality, while physicochemical profiling suggested favourable thermal stability, moderate hydrophilicity, and a slightly alkaline behaviour. Altogether, these findings provide the first structural framework for an almond TAG lipase and highlight immobilization as an effective strategy to stabilize and induce hyperactivation of plant-derived lipases, thereby supporting their potential as eco-friendly biocatalysts for lipid modification, biodiesel production, and other low-carbon biotechnological applications.