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<p>Lipase/phospholipase (Lip/Plip) from thraustochytrids is a bifunctional enzyme capable of hydrolyzing both triglycerides and phospholipids and is characterized by an unusual 150-residue <i>N</i>-terminal domain whose function remains unclear. To elucidate the role of this region, we engineered an <i>N</i>-terminally truncated enzyme (S-270) and compared it with the full-length enzyme (L-420) from <i>Aurantiochytrium</i> sp. MG91 heterologously expressed in <i>Escherichia coli</i>. The present research is limited and directed toward comparing enzyme activities (specific activity, substrate selectivity, and growth-related evaluation) and bioinformatics evidence to offer an extensive overview of the biological effects that occur. The&#xa0;18S rRNA sequence analysis revealed that MG91 isolate is closely related to <i>Aurantiochytrium limacinum</i> and <i>A. mangrovei</i>. Both constructs were successfully expressed in <i>E. coli</i> BL21 (DE3), yielding proteins of ~ 45&#xa0;kDa (L-420) and ~ 30&#xa0;kDa (S-270). Although the substrate specificity remained constant favoring pNP-C12 and sunflower-derived phosphatidylcholine, the&#xa0;S-270 demonstrated a marked enhancement in specific activity, showing a 4–6-fold rise in lipase and a 1.4-fold increase in phospholipase. Consistent with the growth assessments, the&#xa0;S-270 appeared to alleviate the harmful effects Lip/Plip observed in L-420, as evidenced by its normal colony morphology and higher growth relative to the L-420 under IPTG-induced overexpression in <i>E. coli</i>. Nevertheless, the 3D Structural modeling revealed no major conformational differences between L-420 and S-270 apart from the <i>N</i>-terminal membrane-associated region present only in the full-length enzyme. The G-Y-S-R-G lipase motif, containing the catalytic residues Ser311, Asp368, and His382, was annotated and phylogenetic analysis positioned Lip/Plip MG91 within a miscellaneous cluster that encompasses both Y-type and GX-type lipases. Our findings demonstrate that the&#xa0;L-420 exhibits notably higher toxicity in <i>E. coli</i> expression system, as indicated by its diminished expression, decreased specific activity, suppressed cellular growth, and the appearance of smaller colonies compared with S-270. This is the first report showing that removing the <i>N</i>-terminal domain of thraustochytrid Lip/Plip increases its specific activity, highlighting its biotechnological potential.</p>

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Activity Enhancement of a Truncated Lipase-Phospholipase from the Marine Thraustochytrid Aurantiochytrium Sp. MG91

  • 期刊论文

  • 发表日期：

    2025年12月27日

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  • Noer Syahbani,
  • Rika Indri Astuti,
  • Antonius Suwanto

摘要

Lipase/phospholipase (Lip/Plip) from thraustochytrids is a bifunctional enzyme capable of hydrolyzing both triglycerides and phospholipids and is characterized by an unusual 150-residue N-terminal domain whose function remains unclear. To elucidate the role of this region, we engineered an N-terminally truncated enzyme (S-270) and compared it with the full-length enzyme (L-420) from Aurantiochytrium sp. MG91 heterologously expressed in Escherichia coli. The present research is limited and directed toward comparing enzyme activities (specific activity, substrate selectivity, and growth-related evaluation) and bioinformatics evidence to offer an extensive overview of the biological effects that occur. The 18S rRNA sequence analysis revealed that MG91 isolate is closely related to Aurantiochytrium limacinum and A. mangrovei. Both constructs were successfully expressed in E. coli BL21 (DE3), yielding proteins of ~ 45 kDa (L-420) and ~ 30 kDa (S-270). Although the substrate specificity remained constant favoring pNP-C12 and sunflower-derived phosphatidylcholine, the S-270 demonstrated a marked enhancement in specific activity, showing a 4–6-fold rise in lipase and a 1.4-fold increase in phospholipase. Consistent with the growth assessments, the S-270 appeared to alleviate the harmful effects Lip/Plip observed in L-420, as evidenced by its normal colony morphology and higher growth relative to the L-420 under IPTG-induced overexpression in E. coli. Nevertheless, the 3D Structural modeling revealed no major conformational differences between L-420 and S-270 apart from the N-terminal membrane-associated region present only in the full-length enzyme. The G-Y-S-R-G lipase motif, containing the catalytic residues Ser311, Asp368, and His382, was annotated and phylogenetic analysis positioned Lip/Plip MG91 within a miscellaneous cluster that encompasses both Y-type and GX-type lipases. Our findings demonstrate that the L-420 exhibits notably higher toxicity in E. coli expression system, as indicated by its diminished expression, decreased specific activity, suppressed cellular growth, and the appearance of smaller colonies compared with S-270. This is the first report showing that removing the N-terminal domain of thraustochytrid Lip/Plip increases its specific activity, highlighting its biotechnological potential.