Sato_2026_J.Biosci.Bioeng__

Phthalate esters, widely used as plasticizers in plastic manufacturing, are known for their endocrine-disrupting effects. gamma-Oryzanol derivatives, functional lipids composed of sterol and ferulic acid esters, possess diverse biological activities. In this study, we cloned and heterologously expressed a lipase-encoding gene (lipO745) from Aspergillus oryzae RIB40, deleting a 23-amino-acid N-terminal signal peptide, in Pichia pastoris using the pPICZalphaC vector. The resulting recombinant enzyme (rLipO745delta23) was successfully secreted as an active extracellular protein. The purified recombinant lipase exhibited an apparent molecular mass of approximately 65 kDa, as determined using SDS-PAGE. Substrate specificity assays using p-nitrophenyl (pNP) esters (C2-C16) revealed that pNP butyrate (pNP-C4) was hydrolyzed most efficiently, with a specific activity of 369.2 +/- 6.6 nmol.mL(-1) mg(-1). rLipO745delta23 catalyzed the conversion of dibutyl phthalate (DBP) to monobutyl phthalate (MBP) without further degradation to phthalic acid, and exhibited an activity of 7.24 +/- 0.61 nmol.mL(-1) mg(-1) toward DBP. The kinetic parameters (K(m) and k(cat)) were 0.66 +/- 0.1 mM and 37.8 +/- 6.4 s(-1), respectively, for pNP-C4 substrate, and 0.40 +/- 0.05 mM and 1.30 +/- 0.23 s(-1), respectively, for DBP. Moreover, rLipO745delta23 showed detectable hydrolytic activity toward gamma-oryzanol, including cycloartenyl ferulate, a conjugate of ferulic acid and triterpene alcohol. Notably, beta-sitosterol, a major hydrolysate of phytosterol type gamma-oryzanol, did not inhibit enzymatic activity. These findings highlight the potential of rLipO745delta23 as a versatile biocatalyst for the enzymatic degradation of phthalates and gamma-oryzanol derivatives in environmental and industrial applications.