Enzymatic degradation of γ-oryzanol and dibutyl phthalate by Aspergillus oryzae lipase.

Phthalate esters, widely used as plasticizers in plastic manufacturing, are known for their endocrine-disrupting effects. γ-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 pPICZαC vector. The resulting recombinant enzyme (rLipO745Δ23) 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. rLipO745Δ23 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 (Km and kcat) 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, rLipO745Δ23 showed detectable hydrolytic activity toward γ-oryzanol, including cycloartenyl ferulate, a conjugate of ferulic acid and triterpene alcohol. Notably, β-sitosterol, a major hydrolysate of phytosterol type γ-oryzanol, did not inhibit enzymatic activity. These findings highlight the potential of rLipO745Δ23 as a versatile biocatalyst for the enzymatic degradation of phthalates and γ-oryzanol derivatives in environmental and industrial applications.