Identification, purification, and characterization of a thermally stable lipase from rice bran. A new member of the (Phospho) lipase family

Abstract

A thermally stable lipase (EC 3.1.1.3.) was first identified in rice (Oryza sativa) bran, and the enzyme was purified to homogeneity using octyl-Sepharose chromatography. The enzyme was purified to 7.6-fold with the final specific activity of 0.38 μ mol min⁻¹ mg⁻¹ at 80°C using [9,10⁻³H]triolein as a substrate. The purified enzyme was found to be a glycoprotein of 9.4 kD. Enzyme showed a maximum activity at 80°C and at pH 11.0. The protein was biologically active and retained most of its secondary structure even at 90°C as judged by the enzymatic assays and far-ultraviolet circular dichroism spectroscopy, respectively. Differential scanning calorimetric studies indicated that the transition temperature was 76°C and enthalpy 1.3 × 10⁵ Calorie mol⁻¹ at this temperature. The purified lipase also exhibited phospholipase A₂ activity. Colocalization of both the hydrolytic activities in reverse-phase high-performance liquid chromatography and isoelectric focusing showed that the dual activity was associated with a single protein. Further, a direct interaction between both the substrates and the purified protein was demonstrated by photoaffinity labeling, using chemically synthesized analogs of triolein and phosphatidylcholine (PC). Apparent K _m for triolein (6.71 mm) was higher than that for PC (1.02 mm). The enzyme preferentially hydrolyzed thesn⁻² position of PC, whereas it apparently exhibited no positional specificity toward triacylglycerol. Diisopropyl fluorophosphate inhibited both lipase and phospholipase activities of the purified enzyme. This enzyme is a new member from plants in the family of lipases capable of hydrolyzing phospholipids.