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 mol min Ϫ1 mg Ϫ1 at 80°C using [9,10-3 H]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 ϫ 10 5 Calorie mol Ϫ1 at this temperature. The purified lipase also exhibited phospholipase A 2 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 the sn-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.

The kinetics of lipase activity and hydrolytic rancidity of raw, parboiled, and extruded rice bran during storage

“…Bhardwaj, Raju and Rajasekharan (2001) found significant heat stability for the rice bran lipase. Inactivation of the lipases by the heat treatments applied to the bran samples in this study resulted in a gradual fall in the activity of the enzyme during storage.The enzyme was active up to 40 °C, and the activity decreased sharply to 65% at 60 °C, and then it decreased gradually (BHARDWAJ; RAJU; RAJASEKHARAN, 2001). It is likely that the parboiling at 70 °C for 5 hours used in this study was not sufficient to inactivate 100% of lipolytic activity.…”

“…Parameters of the Azuara, Berinstain and Garcia (1992) model obtained for lipase activity in the raw rice bran (RRB), extruded rice bran (ERB), and parboiled rice bran (PRB) stored in low density (15 µm) polyethylene bags at room temperature for 180 days. for hydrolytic rancidity in the treatments of RRB, ERB, and PRB as a function of storage time applying the Azuara, Berinstain and Garcia (1992) model were significant (P < 0.0000) and explained 96%, 91%, and 94% of the responses, respectively.…”

“…The kinetic behavior of the lipase activity and hydrolytic rancidity were modeled according to Azuara, Berinstain and Garcia (1992), Equation 1. Lipase activity (mg caprylic acid 100 mg -1 ) (a) and hydrolytic rancidity (mg caprylic acid 100 mg -1 ) (b) in the raw rice bran (RRB), extruded rice bran (ERB), and parboiled rice bran (PRB) stored at room temperature.…”

“…The experimental data were fitted to the Azuara, Berinstain and Garcia (1992) model (Equation 1) by linear regression. Figure 2b shows the graphical representations of the regressions for the hydrolytic rancidity, while the values calculated for the parameters of the model and the coefficients of determination (R 2 ) are presented in Table 2.…”

“…During prolonged storage, the kinetics of lipase activity can show stabilization at a low level of activity tending to equilibrium after 180 days, and consequently the production of fatty acids stabilizes at the maximum level. If this hypothesis is considered valid, the equation of the Azuara, Berinstain and Garcia (1992) model can be used to determine the velocity constants of the reactions, as well as the points of minimal lipase activity and maximum free fatty acid production.…”