A DIGESTIVE LIPASE OF Pieris brassicae L. (LEPIDOPTERA: PIERIDAE): PURIFICATION, CHARACTERIZATION, AND HOST PLANTS EFFECTS

Abstract

The properties of a digestive lipase from the larval midgut of Pieris brassicae were studied by performing biochemical purification, characterization, effect of host plants, and extracted inhibitors. The purification process revealed a lipase with a purification fold of 42, recovery of 18.12%, molecular weight mass of 72.3 kDa, optimal pH at 11, and optimal temperature at 30°C, as well as stability at the optimal temperature for 12 h. The purified enzyme was inhibited by the ions Na(+), Mn(+), Fe(2+), and Cu(2+) and the inhibitors SDS, EDTA, TTHA, and mercaptoethanol. Ca(2+) and Mg(2+) increased activity of the purified lipase, but urea, PMSF, EGTA, and DTC had no effect on enzymatic activity. Feeding of larvae on three host plants, Trepaeolus majus, Brassica olearcea var. alba, and B. olearcea var. rubra revealed the highest lipase activity on T. majus, but the two varieties of B. olearcea significantly decreased lipase activity. Extraction of a crude inhibitor from two varieties of B. olearcea demonstrated that the crude inhibitor inhibited the purified lipase up to 75%. The inhibitor changed the kinetic parameters of the enzyme by elevating the K(m), as in competitive inhibition. The data suggest a possible role for plant lipase inhibitors in host plant resistance.

Purification and characterization of a digestive lipase in the midgut ofEctomyelois ceratoniaeZeller (Lepidoptera: Pyralidae)

“…Zibaee and Fazeli-Dinan (2012) purified a digestive lipase in the midgut of N. aenescens with a molecular weight of 51 kDa. Zibaee (2012) found that the fourth instar larvae of P. brassicae have a digestive lipase with a molecular weight of 72.3 kDa. Orscelk et al (2007) found a total body lipase of Gryllus campestris L. (Orthoptera: Gryllidae) with a molecular mass of 76.5 kDa.…”

“…Zibaee and Fazeli-Dinan (2012) found an optimal pH of 10 and temperature of 35-40°C for a lipase in the larvae of Naranga aenescens. Zibaee (2012) reported an optimal pH of 8 and 10 for digestive soluble-and membrane-bound lipase in the midgut of Pieris brassicae L. (Lepidoptera: Pieridae). In an unpublished study, optimal pH and temperature for activity of the purified lipase in the midgut of E. integriceps was obtained to be 7 and 25-30°C.…”

“…Inhibition of digestive lipases could form a part of integrated pest management (IPM) programs. Lipase inhibitors cause severe reduction in growth and development and even mortality due to long chain unsaturated fatty acids being essential dietary components (Zibaee 2012). A plant breeding program to increase levels of naturally occurring lipase inhibitors could form a component of an IPM program.…”

“…Homogenates were separately transferred to 1.5 ml centrifuge tubes and centrifuged at 13,000 rpm for 20 min at 4°C. The supernatants were pooled and stored at − 20°C for subsequent analyses (Zibaee 2012).…”

“…Knowledge of insect digestive lipases remains limited since studies have concentrated on fat body lipases. Zibaee et al (2008), Zibaee and FazeliDinan (2012) and Zibaee (2012) have shown activities of digestive lipases in Chilo suppressalis Walker (Lepidoptera: Crambidae), Naranga aenescens Moore (Lepidoptera: Noctuidae) and Pieris brassicae L. (Lepidoptera: Pieridae). Hence, the aims of this study were to purify and characterize a TAG lipase in the midgut of E. ceratoniae by using different biochemical factors including column chromatography, pH, temperature, specific inhibitors and kinetic parameters.…”