Lipase Activity in the Larval Midgut of Rhynchophorus palmarum: Biochemical Characterization and the Effects of Reducing Agents

Abstract

Lipases have key roles in insect lipid acquisition, storage, and mobilization and are also fundamental to many physiological processes in insects. Lipids are an important component of insect diets, where they are hydrolyzed in the midgut lumen, absorbed, and used for the synthesis of complex lipids. The South American palm weevil Rhynchophorus palmarum is one of the most important pests on commercial palm plantations. However, there are few studies about lipid digestion for this insect. In this work, we have described the biochemical characterization of the lipase activity in the posterior midgut of the R. palmarum palm weevil. Lipase activity was highest between the temperatures of 37 °C and 45 °C and at pH 6.5. Lipase activity was also sensitive to variations in salt and calcium concentrations. Lipases have been described structurally as enzymes with the Ser-His-Asp Catalytic Triad, containing an active serine. The serine protease inhibitor PMSF (phenylmethane sulfonyl fluoride) inhibited the lipases from R. palmarum, demonstrating the importance of a serine residue for this activity. The ability of the lipases to hydrolyze p-Nitrophenyl esters with different chain lengths has revealed the activities of a broad range of substrates. The lipase activities of R. palmarum increased in the presence of reduced glutathione (GSH) and dithiothreitol (DTT), while in the presence of oxidized glutathione (GSSG), activities were drastically reduced. To our knowledge, this study has provided the first information about lipase activity in the R. palmarum palm weevil.

Keywords: Rhynchophorus palmarum; digestion; lipase; lipids; midgut.

Figure 1

Time course (A) and the effects of the enzyme concentrations (B) on the lipase activity of the posterior midgut homogenate (crude extract) of R. palmarum larvae. The error bars represent the standard deviation (SD) for three determinations.

Figure 2

The optimum temperature (A) and optimum pH (B) for lipase activity of the posterior midgut homogenate (crude extract) of R. palmarum larvae. The error bars represent the SD for the three determinations.

Figure 3

Effects of NaCl and calcium concentrations on the lipase activities of the posterior midgut homogenate (crude extract) of R. palmarum larvae. The incubations were performed with different NaCl concentrations (A) and with different calcium concentrations (B). The error bars represent the SD for the three determinations (* p < 0.05).

Figure 4

Effects of phenylmethane sulfonyl fluoride (PMSF) on the lipase activities of the posterior midgut homogenate (crude extract) of R. palmarum larvae. The incubations were performed with different PMSF concentrations. The error bars represent the SD for the three determinations (* p < 0.05).

Figure 5

Substrate specificity and the effects of the reducing agents on the lipase activity. The posterior midgut homogenate (crude extract) of R. palmarum was incubated with the p-Nitrophenyl esters with different chain lengths (A). The effects of reducing agents (B) were determined by incubation with different concentrations of reduced glutathione (GSH) (open circles), dithiothreitol (DTT) (open triangles), and oxidized glutathione (GSSG) (filled circles). Lipase activity was measured using C16 pNP-palmitate as substrate. The error bars represent the SD for the three determinations (* p < 0.05).