Revisiting Jatropha curcas Monomeric Esterase: A Dienelactone Hydrolase Compatible with the Electrostatic Catapult Model

Abstract

Jatropha curcas contains seeds with a high oil content, suitable for biodiesel production. After oil extraction, the remaining mass can be a rich source of enzymes. However, data from the literature describing physicochemical characteristics for a monomeric esterase from the J. curcas seed did not fit the electrostatic catapult model for esterases/lipases. We decided to reevaluate this J. curcas esterase and extend its characterization to check this apparent discrepancy and gain insights into the enzyme's potential as a biocatalyst. After anion exchange chromatography and two-dimensional gel electrophoresis, we identified the enzyme as belonging to the dienelactone hydrolase family, characterized by a cysteine as the nucleophile in the catalytic triad. The enzyme displayed a basic optimum hydrolysis pH of 9.0 and an acidic pI range, in contrast to literature data, making it well in line with the electrostatic catapult model. Furthermore, the enzyme showed low hydrolysis activity in an organic solvent-containing medium (isopropanol, acetonitrile, and ethanol), which reverted when recovering in an aqueous reaction mixture. This enzyme can be a valuable tool for hydrolysis reactions of short-chain esters, useful for pharmaceutical intermediates synthesis, due to both its high hydrolytic rate in basic pH and its stability in an organic solvent.

Keywords: Jatropha curcas L.; dienelactone hydrolase; esterase; seed.

Figure 1

Esterase B enrichment with differential ethanol precipitation. After this fractionation step, the EtOH 50–80% fraction was enriched in a 30 kDa band (A) and a single esterase activity region (B). Chain specificity assay (C) shows that esterase B has greater activity towards short-chain esters, decreasing as chain-length increases. *** p < 0.001.

Figure 2

Different esterase B behavior in an anion exchange chromatography. Online absorbance (280 nm) detection was performed (black curves in (A,B)) and each sample was further assayed for esterase activity (red curve in (B)). Resin-bound protein elution was performed by a linear gradient of elution buffer (blue curve in (A)). SDS PAGE analysis shows a single observable band in the flow-through fraction (C) and enrichment in three bands around the 30 kDa marker in the activity peak fractions (D). Band numbering corresponds to the MS protein identification data from Table 2.

Figure 3

Proteomic profile of the EtOH 50–80% (A) and activity peak (B) fractions. The indicated spots were processed and peptides were submitted to mass spectrometry for protein identification (Table 2).

Figure 4

Esterase activity analysis in the EtOH 50–80% fraction with serine (APMSF) and cysteine (iodoacetamide) esterase inhibitors. *** p < 0.001.

Figure 5

Alignment analysis of A. thaliana carboxymethylenebutenolidase and its homologs in J. curcas and R. communis. Both peptides identified during the mass spectrometry analysis are shown within rectangles. The typical dienalactone hydrolase pentapeptide (GxCxGG) is highlighted, as well as the active site residues Cys-78, Asp-126, and His-161 (marked with +). (*) Conserved sites, (:) sites with conservative replacement, (.) sites with semiconservative replacement.

Figure 6

Temperature and pH effects in esterase activity of J. curcas esterase B. (A) Assessment of their effects were done with a central composite rotational design. Higher esterase activity was observed with higher temperatures and pH values, as observed in (B). * p < 0.05 and ** p < 0.01.

Figure 7

Pairwise sequence alignment employed for the comparative modeling of the J. curcas esterase based on the structure of the uncharacterized protein from E. coli O157:H7 str. Sakai (PDB ID: 4ZV9). The sequences have 37% identity and 89% coverage. The active site residues—Cys-78, Asp-126, and His-161- are conserved between target and template. Black filled positions in the sequence alignment represent identical residues. Points represent gaps.

Figure 8

Tridimensional model of J. curcas esterase B developed by comparative modeling. Structures are colored according to the atomic charge for different pH values: 5.5, 8.0, and 9.5. Sticks represent the catalytic triad residues (Cys-78, Asp-126, and His-161) and the residues that had protonation change according to the pH values (Lys-14 and His-43).

Figure 9

Differences in the electrostatic potential of J. curcas esterase B at different pH values (5.5, 8.0, and 9.5). The catalytic triad region (Cys-78, Asp-126, and His-161) is highlighted in green. The molecular surface is colored according to the electrostatic potential, where red, white, and blue correspond to acidic, neutral, and basic potentials.

Figure 10

Esterase activity is not due to proteolytic activity. The azocasein assay (A) could not detect significant peptidase activity in the EtOH 50–80% fraction. (B) Enzymatic analysis in the presence of different peptidase inhibitors corroborated the former finding. Noticeably, the esterase activity of this fraction was enhanced by EDTA addition. *** p < 0.001.

Figure 11

Divalent cations inhibit esterase activity. (A) A dose-response relationship was observed between esterase activity and EDTA. (B) Assaying with different divalent cations corroborated this and the EDTA addition slightly reduced this impact. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 12

Esterase activity is reversibly lowered in the presence of organic solvents. Esterase activity decreased when isopropanol, acetonitrile, and ethanol were added (A) but sample reconstitution in an aqueous reaction mixture reverses this (B).