RESUMEN
We searched for quantitative trait loci (QTL) associated with the palm oil fatty acid composition of mature fruits of the oil palm E. guineensis Jacq. in comparison with its wild relative E. oleifera (H.B.K) Cortés. The oil palm cross LM2T x DA10D between two heterozygous parents was considered in our experiment as an intraspecific representative of E. guineensis. Its QTLs were compared to QTLs published for the same traits in an interspecific Elaeis pseudo-backcross used as an indirect representative of E. oleifera. Few correlations were found in E. guineensis between pulp fatty acid proportions and yield traits, allowing for the rather independent selection of both types of traits. Sixteen QTLs affecting palm oil fatty acid proportions and iodine value were identified in oil palm. The phenotypic variation explained by the detected QTLs was low to medium in E. guineensis, ranging between 10% and 36%. The explained cumulative variation was 29% for palmitic acid C16:0 (one QTL), 68% for stearic acid C18:0 (two QTLs), 50% for oleic acid C18:1 (three QTLs), 25% for linoleic acid C18:2 (one QTL), and 40% (two QTLs) for the iodine value. Good marker co-linearity was observed between the intraspecific and interspecific Simple Sequence Repeat (SSR) linkage maps. Specific QTL regions for several traits were found in each mapping population. Our comparative QTL results in both E. guineensis and interspecific materials strongly suggest that, apart from two common QTL zones, there are two specific QTL regions with major effects, which might be one in E. guineensis, the other in E. oleifera, which are independent of each other and harbor QTLs for several traits, indicating either pleiotropic effects or linkage. Using QTL maps connected by highly transferable SSR markers, our study established a good basis to decipher in the future such hypothesis at the Elaeis genus level.
Asunto(s)
Arecaceae/química , Arecaceae/genética , Ácidos Grasos/química , Aceites de Plantas/química , Sitios de Carácter Cuantitativo/genética , Arecaceae/clasificación , Mapeo Cromosómico/métodos , Cromosomas de las Plantas/genética , Cruzamientos Genéticos , Genes de Plantas/genética , Ligamiento Genético , Pleiotropía Genética , Genotipo , Repeticiones de Microsatélite/genética , Aceite de Palma , Fenotipo , Análisis de Componente Principal , Especificidad de la EspecieRESUMEN
Triacylglycerol (TAG) lipases have been thoroughly characterized in mammals and microorganisms, whereas very little is known about plant TAG lipases. The lipolytic activity occurring in all the laticies is known to be associated with sedimentable particles, and all attempts to solubilize the lipolytic activity of Carica papaya latex have been unsuccessful so far. However, some of the biochemical properties of the lipase from Carica papaya latex (CPL) were determined from the insoluble fraction of the latex. The activity was optimum at a temperature of 37°C and a pH of 9.0, and the specific activities of CPL were found to be 2,000 ± 185 and 256 ± 8 U/g when tributyrin and olive oil were used as substrates, respectively. CPL was found to be active in the absence of any detergent, whereas many lipases require detergent to prevent the occurrence of interfacial denaturation. CPL was inactive in the presence of micellar concentrations of Triton X-100, sodium dodecyl sulfate (SDS) and tetradecyl trimethylammonium bromide (TTAB), and still showed high levels of activity in the presence of sodium taurodeoxycholate (NaTDC) and the zwitterionic Chaps detergent. The effects of various proteases on the lipolytic activity of CPL were studied, and CPL was found to be resistant to treatment with various enzymes, except in the presence of trypsin. All these properties suggest that CPL may be a good candidate for various biotechnological applications.
Asunto(s)
Carica/enzimología , Enzimas Inmovilizadas/metabolismo , Látex/química , Lipasa/química , Detergentes/química , Lipólisis/efectos de los fármacos , Octoxinol/química , Aceite de Oliva , Aceites de Plantas/metabolismo , Dodecil Sulfato de Sodio/química , Especificidad por Sustrato , Ácido Taurodesoxicólico/química , Triglicéridos/metabolismo , TripsinaRESUMEN
An esterase (CpEst) showing high specific activities on tributyrin and short chain vinyl esters was obtained from Carica papaya latex after an extraction step with zwitterionic detergent and sonication, followed by gel filtration chromatography. Although the protein could not be purified to complete homogeneity due to its presence in high molecular mass aggregates, a major protein band with an apparent molecular mass of 41 kDa was obtained by SDS-PAGE. This material was digested with trypsin and the amino acid sequences of the tryptic peptides were determined by LC/ESI/MS/MS. These sequences were used to identify a partial cDNA (679 bp) from expressed sequence tags (ESTs) of C. papaya. Based upon EST sequences, a full-length gene was identified in the genome of C. papaya, with an open reading frame of 1029 bp encoding a protein of 343 amino acid residues, with a theoretical molecular mass of 38 kDa. From sequence analysis, CpEst was identified as a GDSL-motif carboxylester hydrolase belonging to the SGNH protein family and four potential N-glycosylation sites were identified. The putative catalytic triad was localised (Ser(35)-Asp(307)-His(310)) with the nucleophile serine being part of the GDSL-motif. A 3D-model of CpEst was built from known X-ray structures and sequence alignments and the catalytic triad was found to be exposed at the surface of the molecule, thus confirming the results of CpEst inhibition by tetrahydrolipstatin suggesting a direct accessibility of the inhibitor to the active site.
Asunto(s)
Carica/enzimología , Esterasas/química , Esterasas/metabolismo , Látex/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Electroforesis en Gel de Poliacrilamida , Esterasas/antagonistas & inhibidores , Esterasas/aislamiento & purificación , Concentración de Iones de Hidrógeno/efectos de los fármacos , Lactonas/farmacología , Lipasa/antagonistas & inhibidores , Lipólisis/efectos de los fármacos , Espectrometría de Masas , Modelos Moleculares , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Orlistat , Filogenia , Alineación de Secuencia , Análisis de Secuencia de Proteína , Solubilidad/efectos de los fármacos , Especificidad por Sustrato/efectos de los fármacosRESUMEN
Intragastric fat digestion was investigated by analyzing the products of lipolysis and the gastric lipase (HGL) levels of premature infants fed with a formula enriched with medium chain triglycerides (MCT) and those of infants fed with human milk. Infants were fed using a gastric tube and the gastric contents were aspirated twice a day for 5 d, before and at various times after gavage feeding. HGL levels were measured using the pHstat technique. After extraction, lipids were separated and quantified using thin-layer chromatography coupled to a flame ionization detector. Fatty acid methyl esters were analyzed by gas chromatography. HGL concentration increased during digestion, reaching 77.4 +/- 43.1 microg/mL (around 75% of those recorded in adults). Mean HGL output was 115 +/- 43 microg for 3 h and the overall intragastric lipolysis was 6.1 +/- 2.6%. Although the formula was enriched with octanoic and decanoic acid, the main fatty acids released in the stomach were palmitic (C16:0, 17.03 +/- 0.23% wt/wt) and oleic (C18:1 n-9, 28.23 +/- 1.26% wt/wt) acid. Similar results were obtained with infants fed with human milk. MCT supplementation has no quantitative or qualitative effects on the intragastric lipolysis, which is not higher in premature infant than in adults.
Asunto(s)
Fórmulas Infantiles/metabolismo , Recien Nacido Prematuro , Estómago/fisiología , Triglicéridos/metabolismo , Mucosa Gástrica/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Recién NacidoRESUMEN
The enzymatic properties of Plumeria rubra latex have been evaluated for the first time, showing a high activity in both hydrolysis and synthesis reactions, and compared to the biocatalytic behavior of babaco (Vasconcellea x Heilbornii cv.) latex. Both biocatalysts have been optimized by studying the various parameters that influence reaction kinetics. The optimum temperatures for hydrolysis reactions were 50 and 55 degrees C for babaco and Plumeria, respectively. The optimum pH for babaco latex was 7, whereas for Plumeria latex, two optimal pH values (4 and 7) were observed. With regard to esterification and acyl transfer reactions such as alcoholysis and interesterification, the influence of thermodynamic water activity on reaction yields was determined and correlated with water sorption and desorption isotherms. When babaco latex is used as a biocatalyst, optimal synthesis reaction yields are obtained when the enzymatic extract is stabilized at a water activity value of 0.38, which corresponds to a water content of 5.7%. This optimal level of hydration is located on the linear portion of the biocatalyst's sorption isotherm, where the water molecules exhibit high-energy interactions with the protein network. In synthesis reactions (esterification, alcoholysis, and interesterification) biocatalyzed by Plumeria latex, correlation between best reaction yields and water activity cannot be done. Indeed, the sorption isotherm plot has an atypical shape, indicating that water might be trapped in the latex matrix and, consequently, that the water content of the biocatalyst is highly dependent on the hydration history of the latex.
Asunto(s)
Apocynaceae/enzimología , Lipasa/metabolismo , Magnoliopsida/enzimología , Extractos Vegetales/metabolismo , Acilación , Esterificación , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Lípidos/análisis , Aceites de Plantas/metabolismo , Aceite de Girasol , Temperatura , TermodinámicaRESUMEN
In this study, we designed a specific, continuous, and sensitive UV spectrophotometric lipase assay using natural triacylglycerols (TAGs) from the Aleurites fordii seed oil (tung oil). alpha-Eleostearic acid (9,11,13-cis, trans,trans-octadecatrienoic acid) is the main fatty acid component (it accounts for up to 70%) of the TAGs from tung oil. The conjugated triene present in alpha-eleostearic acid constitutes an intrinsic chromophore, which confers strong UV absorption properties on both the free fatty acid and the TAGs from tung oil. The lipase assay is based on the difference between the apparent molar extinction coefficients of the two types of alpha-eleostearic acid present, that which is esterified into TAGs and that which is released into the reaction medium. This difference is responsible for the variations in the UV absorption spectrum of the reaction medium occurring upon enzymatic TAGs hydrolysis. Using the purified lipase from Thermomyces lanuginosa (TLL) and the detergent sodium taurodeoxycholate (NaTDC, 4 mM), it was established that the most suitable method of measuring lipolysis consisted of monitoring the decrease in the OD at 292 nm, which was linear with time and proportional to the amount of lipase added. In order to be able to estimate the specific activity of TLL, we determined an apparent molar extinction coefficient of alpha-eleostearic acid (epsilon = 13,900 M(-1) cm(-1)) under the assay conditions. Amounts of pure TLL as small as 1 ng can be easily detected in the presence of 4 mM NaTDC. Interestingly, the NaTDC concentration can be decreased as far as 0.05 mM. In comparison with other well-known methods of lipase assay, the detection limit of this new method is 100-fold lower than with the pH-stat method and similar to that of a fluorescent assay recently developed at our laboratory.
Asunto(s)
Ascomicetos/enzimología , Euphorbiaceae/química , Lipasa/análisis , Espectrofotometría Ultravioleta/métodos , Triglicéridos/metabolismo , Bacillus subtilis/enzimología , Ácidos Grasos no Esterificados/química , Humanos , Hidrólisis , Cinética , Lipasa/metabolismo , Páncreas/enzimología , Proteínas Recombinantes/metabolismo , Semillas/química , Ácido Taurocólico/química , Ácido Taurocólico/metabolismo , Triglicéridos/químicaRESUMEN
Adsorption and desorption isotherms of two commercial enzyme preparations of papain and bromelain were determined with a Dynamic Vapor System. The Guggenheim-Anderson-deBoer (GAB) modeling of the obtained sorption isotherms allowed the definition of different levels of hydration of those samples. Afterward, these enzyme preparations were used as biocatalysts in water and solvent-free esterification and alcoholysis reactions. The evolution of the obtained fatty acid ester level as a function of the initial hydration level of the biocatalyst, i.e., thermodynamic water activity (a(w)) and water content, was studied. The results show an important correlation between the initial hydration level of the biocatalyst and its catalytic activity during the lipase-catalyzed synthesis reactions. Thus, the Carica papaya lipase (crude papain preparation) catalytic activity is highly dependent on the biocatalyst hydration state. The optimized synthesis reaction yield is obtained when the a(w) value of the enzyme preparation is stabilized at 0.22, which corresponds to 2% water content. This optimal level of hydration occurs on the linear part of the biocatalyst's sorption isotherm, where the water molecules can form a mono- or multiple layer with the protein network. The synthesis reaction yield decreases when the a(w) of the preparation is higher than 0.22, because the excess water molecules modify the system equilibrium leading to the reverse and competitive reaction, i.e., hydrolysis. These results show also that an optimal storage condition for the highly hydrophilic crude papain preparation is a relative humidity strictly lower than 70% to avoid an irreversible structural transition leading to a useless biocatalyst. Concerning the bromelain preparation, no effect of the hydration level on the catalytic activity during esterification reactions was observed. This biocatalyst has too weak a catalytic activity which makes it difficult to observe any differences. Furthermore, the bromelain preparation is far more hydrophobic as it adsorbs only 18 g of water per 100 g of dry material at a(w) around 0.90. No deliquescence of this enzymatic preparation is observed at this a(w) value.