Castor seed triacylglycerols (TAGs) contain 90% ricinoleate (12-hydroxy-oleate) which has numerous industrial applications. Due to the presence of the toxin ricin and potent allergenic 2S albumins in the seed, it is desirable to produce ricinoleate from temperate oilseeds. To identify regulatory genes or genes for enzymes that may up-regulate multiple activities or entire pathways leading to the ricinoleate and TAG synthesis, we have analyzed expression profiles of 12 castor genes involved in fatty acid and TAG synthesis using quantitative reverse transcription-polymerase chain reaction technology. A collection of castor seeds with well-defined developmental stages and morphologies was used to determine the levels of mRNA, ricinoleate and TAG. The synthesis of ricinoleate and TAG occurred when seeds progressed to stages of cellular endosperm development. Concomitantly, most of the genes increased their expression levels, but showed various temporal expression patterns and different maximum inductions ranging from 4- to 43,000-fold. Clustering analysis of the expression data indicated five gene groups with distinct temporal patterns. We identified genes involved in fatty acid biosynthesis and transport that fell into two related clusters with moderate flat-rise or concave-rise patterns, and others that were highly expressed during seed development that displayed either linear-rise or bell-shaped patterns. Castor diacylglycerol acyltransferase 1 was the only gene having a higher expression level in leaf and a declining pattern during cellular endosperm development. The relationships among gene expression, cellular endosperm development and ricinoleate/TAG accumulation are discussed.

The aim of this work was to optimize a supercritical fluid extraction (SFE)/enzymatic reaction process for the determination of the fatty acid composition of castor seeds. A lipase from Candida antarctica (Novozyme 435) was used to catalyze the methanolysis reaction in supercritical carbon dioxide (SC-CO(2)). A Box-Behnken statistical design was used to evaluate effects of various values of pressure (200-400 bar), temperature (40-80 degrees C), methanol concentration (1-5 vol %), and water concentration (0.02-0.18 vol %) on the yield of methylated castor oil. Response surfaces were plotted, and these together with results from some additional experiments produced optimal extraction/reaction conditions for SC-CO(2) at 300 bar and 80 degrees C, with 7 vol % methanol and 0.02 vol % water. These conditions were used for the determination of the castor oil content expressed as fatty acid methyl esters (FAMEs) in castor seeds. The results obtained were similar to those obtained using conventional methodology based on solvent extraction followed by chemical transmethylation. It was concluded that the methodology developed could be used for the determination of castor oil content as well as composition of individual FAMEs in castor seeds.

The objective of this study was to find the optimal parameters for lipase-catalyzed methanolysis of triricinolein to produce 1,2(2,3)-diricinolein. Four different immobilized lipases were tested, Candida antarctica type B (CALB), Rhizomucor miehei (RML), Pseudomonas cepacia (PCL), and Penicillium roquefortii (PRL). n-Hexane and diisopropyl ether (DIPE) were examined as reaction media at three different water activities (a(w)), 0.11, 0.53, and 0.97. The consumption of triricinolein and the formation of 1,2(2,3)-diricinolein, methyl ricinoleate, and ricinoleic acid were followed for up to 48 h. PRL gave the highest yield of 1,2(2,3)-diricinolein. Moreover, this lipase showed the highest specificity for the studied reaction, i.e., high selectivity for the reaction with triricinolein but low for 1,2(2,3)-diricinolein. Recoveries of 93 and 88% DAG were obtained using PRL in DIPE at a(w) of 0.11 and 0.53, respectively. Further, NMR studies showed that a higher purity of the 1,2(2,3)-isomer vs. the 1,3-isomer was achieved at higher a(w) (88% at a(w) = 0.53), compared to lower a(w) (71% at a(w) = 0.11). The DAG obtained was acylated by the DAG acyltransferase from Arabidopsis thaliana. Therefore, this enzymatic product is a useful enzyme substrate for lipid biosynthesis. Accordingly, the use of PRL in DIPE at a(w) 0.53 is considered optimal for the synthesis of 1,2(2,3)-diricinolein from triricinolein.

Because castor produces oil with a high content of hydroxyl FA (90% ricinoleate), we were interested in determining the flexibility of castor seed microsomes in incorporating other hydroxyl FA into castor oil. To this end, we incubated the [ 14 C]-labeled 12:0 FA laurate (La), 11-hydroxylaurate, and 12-hydroxylaurate with castor microsomes that were capable of synthesizing castor oil. The molecular species of PC and acylglycerols (AG) incorporating these nonendogenous FA of castor were identified by reversed-phase C 8 and C 18 HPLC, respectively. [ 14 C]Laurate was incorporated into the molecular species of PC and AG at levels of 10 and 4%, respectively, that of [ 14 C]ricinoleate. Similar to those from the incorporation of six [ 14 C]FA reported previously [ricinoleate (R), oleate (O), linoleate (L), linolenate (Ln), stearate (S), and palmitate (P)], the molecular species of PC incorporating [ 14 C]laureate were LLa-PC > PLa-PC > OLa-PC > LnLa-PC > SLa-PC > RLa-PC. The molecular species of AG incorporating [ 14 C]laurate were RRLa > LaLa > RLa > RLLa > ROLa > LOLa > LLLa > LLa > LLnLa > RSLa > OOLa. The retention times for lipids incorporating laurate were similar to those of lipids incorporating linolenate, because the equivalent carbon numbers of laurate and linolenate are the same. Relative retention times of the molecular species of PC and AG containing laurate are also reported here. The incorporation of 11-hydroxylaurate and 12-hydroxylaurate into PC and AG was not detected. Paper no. J10984 in JAOCS 82, 495-499 (2005).The presence of a hydroxyl group on ricinoleate (12-hydroxyoleate, C 18 long-chain FA) underlies many industrial uses such as the manufacture of lithium grease, plastics, coatings, and cosmetics. Castor oil contains 90% of its FA as ricinoleate (1) and is the only commercial source of ricinoleate. We have recently studied the incorporation of six endogenous long-chain FA-ricinoleate (R), stearate (S), oleate (O), linoleate (L), linolenate (Ln), and palmitate (P)-into PC and acylglycerols (AG) in castor microsomes (2,3). Among the six FA, ricinoleate was incorporated into TAG most effectively. Since the position of the hydroxyl group on ricinoleate (C 18 ) is the same as on 12-hydroxylaurate (C 12 ), castor microsomal incorporation of radiolabeled laurate (La), 11-hydroxylaurate, and 12-hydroxylaurate into PC and AG was of interest to determine the flexibility of castor in producing other hydroxyl FA. Laurate is mainly found in lauric oils such as coconut and palm kernel and is used in food products and in the manufacture of soaps and detergents. The mediumchain hydroxyl FA 11-hydroxylauric acid and 12-hydroxylauric acid can be used in the manufacture of polymers. EXPERIMENTAL PROCEDURESMicrosomal incubation. Microsomes from castor bean were prepared as described previously, and microsomal incubations were scaled up 10 times with the addition of CoA-SH to generate suitable amounts of material for analysis (4,5). The incubation mixture, in a total volume of 10 mL, included sodium phosphate buf...