RESUMO
When pDHA4, a vector carrying all five pfaA-pfaE genes responsible for docosahexaenoic acid (DHA; 22:6) biosynthesis in Moritella marina MP-1, was coexpressed in Escherichia coli with the individual pfaA-pfaD genes for eicosapentaenoic acid (EPA; 20:5) biosynthesis from Shewanella pneumatophori SCRC-2738, both polyunsaturated fatty acids were synthesized only in the recombinant carrying pfaB for EPA synthesis. Escherichia coli coexpressing a deleted construct comprising pfaA, pfaC, pfaD and pfaE for EPA and pfaB for DHA produced EPA and DHA. Both EPA and DHA were detected in bacteria that inherently contained pfa genes for DHA. These results suggest that PfaB is the key enzyme determining the final product in EPA or DHA biosynthesis.
Assuntos
Proteínas de Bactérias/metabolismo , Ácidos Docosa-Hexaenoicos/química , Ácido Eicosapentaenoico/química , Ácidos Graxos Insaturados/biossíntese , Regulação Bacteriana da Expressão Gênica , Moritella/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Ácidos Docosa-Hexaenoicos/metabolismo , Ácido Eicosapentaenoico/biossíntese , Ácidos Graxos Insaturados/química , Regulação da Expressão Gênica , Moritella/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
An original gas chromatography/mass spectrometry method for quantifying trace amounts of ricinoleic acid (12-hydroxy-cis-9-octadecenoic acid) is detailed. Data are presented on trace amounts of ricinoleic acid found in several common vegetable oils and oils extracted from common oil seeds: e.g., ca. 30 ppm in commercial olive oil was the lowest amount; and ca. 2,690 ppm in oil extracted from cottonseeds was the highest amount.
Assuntos
Óleos de Plantas/química , Ácidos Ricinoleicos/análise , Sementes/química , Calibragem , Cromatografia Gasosa-Espectrometria de MassasRESUMO
It was found that weakly polar columns, routinely used in capillary GC for analyzing sterols, food additives, etc., can also be used for separating fatty acid methyl esters (FAMEs). On these columns, FAMEs elute in the order of their unsaturation. The equivalent chain-length value of methyl 22:6 is below 23.00. This means FAMEs within a carbon chain length, having up to six double bonds, elute before the next (one carbon longer) saturated FAME elutes. Peak identification is easy. Weakly polar columns are compatible in both GC and GC/MS systems.
Assuntos
Cromatografia Gasosa/métodos , Ácidos Graxos/química , Ácidos Graxos/isolamento & purificação , Cromatografia Gasosa/instrumentação , Ésteres , Cromatografia Gasosa-Espectrometria de MassasRESUMO
A convenient method was developed for preparation of FAME in small amounts from glycerolipids of blood or breast milk. Initially, 0.04-0.06 mL blood or breast milk was spotted onto a small piece of filter paper (1.5 x 1.5 cm) that had been washed with acetone containing 0.05% 2,6-di-tert-butyl-p-cresol (BHT). Each piece, once it had dried, was put in a small test tube, to which 2 mL hexane and 0.2 mL 2 M KOH/methanol were added. After vigorous mixing or sonication for 2 min at room temperature, the solution was neutralized or acidified by the addition of a few drops of acetic acid. To the solution was added 2 mL H2O, and then the hexane layer that separated was concentrated to dryness in vacuo. The FAME obtained were analyzed by GC. The method was applicable to the analysis of a large number of blood and breast milk samples, and the arachidonate/(eicosapentaenoate + docosahexaenoate) ratios could be determined rapidly.
