Identification of regioisomers and enantiomers of triacylglycerols in different yeasts using reversed‐ and chiral‐phase LC–MS

Abstract

LC with atmospheric pressure chemical ionization (ACPI) MS with RP and chiral phase was used for separation of triacylglycerols (TAGs) from yeasts of the genera Candida, Kluyveromyces, Rhodotorula, Saccharomyces, Torulospora, Trichosporon, and Yarrowia. Chiral LC-APCI-MS is based on using two columns in series packed with a 3,5-dimethylphenyl carbamate modified β-cyclodextrin chiral phase. All regioisomers and enantiomers of TAGs containing one to five double bonds were separated. Molecular species of TAGs, i.e. regioisomers and enantiomers, were identified and quantified by MS/MS. Among the 94 identified TAGs, the most abundant were triolein, oleopalmitoleoolein, and dipalmitoleoolein. In strains producing palmitoleic acid in amounts >25% of total fatty acids (FAs), this acid, or unsaturated FA is bound in sn-1. In strains containing palmitoleic acid at 10-25% total FAs this acid is mainly bound in sn-3, saturated FA being bound in sn-1. Strains containing <10% palmitoleic acid form preferentially symmetrical TAGs.

New yeast-based approaches in production of palmitoleic acid

“…It is however true that our changes in culture conditions were substantial; for instance, the use of VFA resulted in the formation of heptadecenoic acid (17:1) (Rezanka et al, 2015). As reported in the literature (Rezanka et al, 2013a;Zheng et al, 2012), the synthesis of FA is strongly influenced by the presence of propionic acid as a substrate. Its presence caused an increase in heptadecenoic acid (17:1) to more than 30% of total FA in K. polysporus, T. delbrueckii, C. krusei, Y. lipolytica and T. cutaneum.…”

Separation of Enantiomeric Triacylglycerols by Chiral‐Phase HPLC

“…The organisms used included snow alga-Chloromonas pichinchae [19] and yeast-Kluyveromyces polysporus [13]. The alcohol-water mixture of the frozen cells was cooled, one part chloroform was added and the lipids were extracted for 30 min.…”

“…TAG (*1 mg/mL in methanol) were chromatographed on two Astec cyclobond TM I 2000 DMP (3,5-dimethyl-phenylcarbamate modified b-cyclodextrin) chiral LC columns (5 lm, 25 cm 9 4.6 mm) connected in series, from Sigma. Mobile phase was a gradient from 90 % of A and 10 % of B at 0 min to 50 % of A and 50 % of B during 180 min, and then isocratically for 30 min, where A is hexane and B is hexane-2-propanol (99:2, v/v) mixture [13]. The flow rate, column temperature, and injection volume were 0.5 mL/min, 25°C, and 10 lL, respectively.…”

“…Their selectivity can be improved by using, e.g., tris-(3,5-dimethyl-phenylcarbamate) covalently bound to cyclodextrin or cellulose. Owing to the different conformation of cyclodextrin (cyclic conformation) and cellulose (linear conformation) the two chiral stationary phases equipped with the same chiral selector exhibit different enantioselectivities [12], modified cyclodextrin showing a higher chiral selectivity than cellulose [5,[13][14][15].…”

The Updated Bottom Up Solution Applied to Atmospheric Pressure Photoionization and Electrospray Ionization Mass Spectrometry

“…Similarly, in Type III TAG, although the FA in the sn-2 position can be identified, ABC cannot be distinguished from its enantiomer CBA. Enantiomers can only be differentiated using chiral chromatography (CC) [15][16][17][18]. Even so, not all isomers may be separable using CC, depending on the column and conditions used.…”