Oleate Hydratase from Lactobacillus rhamnosus ATCC 53103: A FADH2-Dependent Enzyme with Remarkable Industrial Potential

Recently, we described the preparation of the recombinant oleate hydratase from Lactobacillus rhamnosus ATCC 53103. We observed that the purified C-terminal His-tagged enzyme was completely inactive and the catalytic activity was partially restored only in presence of a large amount of flavin adenine dinucleotide (FAD). In the present work, we assess that this hydratase in the presence of the reduced form of flavin adenine dinucleotide (FADH2) is at least one hundred times as active as in the presence of the same concentration of FAD. By means of two different biochemical processes, we demonstrated unambiguously that oleate hydratase from Lactobacillus rhamnosus ATCC 53103 is a FADH2-dependent enzyme. As a first relevant application of this discovery, we devised a preparative procedure for the stereoselective synthesis of (R)-10-hydroxystearic acid. Accordingly, the hydration of oleic acid (up to 50 g/L) is performed on a multigram scale using the recombinant hydratase and FADH2 generated in situ as cofactor. The produced (R)-10-hydroxystearic acid (ee > 97%) precipitates from the reaction solvent (water/glycerol/ethanol) and is conveniently recovered by simple filtration (>90% yield).

Investigation of an Organogel by Micro-Differential Scanning Calorimetry: Quantitative Relationship between the Shapes of the Thermograms and the Phase Diagram

The phase diagrams of organogels are necessary for applications and fundamental aspects, for instance to understand their thermodynamics. Differential scanning calorimetry is one of the techniques implemented to map these diagrams. The thermograms of organogels upon heating show broad endotherms, increasing gradually to a maximum, at a temperature Tmax, and decreasing back to the baseline, sometimes 10 °C above. This broadening can lead to uncertainty in determining the molar enthalpies and the melting temperatures Tm of the gels. Herein, we have measured the thermograms of the 12-hydroxystearic acid/nitrobenzene gels for weight fractions ranging from 0.0015 to 0.04. Compared with transition temperatures measured by other techniques, the inflection points of the thermograms provide a measurement of Tm with less bias than Tmax. The phase diagram explains why the molar melting enthalpies derived from the thermograms for samples of low concentration are lower than expected. The shapes of the heat flows below the peak correlate quantitatively with the diagrams: after suitable correction and normalization, the integral curves superimpose with the phase diagram in their ascending branch and reach a plateau when the gel is fully melted. The shape of the thermograms upon cooling is also qualitatively explained within the frame of the diagrams.

(R)-10-Hydroxystearic Acid: Crystals vs. Organogel

The chiral (R)-10-hydroxystearic acid ((R)-10-HSA) is a positional homologue of both (R)-12-HSA and (R)-9-HSA with the OH group in an intermediate position. While (R)-12-HSA is one of the best-known low-molecular-weight organogelators, (R)-9-HSA is not, but it forms crystals in several solvents. With the aim to gain information on the structural role of hydrogen-bonding interactions of the carbinol OH groups, we investigated the behavior of (R)-10-HSA in various solvents. This isomer displays an intermediate behavior between (R)-9 and (R)-12-HSA, producing a stable gel exclusively in paraffin oil, while it crystallizes in other organic solvents. Here, we report the X-ray structure of a single crystal of (R)-10-HSA as well as some structural information on its polymorphism, obtained through X-ray Powder Diffraction (XRPD) and Infrared Spectroscopy (IR). This case study provides new elements to elucidate the structural determinants of the microscopic architectures that lead to the formation of organogels of stearic acid derivatives.

A Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) Method for the Determination of Free Hydroxy Fatty Acids in Cow and Goat Milk

A liquid chromatography–high resolution mass spectrometry (LC-HRMS) method for the direct determination of various saturated hydroxy fatty acids (HFAs) in milk was developed for the first time. The method involves mild sample preparation conditions, avoids time-consuming derivatization procedures, and permits the simultaneous determination of 19 free HFAs in a single 10-min run. This method was validated and applied in 17 cow milk and 12 goat milk samples. This work revealed the existence of various previously unrecognized hydroxylated positional isomers of palmitic acid and stearic acid in both cow and goat milk, expanding our knowledge on the lipidome of milk. The most abundant free HFAs in cow milk were proven to be 7-hydroxystearic acid (7HSA) and 10-hydroxystearic acid (10HSA) (mean content values of 175.1 ± 3.4 µg/mL and 72.4 ± 6.1 µg/mL in fresh milk, respectively). The contents of 7HSA in cow milk seem to be substantially higher than those in goat milk.