A Thermostable Fatty Acid Hydratase from <i>Marinitoga Piezophila</i> with Low Temperature Optimum and Broad Product Scope

Hydroxy fatty acids (HFAs) are valuable derivatives of fatty acids (FAs) with interesting bioactivities. Moreover, they are used in materials industry as additives, starting materials and surfactants. HFAs can be produced from FAs either by hydroxylation or by hydration reaction, if FA is unsaturated, using chemical or enzymatic methods. FA hydratases (FAHs) are promising biocatalysts for HFA synthesis thanks to their non-redox nature, high efficiency and excellent selectivity. Although FAHs are relatively more stable compared to other enzymes like monooxygenases, their tolerance to high temperature and organic solvents is limited. In this study, we characterized a rare thermostable FAH ortholog through database gene mining. This enzyme from Marinitoga Piezophila, a thermo-piezophilic organism, displayed novel properties, including broad substrate scope, broad pH range, unique regioselectivity and excellent thermostability (retaining full activity after 30 min incubation at 70 °C); however, quite interestingly, its temperature optimum was at 20 °C. Although kinetic parameters indicate a less efficient enzyme compared to some other FAHs, the enzyme can reach over 90% conversion within 24 h at a 100 mL scale reaction containing 1.75 mM substrate. Furthermore, mutagenesis of key active-site residues indicated a possibly different reaction mechanism compared to earlier proposed mechanisms.