Immobilization of recombinant Thermomyces lanuginosus lipase (designated as rPichia/lip) was carried out by moisture capacity impregnation of mesoporous silica granules followed by drying, and forcible adsorption of enzyme occurred. Eventually prepared lipase-active heterogeneous biocatalysts were systematically studied for enzymatic esterification performed at ambient conditions (20 ± 2 °C, 1 bar) in unconventional anhydrous media of organic solvents such as hexane and diethyl ether. The saturated fatty acids differing in the number of carbon atoms (C2–C10, C18), and aliphatic alcohols differing in the structure of the molecules, namely both the number of carbon atoms (C2–12, C16), and the isomerism of the carbon skeleton (n- and iso-), and OH-group position (prim-, sec-, tert-) were studied as substrates for enzymatic esterification. The specificity of the heterogeneous enzymatic esterification was determined by comparing the reaction rates for various pairs of substrates; and the matrix of relative units of activities was composed. The immobilized on silica rPichia/lip was found to have sufficiently wide specificity toward saturated fatty acids and aliphatic alcohols. High reaction rates were measured in esterification of fatty acids and primary n- and iso-aliphatic alcohols possessing more than four carbon atoms in the molecules. The enanthic acid (heptanoic, C7:0) reacted with butanol (C4) with the highest rate; and the kinetic parameters such as Michaelis constant (KM) for acid and maximal reaction rate (Vmax) were determined under the studied conditions of esterification. Substrates containing aromatic residues did not participate in esterification. The lipase-active heterogeneous biocatalysts possessed considerably high operational stability, and the catalytic activity was completely retained for several tens of reaction cycles in a periodic batch process of low-temperature synthesis of various fatty acid esters.
Immobilization by impregnation Lipase Heterogeneous biocatalysts Esterification of fatty acids
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Authors are grateful to Maria B. Pykhtina for cultivation of the rPichia/lip strain-producer for this research. This work was conducted within the framework of the budget Project No. AAAA-A17-117,041,710,075-0.