Lipase-active heterogeneous biocatalysts for enzymatic synthesis of short-chain aroma esters

Abstract

The lipase-active heterogeneous biocatalysts were prepared by adsorptive immobilization of Thermomyces lanuginosus lipase (designated as rPichia/lip) produced by authoring recombinant Pichia pastoris strain. Biocatalysts of LipoCarb type were prepared by spontaneous adsorption of rPichia/lip on the authoring macroporous carbon aerogel. Biocatalysts of LipoSil type were prepared by forced adsorption of rPichia/lip on mesoporous silica. The prepared biocatalysts were studied carefully in the esterification of short-chain fatty acids (butyric C4, C5–C7) and aliphatic alcohols (ethanol C2, C3–C5, C8) in order to produce valuable fruity-smelling esters. Biocatalytic properties, such as enzymatic activity, substrates specificity, and operational stability, were found to depend on chemical nature of both the adsorbents and organic solvents, primarily on their polarity. When comparing the activity in the synthesis of n-butyl heptanoate, it was found that the LipoCarb biocatalysts demonstrated activity an order of magnitude higher than LipoSil type. The Michaelis-Menten kinetic constants differed by a factor of 30, e.g. 1.1·102 s−1 and 3.7 s−1, respectively. The operational stability of the lipase-active biocatalysts was sufficiently high; rPichia/lip adsorbed on carbon aerogel retained till 96% of the initial activity after 29 reaction cycles, whereas rPichia/lip adsorbed on silica completely retained the activity after 38 reaction cycles of esterification under optimal reaction conditions selected. The prepared biocatalysts of LipoCarb and LipoSil types were promising for the Green enzymatic synthesis of fragrance and aroma esters under very mild ambient conditions (20±2 °C, 1 bar).