Modulation of the Catalytic Properties of Immobilized Recombinant Lipase from Thermomyces lanuginosus in the Reaction of Esterification by the Selection of an Adsorbent

Biocatalysts with lipase activity (BLAs) were prepared by adsorptive immobilization of recombinant lipase (rPichia/lip) from thermophilic microscopic fungi Thermomyces lanuginosus produced by a genetically engineered strain of methylotrophic yeast Komagataella phafii (Pichia pastoris). Supports with different physicochemical properties were used as adsorbents: mesoporous hydrophilic silica (SiO₂) and macroporous hydrophobic carbon aerogel (MCA). The enzymatic activity, substrate specificity and operational stability of BLAs were studied in the esterification of saturated fatty acids with aliphatic alcohols differing in the number of carbon atoms in the molecule from 2 to 18. Matrices of relative activities were compiled for more than 60 pairs of substrates, an acid and an alcohol, by comparing the reaction rates of the esterification under identical conditions, which allowed us to reveal differences in the specificity of adsorbed lipase depending on the chemical nature of the support. It was found that for both types of biocatalysts, rPichia/lip on SiO₂ (PLSi) and rPichia/lip on MCA (PLC), the maximum reaction rate was observed under esterification of heptanoic acid (C₇) with butyl alcohol (C₄). Under the same conditions of the synthesis of esters (20 ± 2°C, 1 bar, a mixture of hexane and diethyl ether as an organic solvent), including the synthesis of butylheptanoate, rPichia/lip adsorbed on silica showed an order of magnitude lower activity than lipase adsorbed on carbon aerogel. The catalytic constants, equal to 3.7 s–¹ and 1.1 × 10² s–¹, respectively, differed by 30 times. It was found that esters of short chain fatty acids C₄–C₇ and ethyl alcohol C₂ were synthesized 2–3 times faster using the hydrophobic PLC type than using the hydrophilic PLSi type of BLAs. At the same time, esters of high-molecular-weight acids С₉, C₁₀, С₁₈ and alcohols С₈–С₁₆ with pronounced hydrophobicity were synthesized 1.5–2 times faster using of PLSi type BLAs. The operational stability of the biocatalysts was quite high: the prepared BLAs retained 82–99% of their initial activity after more than 30 reaction cycles, while the duration of each cycle to reach an acid conversion above 85% was several hours (4–6 h).