The Amano lipase from Pseudomonas fluorescens (L-AK) was covalently immobilized on various carbon nanomaterials (functionalized single-walled carbon nanotubes and graphene oxide) and tested for biodiesel production. Using the most active lipase preparation (covalently immobilized L-AK on SwCNT_NH2 derivatized with glycerol diglycidyl ether) under optimal conditions, quasi-complete conversion (>99%) of sunflower oil was obtained after only 4 h reaction time. Moreover, the biocatalyst maintained more than 99% of its initial activity in the batch system after multiple recycling experiments.

Phenylalanine ammonia-lyases (PALs) catalyse the non-oxidative deamination of L-phenylalanine to trans-cinnamic acid, while in the presence of high ammonia concentration, the synthetically attractive reverse reaction occurs. Although they have been intensively studied, the wider application of PALs for the large scale synthesis of non-natural amino acids is still rather limited, mainly due to the decreased operational stability of PALs under the high ammonia concentration conditions of ammonia addition. Herein, we describe the development of a highly stable and active immobilized PAL-biocatalyst obtained through site-specific covalent immobilization onto single-walled carbon nanotubes (SWCNTs), employing maleimide/thiol coupling of engineered enzymes containing surficial Cys residues. The immobilization method afforded robust biocatalysts (by strong covalent attachment to the support) and allowed modulation of enzymatic activity (by proper selection of binding site, controlling the orientation of the enzyme attached to the support). The novel biocatalysts were investigated in PAL-catalyzed reactions, focusing on the synthetically challenging ammonia addition reaction. The optimization of the immobilization (enzyme load) and reaction conditions (substrate : biocatalyst ratio, ammonia source, reaction temperature) involving the best performing biocatalyst SWCNT NH₂ -SS-PcPAL was performed. The biocatalyst, under the optimal reaction conditions, showed high catalytic efficiency, providing excellent conversion (c ∼90% in 10 h) of cinnamic acid into L-Phe, and more importantly, possesses high operational stability, maintaining its high efficiency over >7 reaction cycles. Moreover, the site-specifically immobilized PcPAL L134A/S614C and PcPAL I460V/S614C variants were successfully applied in the synthesis of several L-phenylalanine analogues of high synthetic value, providing perspectives for the efficient replacement of classical synthetic methods for L-phenylalanines with a mild, selective and eco-friendly enzymatic alternative.

Tailored immobilized lipases from Candida antarctica B and Pseudomonas fluorescens, with improved thermal and operational stability, were prepared through fine tuning of the structure of the sol–gel matrix, using various binary or ternary precursor mixtures for the EKR of various chiral heteroaromatic secondary alcohols with benzofuran, benzo[b]thiophen, phenothiazine and 2-phenylthiazol moieties. The operational stability in batch process was studied for five selected systems by performing reuse experiments, using the conversion, enantiomeric excesses and enantiomeric ratio as parameters, demonstrating the dependence of the sol–gel lipase preparate performance on the structure of both biocatalyst and substrate. The resolution of the benzofuranic substrates with the best performing biocatalysts was studied in continuous-flow mode, using the productivity as a criterion. The specific reaction rates under continuous-flow operation (r_flow) were higher than those obtained in batch mode (r_batch) in both cases, sustaining its usefulness for further process development.

A new and efficient lipase-mediated kinetic resolution (KR)–click-reaction-based procedure is presented for the production of both enantiomers of various 1-(hetero)aromatic ethanols applying enzymatic acylation with 2-(prop-2-yn-1-yloxy)acetyl esters. Click-reaction-based in situ derivatization of the (R)-2-(prop-2-yn-1-yloxy)acetate produced from the reacting enantiomer in the KR step enabled facile and efficient separation of the derivatized (R)-ester and the (S)-alcohol by simple extractions and pH adjustments from the reaction mixture. A mild and efficient enzymatic ethanolysis of the derivatized (R)-ester allowed almost quantitative recovery of the (R)-alcohol without a detectable decrease in enantiopurity. The process could provide, from racemates of various 1-(hetero)aromatic ethanols, almost quantitatively both enantiomers without chromatographic separation of the KR products.