Production of Thermostable Lipase by Thermomyces lanuginosus on Solid-State Fermentation: Selective Hydrolysis of Sardine Oil

A naturally immobilized biocatalyst with lipase activity was produced by Thermomyces lanuginosus on solid-state fermentation with perlite as inert support. Maxima lipase activities (22 and 120 U/g of dry matter, using p-nitrophenyl octanoate and trioctanoine, respectively, as substrates) were obtained after 72 h of solid culture, remaining nearly constant up to 120 h. Maxima lipase activity was found at 60 to 85 °C and pH 10. The biocatalyst was stable at 60 °C for at least 4 h of incubation and a pH from 7 to 10. Energy values of activation and deactivation of lipase were of 26 and 6.7 kJ/mol, respectively. The biocatalyst shows high selectivity for the release of the omega-3 polyunsaturated fatty acids, eicosapentaenoic (EPA) and docosahexaenoic acids (DHA), during the hydrolysis of sardine oil. The EPA/DHA ratio (16:6) released by this biocatalyst was superior to that obtained with the commercial preparations of T. lanuginosus.     

A Semi-Analytical Solution for Countercurrent Spontaneous Imbibition in Water-Wet Fractured Reservoirs

Transport in Porous Media - Countercurrent spontaneous imbibition (SI) is an important flow mechanism for oil recovery in fractured reservoirs during waterflooding. SI plays a key role in the...     

Co-immobilized Phosphorylated Cofactors and Enzymes as Self-Sufficient Heterogeneous Biocatalysts for Chemical Processes

Enzyme cofactors play a major role in biocatalysis, as many enzymes require them to catalyze highly valuable reactions in organic synthesis. However, the cofactor recycling is often a hurdle to implement enzymes at the industrial level. The fabrication of heterogeneous biocatalysts co-immobilizing phosphorylated cofactors (PLP, FAD⁺, and NAD⁺) and enzymes onto the same solid material is reported to perform chemical reactions without exogeneous addition of cofactors in aqueous media. In these self-sufficient heterogeneous biocatalysts, the immobilized enzymes are catalytically active and the immobilized cofactors catalytically available and retained into the solid phase for several reaction cycles. Finally, we have applied a NAD⁺-dependent heterogeneous biocatalyst to continuous flow asymmetric reduction of prochiral ketones, thus demonstrating the robustness of this approach for large scale biotransformations.     

Selective oxidation of alkyl and aryl glyceryl monoethers catalysed by an engineered and immobilised glycerol dehydrogenase

Enzymes acting over glyceryl ethers are scarce in living cells, and consequently biocatalytic transformations of these molecules are rare despite their interest for industrial chemistry. In this work, we have engineered and immobilised a glycerol dehydrogenase from Bacillus stearothermophilus (BsGlyDH) to accept a battery of alkyl/aryl glyceryl monoethers and catalyse their enantioselective oxidation to yield the corresponding 3-alkoxy/aryloxy-1-hydroxyacetones. QM/MM computational studies decipher the key role of D123 in the oxidation catalytic mechanism, and reveal that this enzyme is highly enantioselective towards S-isomers (ee > 99%). Through structure-guided site-selective mutagenesis, we find that the mutation L252A sculpts the active site to accommodate a productive configuration of 3-monoalkyl glycerols. This mutation enhances the k_cat 163-fold towards 3-ethoxypropan-1,2-diol, resulting in a specific activity similar to the one found for the wild-type towards glycerol. Furthermore, we immobilised the L252A variant to intensify the process, demonstrating the reusability and increasing the operational stability of the resulting heterogeneous biocatalyst. Finally, we manage to integrate this immobilised enzyme into a one-pot chemoenzymatic process to convert glycidol and ethanol into 3-ethoxy-1-hydroxyacetone and (R)-3-ethoxypropan-1,2-diol, without affecting the oxidation activity. These results thus expand the uses of engineered glycerol dehydrogenases in applied biocatalysis for the kinetic resolution of glycerol ethers and the manufacturing of substituted hydroxyacetones.

Design and fabrication of a robust heterogeneous biocatalyts for the selective oxidation of alkyl/aryl glyceryl monoethers through the engineering and immobilization of glycerol dehydrogenases.     

Metal substrate catalysis in the confined space for platinum drug delivery

Catalysis-based approaches for the activation of anticancer agents hold considerable promise. These principally rely on the use of metal catalysts capable of deprotecting inactive precursors of organic drugs or transforming key biomolecules available in the cellular environment. Nevertheless, the efficiency of most of the schemes described so far is rather low, limiting the benefits of catalytic amplification as strategy for controlling the therapeutic effects of anticancer compounds. In the work presented here, we show that flavin reactivity within a hydrogel matrix provides a viable solution for the efficient catalytic activation and delivery of cisplatin, a worldwide clinically-approved inorganic chemotherapy agent. This is achieved by ionically adsorbing a flavin catalyst and a Pt(iv) prodrug as substrate into porous amino-functionalized agarose beads. The hydrogel chassis supplies high local concentrations of electron donating groups/molecules in the surrounding of the catalyst, ultimately boosting substrate conversion rates (TOF >200 min⁻¹) and enabling controlled liberation of the drug by light or chemical stimuli. Overall, this approach can afford platforms for the efficient delivery of platinum drugs as demonstrated herein by using a transdermal diffusion model simulating the human skin.

Loading of a flavin catalyst and Pt prodrug onto a hydrogel affords biomaterials for the catalytic generation and delivery of cisplatin upon light irradiation or addition of electron donors. Confinement boosts the turnover frequency of the flavin.