A systematic study of the effects of hydrophilic ionic liquids concentration and nature (alkyl chain length and type of anion) on the activity of Candida antarctica lipase B is here reported. The increase in the concentration of the studied ionic liquids is shown to cause a decrease of the enzyme activity, but the effect is dependent on the ionic liquid used. This behavior is partially due to the ionic liquid impact on the thermodynamic water activity, but direct interactions between the hydrophilic ionic liquid and the enzyme are also disclosed. Cations with longer alkyl chains decrease the enzyme activity by obstruction of its non-polar active site, while direct interactions established between the enzyme and the anions, dominated by dispersion forces and hydrogen-bonding, contribute also for the loss of activity observed.

Biocatalysis is nowadays considered as one of the most important tools in green chemistry. The elimination of multiple steps involved in some of the most complex chemical synthesis, reducing the amounts of wastes and hazards, thus increasing the reaction yields and decreasing the intrinsic costs, are the major advantages of biocatalysis. This work aims at improving the enzymatic hydrolysis of olive oil to produce valuable fatty acids through emulsion systems formed by long alkyl chain ionic liquids (ILs). The optimization of the emulsion and the best conditions to maximize the production of fatty acids were investigated. The stability of the emulsion was characterized considering the effect of several parameters, namely, the IL and its concentration and different water/olive oil volumetric ratios. ILs from the imidazolium and phosphonium families were evaluated. The results suggest that the ILs effect on the hydrolysis performance varies with the water concentration and the emulsion system formed, that is, water-in-oil or oil-in-water emulsion. Although at low water concentrations, the presence of ILs does not present any advantages for the hydrolysis reaction, at high water contents (in oil-in-water emulsions), the imidazolium-based IL acts as an enhancer of the lipase catalytic capacity, super-activating 1.8 times the enzyme, and consequently promoting the complete hydrolysis of the olive oil for the highest water contents [85% (v/v)].

Actinobacteria of the genus Amycolatopsis are important for antibiotic production and other valuable biotechnological applications such as bioconversion or bioremediation. Despite their importance, tools and methods for their genetic manipulation are less developed than in other actinobacteria such as Streptomyces. We report here the use of the pSAM2 site-specific recombination system to delete antibiotic resistance cassettes used in gene replacement experiments or to create large genomic deletions. For this purpose, we constructed a shuttle vector, replicating in Escherichia coli and Amycolatopsis, expressing the integrase and the excisionase from the Streptomyces integrative and conjugative element pSAM2. These proteins are sufficient for site-specific recombination between the attachment sites attL and attR. We also constructed two plasmids, replicative in E. coli but not in Amycolatopsis, for the integration of the attL and attR sites on each side of a large region targeted for deletion. We exemplified the use of these tools in Amycolatopsis mediterranei by obtaining with high efficiency a marker-free deletion of one single gene in the rifamycin biosynthetic gene cluster or of the entire 90-kb cluster. These robust and simple tools enrich the toolbox for genome engineering in Amycolatopsis.