Cascade reactions have been described as efficient and universal tools, and are of substantial interest in synthetic organic chemistry. This review article provides an overview of the novel and recent achievements in enzyme cascade processes catalyzed by multi-enzymatic or chemoenzymatic systems. The examples here selected collect the advances related to the application of the sequential use of enzymes in natural or genetically modified combination; second, the important combination of enzymes and metal complex systems, and finally we described the application of biocatalytic biohybrid systems on in situ catalytic solid-phase as a novel strategy. Examples of efficient and interesting enzymatic catalytic cascade processes in organic chemistry, in the production of important industrial products, such as the designing of novel biosensors or bio-chemocatalytic systems for medicinal chemistry application, are discussed

The effects of the reaction medium and substrate concentration were studied on the selectivity of Novozym 435 using the asymmetric hydrolysis of dimethyl-3-phenylglutarate as a model reaction. Results show that the use of choline chloride ChCl:urea/phosphate buffer 50% (v/v) as a reaction medium increased the selectivity of Novozym 435 by 16% (e.e = 88%) with respect to the one in 100% phosphate buffer (e.e = 76%). Best results were obtained when high substrate concentrations (well above the solubility limit, 27-fold) and ChCl:urea/phosphate buffer 50% (v/v) as reaction medium at pH 7 and 30 °C were used. Under such conditions, the R-monoester was produced with an enantiomeric purity of 99%. Novozym 435 was more stable in ChCl:urea/phosphate buffer 50% (v/v) than in phosphate buffer, retaining a 50% of its initial activity after 27 h of incubation at pH 7 and 40 °C. Results suggest that the use of deep eutectic solvents (ChCl:urea/phosphate buffer) in an heterogeneous reaction system (high substrate concentration) is a viable and promising strategy for the synthesis of chiral drugs from highly hydrophobic substrates.

Defatted rapeseed meal (DRM) is a sub-valorized agro-industrial by-product, with a high protein content whose peptides could have potential anticancer activity against cancer cell lines. The objective of the present study is to obtain an enzymatic hydrolysate of rapeseed protein that inhibits proliferation on a breast cancer cell line (MCF-7), but not healthy human fibroblast cells. The DRM was solubilized in an alkaline medium to obtain an alkaline rapeseed extract (RAE). Acid precipitation of the proteins contained in RAE recovered a rapeseed protein isolate (RPI). To produce protein hydrolysates, two alkaline protease and different enzyme/substrate ratios were used. All the protein hydrolysates showed antiproliferative activity on MCF-7 cells. However, only the hydrolysate recovered from the enzymatic hydrolysis of RPI (Degree of hydrolysis (DH ) between 8.5 and 9% (DH1)) did not affect human fibroblast cells, inhibiting 83.9% of MCF-7 cells’ proliferation and showing a mass yield of 22.9% (based on the initial DRM). The SDS-PAGE gel revealed that DH1 was composed mainly of 10 kDa peptides and, to a lesser extent, 5 and 2 kDa. It is concluded that DH1 is a promising peptide extract for future research as a putative anti-breast cancer agent.

Oligosaccharide and peptide extracts obtained separately from defatted rapeseed meal (DRM) have shown antiproliferative activities on the MCF-7 breast cancer cell line. However, oligosaccharide extracts were not tested on human fibroblasts and have low yields. The objective of the present study was to combine two antiproliferative extracts, the peptides and oligosaccharides, that were obtained independently with commercial enzymes from DRM, allowing improvement of the mass yield and antiproliferative activity. The DRM was solubilized in an alkaline medium to obtain an insoluble meal residue (IMR) and an alkaline extract (RAE). To produce the oligosaccharide extract from IMR, three enzymes and different enzyme/substrate ratios were used. The oligosaccharide extract (molecular weight <30 kDa) recovered with the commercial enzyme. Endogalacturonase showed an 80% inhibition on MCF-7 cells at 20 mg/mL. The combination of this oligosaccharide extract with the peptide extract (obtained with Alkalase 2.4 L from a RAE at 10 mg/mL) inhibited 84.3% of MCF-7 cells proliferation at a concentration of 20 mg/mL, exhibiting no cytotoxic effects on fibroblasts. The mass yield of the extract pool was 27.07% (based on initial DRM). It can be concluded that a mixture of antiproliferative extracts was produced from DRM which was selective against MCF-7 cells.

Different materials containing carboxylic groups have been functionalized with geranyl-amine molecules by using an EDC/NHS strategy. Chemical modification of the support was confirmed by XRD, UV-spectrophotometer, and FT-IR. This geranyl-functionalized material was successfully applied for four different strategies of site-selective immobilization of proteins at room temperature and aqueous media. A reversible hydrophobic immobilization of proteins (lipases, phosphoglucosidases, or tyrosinase) was performed in neutral pH in yields from 40 to >99%. An increase of the activity in the case of lipases was observed from a range of 2 to 4 times with respect to the initial activity in solution. When chemically or genetically functionalized cysteine enzymes were used, the covalent immobilization, via a selective thiol-alkene reaction, was observed in the presence of geranyl support at pH 8 in lipases in the presence of detergent (to avoid the previous hydrophobic interactions). Covalent attachment was confirmed with no release of protein after immobilization by incubation with hydrophobic molecules. In the case of a selenium-containing enzyme produced by the selenomethionine pathway, the selective immobilization was successfully yielded at acidic pH (pH 5) (89%) much better than at pH 8. In addition, when an azido-enzyme was produced by the azide–homoalanine pathway, the selective immobilization was successful at pH 6 and in the presence of CuI for the click chemistry reaction.