By mimicking the role of human liver P450 monooxygenases, fungal unspecific peroxygenases (UPOs) can perform a range of highly selective oxyfunctionalization reactions on pharmacological compounds, including O-dealkylations and hydroxylations, thereby simulating drug metabolism. Here we have benchmarked human drug metabolite (HDM) synthesis by several evolved UPO mutants, focusing on dextromethorphan, naproxen and tolbutamide. The HDM from dextromethorphan was prepared at the semi-preparative scale as a proof of production. The structural analysis of mutations involved in the synthesis of HDMs highlights the heme access channel as the main feature on which to focus when designing evolved UPOs. These variants are becoming emergent tools for the cost-effective synthesis of HDMs from next-generation drugs.

Summary Metschnikowia reukaufii is a widespread yeast able to grow in the plants’ floral nectaries, an environment of extreme conditions with sucrose concentrations exceeding 400 g l−1, which led us into the search for enzymatic activities involved in this sugar use/transformation. New oligosaccharides were produced by transglucosylation processes employing M. reukaufii cell extracts in overload‐sucrose reactions. These products were purified and structurally characterized by MS‐ESI and NMR techniques. The reaction mixture included new sugars showing a great variety of glycosidic bonds including α‐(1→1), α‐(1→3) and α‐(1→6) linkages. The main product synthesized was the trisaccharide isomelezitose, whose maximum concentration reached 81 g l−1, the highest amount reported for any unmodified enzyme or microbial extract. In addition, 51 g l−1 of the disaccharide trehalulose was also produced. Both sugars show potential nutraceutical and prebiotic properties. Interestingly, the sugar mixture obtained in the biosynthetic reactions also contained oligosaccharides such as esculose, a rare trisaccharide with no previous NMR structure elucidation, as well as erlose, melezitose and theanderose. All the sugars produced are naturally found in honey. These compounds are of biotechnological interest due to their potential food, cosmeceutical and pharmaceutical applications.

We have developed a sustainable three-stage process for the revaluation of cheese whey permeate into D-tagatose, a rare sugar with functional properties used as sweetener. The experimental conditions (pH, temperature, cofactors, etc.) for each step were independently optimized. In the first step, concentrated whey containing 180–200 g/L of lactose was fully hydrolyzed by β-galactosidase from Bifidobacterium bifidum (Saphera®) in 3 h at 45 °C. Secondly, glucose was selectively removed by treatment with Pichia pastoris cells for 3 h at 30 °C. The best results were obtained with 350 mg of cells (previously grown for 16 h) per mL of solution. Finally, L-arabinose isomerase US100 from Bacillus stearothermophilus was employed to isomerize D-galactose into D-tagatose at pH 7.5 and 65 °C, in presence of 0.5 mM MnSO4. After 7 h, the concentration of D-tagatose was approximately 30 g/L (33.3% yield, referred to the initial D-galactose present in whey). The proposed integrated process takes place under mild conditions (neutral pH, moderate temperatures) in a short time (13 h), yielding a glucose-free syrup containing D-tagatose and galactose in a ratio 1:2 (w/w).

The transglycosylation activity of a novel commercial β-galactosidase from Bifidobacterium bifidum (Saphera) was evaluated. The optimal conditions of operation of this enzyme, measured with o-nitrophenyl-β-D-galactopyranoside, were 40 °C and pH around 6.0. Although at low lactose concentrations the character of this enzyme was basically hydrolytic, an increase of lactose concentration to 400 g/L resulted in a significant formation (107.2 g/L, 27% yield) of prebiotic galactooligosaccharides (GOS).The maximum amount of GOS was obtained at a lactose conversion of approximately 90%, which contrasts with other β-galactosidases, for which the highest GOS yield is achieved at 40-50% lactose conversion. Using HPAEC-PAD, semipreparative HPLC-HILIC, MS, 1D and 2D NMR, we determined the structure of most of the GOS synthesized by this enzyme.The main identified products were Gal-β(13)-Gal-β(14)-Glc (3´-O-Beta-galactosyllactose), Gal-β(1→6)-Glc (allolactose), Gal-β(13)-Glc (3-galactosyl-glucose), Galβ(1→3)-Gal (3-galactobiose) and the tetrasaccharide Gal-β(13)-Gal-β(13)-Galβ(14)-Glc. In general, the B. bifidum β-galactosidase showed a tendency to form β(13) linkages followed by β(16), and more scarcely β(14).

The β-fructofuranosidase (Xd-INV) from the basidiomycota yeast Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) is unique in its ability to synthesize neo-fructooligosaccharides (neo-FOS). In order to facilitate its industrial application, the recombinant enzyme expressed in Pichia pastoris (pXd-INV) was immobilized by entrapment in polyvinyl alcohol (PVA) hydrogels. The encapsulation efficiency exceeded 80%. The PVA lenticular particles of immobilized pXd-INV were stable up to approximately 40 • C. Using 600 g/L sucrose, the immobilized biocatalyst synthesized 18.9% (w/w) FOS (59.1 g/L of neokestose, 30.2 g/L of 1-kestose, 11.6 g/L of neonystose and 12.6 g/L of blastose). The operational stability of PVA-immobilized biocatalyst was assayed in a batch reactor at 30 • C. The enzyme preserved its initial activity during at least 7 cycles of 26 h.