Gijs J. C. Vreeke

The application of potato proteins in food products is limited by the development of off-flavour components resulting from, amongst others, the lipase activity of patatin. Currently, a comprehensive study about patatin's specificity towards different lipid classes is lacking. Furthermore, it is unclear why patatin has two chain length optima (C10 and C16) towards model substrates such as p-nitrophenol esters. Therefore, this study systematically investigated the lipase activity of patatin towards various substrate classes under identical experimental conditions. Activity was highest on monoacylglycerols (MAGs ~ 2 U mg-1), followed by diacylglycerols (DAGs ~ 0.7 U mg-1) and phospholipids (~ 0.2 U mg-1). Triacylglycerols (TAGs) with chain lengths ≤ C10 were converted less efficiently and activity on longer chain TAGs (>C10) was negligible. Molecular docking using a modelled dimeric patatin structure suggested two distinct favourable substrate orientations inside patatin’s binding site, which may explain the activity against long-chain acylglycerols. Further, patatin activity was about seven times higher on polyunsaturated MAG (C18:3) compared to saturated MAG (C18:0), likely due to the highly flexible nature of the unsaturated acyl chain. The results provide a systematic overview of patatin’s substrate specificity and give valuable information to manufacturers when using potato protein in food applications.

The growing production volume of the meat industry has increased the need for revalorization of meat by-products to reduce economic and environmental impacts. Enzymatic hydrolysis of protein-rich meat by-products is an effective strategy for producing hydrolyzates with bioactive potential. Combining sequential enzymatic hydrolysis with γ-glutamyl transpeptidase activity can promote the formation of γ-glutamyl peptides associated with koku perception, a sensory attribute that increases taste intensity, continuity, and palatability. This study aimed to develop porcine liver hydrolyzates enriched in koku-related peptides through enzymatic hydrolysis followed by post-treatment with the transpeptidase Protana Uboost. Substrate specificity assays showed that a 0.2 U/mL enzyme concentration maximized γ-glutamyl dipeptide formation. Sequential hydrolysis using Alcalase and Protana Prime followed by Protana Uboost post-treatment generated the highest levels of koku-related peptides. Moreover, post-treatment significantly enhanced antioxidant capacity in the resulting hydrolyzates, supporting their potential as a functional ingredient.

Dimethylallyl tryptophan synthases (DMATSs) are aromatic prenyltransferases that catalyze the transfer of a prenyl moiety from a donor to an aromatic acceptor during the biosynthesis of microbial secondary metabolites. Due to their broad substrate scope, DMATSs are anticipated as biotechnological tools for producing bioactive prenylated aromatic compounds. Our study explored the substrate scope and product profile of a recombinant RePT, a novel DMATS from the thermophilic fungus Rasamsonia emersonii. Among a variety of aromatic substrates, RePT showed the highest substrate conversion for l-tryptophan and l-tyrosine (> 90%), yielding two mono-prenylated products in both cases. Nine phenolics from diverse phenolic subclasses were notably converted (> 10%), of which the stilbenes oxyresveratrol, piceatannol, pinostilbene, and resveratrol were the best acceptors (37–55% conversion). The position of prenylation was determined using NMR spectroscopy or annotated using MS² fragmentation patterns, demonstrating that RePT mainly catalyzed mono-O-prenylation on the hydroxylated aromatic substrates. On l-tryptophan, a non-hydroxylated substrate, it preferentially catalyzed C7 prenylation with reverse N1 prenylation as a secondary reaction. Moreover, RePT also possessed substrate-dependent organic solvent tolerance in the presence of 20% (v/v) methanol or DMSO, where a significant conversion (> 90%) was maintained. Our study demonstrates the potential of RePT as a biocatalyst for the production of bioactive prenylated aromatic amino acids, stilbenes, and various phenolic compounds. Key points: • RePT catalyzes prenylation of diverse aromatic substrates. • RePT enables O-prenylation of phenolics, especially stilbenes. • The novel RePT remains active in 20% methanol or DMSO. Graphical