Challenging DESs and ILs in the valorization of food waste: a case study.

In this study, the efficacy of two of the best performing green solvents for the fractionation of lignocellulosic biomass, cholinium arginate (ChArg) as biobased ionic liquid (Bio-IL) and ChCl:lactic acid (ChCl:LA, 1:10) as natural deep eutectic solvent (NADES), was investigated and compared in the pretreatment of an agri-food industry waste, apple fibers (90°C for 1 h). For the sake of comparison, 1-butyl-3-methylimidazolium acetate (BMIM OAc) as one of the best IL able to dissolve cellulose was also used. After the pretreatment, two fractions were obtained in each case. The results gathered through FTIR and TG analyses of the two materials and the subsequent DNS assay performed after enzymatic treatment led to identify ChArg as the best medium to delignify and remove waxes, present on the starting apple fibers, thus producing a material substantially enriched in cellulose (CRM). Conversely, ChCl:LA did not provide satisfactorily results using these mild conditions, while BMIM OAc showed intermediate performance probably on account of the reduced crystallinity of cellulose after the dissolution-regeneration process. To corroborate the obtained data, FTIR and TG analyses were also performed on the residues collected after the enzymatic hydrolysis. At the end of the pretreatment, ChArg was also quantitatively recovered without significant alterations.

An in vitro Coupled Assay for PEPC with Control of Bicarbonate Concentration.

Phosphoenolpyruvate carboxylase (PEPC) catalyzes a critical step in carbon metabolism in plants and bacteria, the irreversible reaction between bicarbonate and phosphoenolpyruvate to produce the C4 compound oxaloacetate. This enzyme is particularly important in the context of C4 photosynthesis, where it is the initial carbon-fixing enzyme. Many studies have used kinetic approaches to characterize the properties of PEPCs from different species, different post-translational states, and after mutagenesis. Most of these studies have worked at a fixed saturating concentration of bicarbonate. Controlling the concentration of bicarbonate is difficult at low concentrations because of equilibration with atmospheric CO2. We describe here a simple, repeatable, and gas-tight assay system for PEPC that allows bicarbonate concentrations to be controlled above ca. 50 µM.

Kinetic Modifications of C4 PEPC Are Qualitatively Convergent, but Larger in Panicum Than in Flaveria.

C4 photosynthesis results from a set of anatomical features and biochemical components that act together to concentrate CO2 within the leaf and boost productivity. This complex trait evolved independently many times, resulting in various realizations of the phenotype, but in all C4 plants the primary fixation of atmospheric carbon is catalyzed by phosphoenolpyruvate carboxylase. Comparisons of C4 and non-C4 PEPC from a few closely related species suggested that the enzyme was modified to meet the demands of the C4 cycle. However, very few C4 groups have been investigated, hampering general conclusions. To test the hypothesis that distant C4 lineages underwent convergent biochemical changes, we compare the kinetic variation between C4 and non-C4 PEPC from a previously assessed young lineage (Flaveria, Asteraceae) with those from an older lineage found within the distantly related grass family (Panicum). Despite the evolutionary distance, the kinetic changes between the non-C4 and C4 PEPC are qualitatively similar, with a decrease in sensitivity for inhibitors, an increased specificity (k cat/K m) for bicarbonate, and a decreased specificity (k cat/K m) for PEP. The differences are more pronounced in the older lineage Panicum, which might indicate that optimization of PEPC for the C4 context increases with evolutionary time.

Engineering novel S-glycosidase activity into extremo-adapted ß-glucosidase by rational design.

The breakdown of sulphur glycosidic bonds in thioglycosides can produce isothiocyanate, a chemoprotective agent linked to the prevention of cancers; however, only a handful of enzymes have been identified that are k0nown to catalyse this reaction. Structural studies of the myrosinase enzyme, which is capable of hydrolysing the thioglycosidic bond, have identified residues that may play important roles in sulphur bond specific activity. Using rational design, two extremo-adapted ß-glycosidases from the species Thermus nonproteolyticus (TnoGH1) and Halothermothrix orenii (HorGH1) were engineered towards thioglycoside substrates. Twelve variants, six for TnoGH1and six for HorGH1, were assayed for activity. Remarkable enhancement of the specificity (kcat/KM) of TnoGH1 and HorGH1 towards ß-thioglycoside was observed in the single mutants TnoGH1-V287R (2500 M-1 s-1) and HorGH1-M229R (13,260 M-1 s-1) which showed a 3-fold increase with no loss in turnover rate when compared with the wild-type enzymes. Thus, the role of arginine is key to induce ß-thioglycosidase activity. Thorough kinetic investigation of the different mutants has shed light on the mechanism of ß-glycosidases when acting on the native substrate.Key Points •Key residues were identified in the active site of Brevicoryne brassicae myrosinase. •Rationally designed mutations were introduced into two extremo-adapted ß-glycosidases. •ß-glycosidases mutants exhibited improved activity against thioglycosidic bonds. •The mutation to arginine in the active site yielded the best variant.

Insight into the structural and biological relevance of the T/R transition of the N-terminus of the B-chain in human insulin.

The N-terminus of the B-chain of insulin may adopt two alternative conformations designated as the T- and R-states. Despite the recent structural insight into insulin-insulin receptor (IR) complexes, the physiological relevance of the T/R transition is still unclear. Hence, this study focused on the rational design, synthesis, and characterization of human insulin analogues structurally locked in expected R- or T-states. Sites B3, B5, and B8, capable of affecting the conformation of the N-terminus of the B-chain, were subjects of rational substitutions with amino acids with specific allowed and disallowed dihedral φ and ψ main-chain angles. α-Aminoisobutyric acid was systematically incorporated into positions B3, B5, and B8 for stabilization of the R-state, and N-methylalanine and d-proline amino acids were introduced at position B8 for stabilization of the T-state. IR affinities of the analogues were compared and correlated with their T/R transition ability and analyzed against their crystal and nuclear magnetic resonance structures. Our data revealed that (i) the T-like state is indeed important for the folding efficiency of (pro)insulin, (ii) the R-state is most probably incompatible with an active form of insulin, (iii) the R-state cannot be induced or stabilized by a single substitution at a specific site, and (iv) the B1-B8 segment is capable of folding into a variety of low-affinity T-like states. Therefore, we conclude that the active conformation of the N-terminus of the B-chain must be different from the "classical" T-state and that a substantial flexibility of the B1-B8 segment, where GlyB8 plays a key role, is a crucial prerequisite for an efficient insulin-IR interaction.