Structural diversity and substrate preferences of three tannase enzymes encoded by the anaerobic bacterium Clostridium butyricum.

Tannins are secondary metabolites that are enriched in the bark, roots, and knots in trees and are known to hinder microbial attack. The biological degradation of water-soluble gallotannins, such as tannic acid, is initiated by tannase enzymes (EC 3.1.1.20), which are esterases able to liberate gallic acid from aromatic-sugar complexes. However, only few tannases have previously been studied in detail. Here, for the first time, we biochemically and structurally characterize three tannases from a single organism, the anaerobic bacterium Clostridium butyricum, which inhabits both soil and gut environments. The enzymes were named CbTan1-3, and we show that each one exhibits a unique substrate preference on a range of galloyl ester model substrates; CbTan1 and 3 demonstrated preference toward galloyl esters linked to glucose, while CbTan2 was more promiscuous. All enzymes were also active on oak bark extractives. Furthermore, we solved the crystal structure of CbTan2 and produced homology models for CbTan1 and 3. In each structure, the catalytic triad and gallate-binding regions in the core domain were found in very similar positions in the active site compared with other bacterial tannases, suggesting a similar mechanism of action among these enzymes, though large inserts in each enzyme showcase overall structural diversity. In conclusion, the varied structural features and substrate specificities of the C. butyricum tannases indicate that they have different biological roles and could further be used in development of new valorization strategies for renewable plant biomass.

Characterization and demonstration of drug compound ring-chain tautomer formation and its impacts on quality control.

Unknown chromatographic peaks, potential impurities, were observed in a series of related compounds. This led to the identification and characterization of tautomeric equilibria. Structural elucidation was required to understand the potential impurity profile, thus impacting method development for quality control. In this work, characterization of the chemical structures, AZ13581258 and AZD5718, and equilibria of the tautomeric forms was performed using a range of advanced analytical techniques such as preparative chromatography, nuclear magnetic resonance (NMR), chromatographic detection by mass spectrometry (MS), MSMS, and ultraviolet spectroscopy (UV). Predictions using density functional theory (DFT) further explains and confirms the tautomer equilibria through predictions of reaction barrier energies, UV-spectra and NMR data. These investigations led to fully understand the impurity profile and to the development of a quality control method for AZD5718 drug substance and drug product. In conclusion, ring-chain tautomeric structures are predominately formed under acidic conditions, and the additional peaks observed in LC during organic impurity determination were found to originate from ring-chain closed tautomers in equilibria with the parent open form compound. Hence, the closed and open tautomer forms should all be considered as the same compound.

Investigation of robustness for supercritical fluid chromatography separation of peptides: Isocratic vs gradient mode.

We investigated and compared the robustness of supercritical fluid chromatography (SFC) separations of the peptide gramicidin, using either isocratic or gradient elution. This was done using design of experiments in a design space of co-solvent fraction, water mass fraction in co-solvent, pressure, and temperature. The density of the eluent (CO2-MeOH-H2O) was experimentally determined using a Coriolis mass flow meter to calculate the volumetric flow rate required by the design. For both retention models, the most important factor was the total co-solvent fraction and water mass fraction in co-solvent. Comparing the elution modes, we found that gradient elution was more than three times more robust than isocratic elution. We also observed a relationship between the sensitivity to changes and the gradient steepness and used this to draw general conclusions beyond the studied experimental system. To test the robustness in a practical context, both the isocratic and gradient separations were transferred to another laboratory. The gradient elution was highly reproducible between laboratories, whereas the isocratic system was not. Using measurements of the actual operational conditions (not the set system conditions), the isocratic deviation was quantitatively explained using the retention model. The findings indicate the benefits of using gradient elution in SFC as well as the importance of measuring the actual operational conditions to be able to explain observed differences between laboratories when conducting method transfer.

The importance of ion-pairing in peptide purification by reversed-phase liquid chromatography.

The adsorption mechanism for three peptides was studied under overloaded conditions through adsorption isotherm measurements in the presence of an ion-pairing reagent, trifluoroacetic acid (TFA), on an end-capped C18-bonded stationary phase. The overall aim of the study was to obtain a better understanding of how the acetonitrile and the TFA fractions in the eluent affected the overloaded elution profiles and the selectivity between peptides using mechanistic modelling and multivariate design of experiments. When studying the effect of TFA, direct evidence for ion pair formation between a peptide and TFA in acetonitrile-water solutions was provided by fluorine-proton nuclear Overhauser NMR enhancement experiments and the adsorption of TFA on the stationary phase was measured by frontal analysis. The adsorption isotherms for each peptide were then determined by the inverse method at eight TFA concentrations ranging from 2.6mM to 37.3mM (0.02-0.29vol-%) in isocratic elution. The equilibrium between the peptide ion and the peptide-TFA complex was modelled by coupling the mass-balance to reaction kinetics and determining separate adsorption isotherms for the two species. We found that a Langmuir isotherm described the elution profile of peptide-TFA complex well while the peptide ion was described by a bi-Langmuir adsorption isotherm since it exhibited strong secondary interactions. The elution profiles had an unfavorable shape at low TFA concentrations consisting of a spike in their front and a long tailing rear due to the secondary interactions for the peptide ion having very low saturation capacity. The acetonitrile dependence on the adsorption isotherms was studied by determination of adsorption isotherms directly from elution profiles obtained in gradient elution which enabled a broad acetonitrile interval to be studied. Here, it was found that the column saturation capacity was quickly reached at very low acetonitrile fractions and that there were significant variations in adsorption with the molecular weight. Finally, practical implications for method development are discussed based on an experimental design where gradient slope and TFA concentrations are used as factors.