Physical and Oxidative Stability of Emulsions Stabilized with Fractionated Potato Protein Hydrolysates Obtained from Starch Production Side Stream.

This work studies the emulsifying and antioxidant properties of potato protein hydrolysates (PPHs) fractions obtained through enzymatic hydrolysis of potato protein using trypsin followed by ultrafiltration. Unfractionated (PPH1) and fractionated (PPH2 as >10 kDa, PPH3 as 10-5 kDa, PPH4 as 5-0.8 kDa, and PPH5 as <0.8 kDa) protein hydrolysates were evaluated. Pendant drop tensiometry and dilatational rheology were applied for determining the ability of PPHs to reduce interfacial tension and affect the viscoelasticity of the interfacial films at the oil-water interface. Peptides >10 kDa showed the highest ability to decrease oil-water interfacial tension. All PPH fractions predominantly provided elastic, weak, and easily stretchable interfaces. PPH2 provided a more rigid interfacial layer than the other hydrolysates. Radical scavenging and metal chelating activities of PPHs were also tested and the highest activities were provided by the unfractionated hydrolysate and the fractions with peptides >5 kDa. Furthermore, the ability of PPHs to form physically and oxidatively stable 5% fish oil-in-water emulsions (pH 7) was investigated during 8-day storage at 20 °C. Our results generally show that the fractions with peptides >5 kDa provided the highest physicochemical stability, followed by the fraction with peptides between 5 and 0.8 kDa. Lastly, promising sensory results with mostly mild attributes were obtained even at protein concentration levels that are higher than needed to obtain functional properties. The more prominent attributes (e.g., bitterness and astringency) were within an acceptable range for PPH3 and PPH4.

Engineering Bifidobacterium longum Endo-α-N-acetylgalactosaminidase for Neu5Acα2-3Galß1-3GalNAc reactivity on Fetuin.

Endo-α-N-acetylgalactosaminidase from Bifidobacterium longum (EngBF) belongs to the glycoside hydrolase family GH101 and has a strict preference towards the mucin type glycan, Galß1-3GalNAc, which is O-linked to serine or threonine residues on glycopeptides and -proteins. While other enzymes of the GH101 family exhibit a wider substrate spectrum, no GH101 member has until recently been reported to process the α2-3 sialidated mucin glycan, Neu5Acα2-3Galß1-3GalNAc. However, work published by others (ACS Chem Biol 2021, 16, 2004-2015) during the preparation of the present manuscript demonstrated that the enzymes from several bacteria are able to hydrolyze this glycan from the fluorophore, methylumbelliferyl. Based on molecular docking using the EngBF homolog, EngSP from Streptococcus pneumoniae, substitution of active site amino acid residues with the potential to allow for accommodation of Neu5Acα2-3Galß1-3GalNAc were identified. Based on this analysis, the mutant EngBF variants W750A, Q894A, K1199A, E1294A and D1295A were prepared and tested, for activity towards the Neu5Acα2-3Galß1-3GalNAc O-linked glycan present on bovine fetuin. Among the mutant EngBF variants listed above, only E1294A was shown to release Neu5Acα2-3Galß1-3GalNAc from fetuin, which subsequently was also demonstrated for the substitutions: E1294 M, E1294H and E1294K. In addition, the kcat/KM of the EngBF variants for cleavage of the Neu5Acα2-3Galß1-3GalNAc glycan increased between 5 and 70 times from pH 4.5 to pH 6.0.

Identification of a Catalytic Nucleophile-Activating Network in the endo-α-N-Acetylgalactosaminidase of Family GH101.

Bifidobacterium longum endo-α-N-acetylgalactosaminidase (GH101), EngBF, is highly specific toward the mucin Core 1 glycan, Galß1-3GalNAc. Apart from the side chains involved in the retaining mechanism of EngBF, Asp-682 is important for the activity. In the crystal structures of both EngBF and EngSP (from Streptococcus pneumoniae), we identified a conserved water molecule in proximity to Asp-682 and the homologue residue in EngSP. The water molecule also coordinates the catalytic nucleophile and three other residues conserved in GH101 enzymes; in EngBF, these residues are His-685, His-718, and Asn-720. With casein-glycomacropeptide as the substrate, the importance of Asp-682 was confirmed by the lack of a detectable activity for the D682N enzyme. The enzyme variants, H685A, H718A, H685Q, and H718Q, all displayed only a modestly reduction in kcat of up to 15 fold for the H718A variant. However, the double-substituted variants, H685A/H718A and H685Q/H718Q, had a greatly reduced kcat value by about 200 fold compared to that of wild-type EngBF. With the synthetic substrate, Galß(1-3)GalNAcα1-para-nitrophenol, kcat of the double-substituted variants was only up to 30-fold reduced and was found to increase with pH. Compared to the pre-steady-state kinetics of wild-type EngBF, a burst of about the size of the enzyme concentration was absent with the double-substituted EngBF variants, indicating that the nucleophilic attack had become at least as slow as the hydrolysis of the enzyme intermediate. Together, the results indicate that not only Asp-682 but also the entire conserved network of His-685, His-718, and what we suggest is a catalytic water molecule is important in the activation of the catalytic nucleophile.

Steady state kinetic analysis of O-linked GalNAc glycan release catalyzed by endo-α-N-acetylgalactosaminidase.

Often glycosidase assays are based on small-molecule compounds where a glycan of interest is linked to a chromophore allowing for easy detection of cleavage of the glycoside bond. However, such compounds only resemble part of the more complex substrate molecule for enzymes acting on glycoconjugates of glycopeptides or glycoproteins. Nonetheless, the advantage is obvious as enzyme activity is readily recorded and kinetic parameters easily obtained. This is not often the case with glycopeptides or glycoproteins as these may reveal increased complexity in terms of heterogeneity in protein-glycan stoichiometry and restricted enzyme accessibility. However, a kinetic analysis of glycan release from glycopeptides could provide information complementary to that of small-molecule substrates, especially if providing kinetic parameters that are immediately comparable. We have characterized the steady state kinetics of wild type and mutant variants of Bifidobacterium longum endo-α-N-acetylgalactosaminidase, by recording the enzymatic release of Galß(1-3)GalNAc from bovine glycomacropeptide pre-treated with sialidase to remove sialic acid units. Differences between previously reported kinetic constants obtained with synthetic substrates and those obtained in the present work demonstrate an influence of the peptide moiety on the kinetic properties of endo-α-N-acetylgalactosaminidase. The devised assay and data handling method determines the accessible substrate concentration as well as the steady state kinetic parameters, KM and kcat, for glycoconjugates of glycopeptides described by the same units as obtained from using small-molecule substrates and thus allows for a direct comparison.

Mutational analysis of divalent metal ion binding in the active site of class II α-mannosidase from Sulfolobus solfataricus.

Mutational analysis of Sulfolobus solfataricus class II α-mannosidase was focused on side chains that interact with the hydroxyls of the -1 mannosyl of the substrate (Asp-534) or form ligands to the active site divalent metal ion (His-228 and His-533) judged from crystal structures of homologous enzymes. D534A and D534N appeared to be completely inactive. When compared to the wild-type enzyme, the mutant enzymes in general showed only small changes in K(M) for the substrate, p-nitrophenyl-α-mannoside, but elevated activation constants, K(A), for the divalent metal ion (Co²âº, Zn²âº, Mn²âº, or Cd²âº). Some mutant enzyme forms displayed an altered preference for the metal ion compared to that of the wild type-enzyme. Furthermore, the H228Q, H533E, and H533Q enzymes were inhibited at increasing Zn²âº concentrations. The catalytic rate was reduced for all enzymes compared to that of the wild-type enzyme, although less dramatically with some activating metal ions. No major differences in the pH dependence between wild-type and mutant enzymes were found in the presence of different metal ions. The pH optimum was 5, but enzyme instability was observed at pH <4.5; therefore, only the basic limb of the bell-shaped pH profile was analyzed.