Polymerizing Like Mussels Do: Toward Synthetic Mussel Foot Proteins and Resistant Glues

A novel strategy to generate adhesive protein analogues by enzyme-induced polymerization of peptides is reported. Peptide polymerization relies on tyrosinase oxidation of tyrosine residues to Dopaquinones, which rapidly form cysteinyldopa-moieties with free thiols from cysteine residues, thereby linking unimers and generating adhesive polymers. The resulting artificial protein analogues show strong adsorption to different surfaces, even resisting hypersaline conditions. Remarkable adhesion energies of up to 10.9 mJ m−2 are found in single adhesion events and average values are superior to those reported for mussel foot proteins that constitute the gluing interfaces

Comprehensive Polyphenolic Profiling of Nine Distinct Plants and Edible Mushrooms by Targeted and Untargeted LC-(HR)MS(/MS)

Polyphenols are a broad molecular family whose presence in an organism is mostly only partially known. They are secondary metabolites exhibiting antioxidant properties and are involved in a wide range of biological actions. Moreover, polyphenol oxidases, metalloenzymes present in various plants and fungi, play a role in metabolic pathways by catalyzing the hydroxylation and oxidation of polyphenols. Functional studies on these enzymes are greatly hampered by the lack of information on the polyphenols present in a specific organism, so the physiological substrate is unknown for most polyphenol oxidases. Therefore, seven commercially available fungal and two plant species were examined in detail for the presence of polyphenols using an LC-HRMS-based suspect screening workflow. A total of 401 features were annotated, composed of 221 flavonoids and 180 non-flavonoids. As 64% of the flavonoids were conjugated with a glycone, (semi-)quantification was applied when a reference standard of the aglycone was available. Isomers of hesperetin-O-glucoside were ubiquitous in all investigated samples. The samples were then additionally investigated by a quantitative, targeted LC-MS/MS assay that covers 90 polyphenols from 13 distinct polyphenol classes. The targeted assay showed phenolic acids as prevalent analytes. Overall, this pilot study provides new insights into the complex polyphenol profiles of nine mushroom and plant species

Immobilization of Agaricus bisporus Polyphenol Oxidase 4 on mesoporous silica : Towards mimicking key enzymatic processes in peat soils

Hypothesis: The use of immobilized enzyme-type biocatalysts to mimic specific processes in soil can be considered one of the most promising alternatives to overcome the difficulties behind the structural elucidation of riverine humic-derived iron-complexes. Herein, we propose that the immobilization of the functional mushroom tyrosinase, Agaricus bisporus Polyphenol Oxidase 4 (AbPPO4) on mesoporous SBA-15-type silica could contribute to the study of small aquatic humic ligands such as phenols. Experiments: The silica support was functionalized with amino-groups in order to investigate the impact of surface charge on the tyrosinase loading efficiency as well as on the catalytic performance of adsorbed AbPPO4. The oxidation of various phenols was catalyzed by the AbPPO4-loaded bioconjugates, yielding high levels of conversion and confirming the retention of enzyme activity after immobilization. The structures of the oxidized products were elucidated by integrating chromatographic and spectroscopic techniques. We also evaluated the stability of the immobilized enzyme over a wide range of pH values, temperatures, storage-times and sequential catalytic cycles. Findings: This is the first report where the latent AbPPO4 is confined within silica mesopores. The improved catalytic performance of the adsorbed AbPPO4 shows the potential use of these silica-based mesoporous biocatalysts for the preparation of a column-type bioreactor for in situ identification of soil samples

The Structure of a Plant Tyrosinase from Walnut Leaves Reveals the Importance of 'Substrate-Guiding Residues' for Enzymatic Specificity

Tyrosinases and catechol oxidases are members of the class of type III copper enzymes. While tyrosinases accept both mono- and o-diphenols as substrates, only the latter substrate is converted by catechol oxidases. Researchers have been working for decades to elucidate the monophenolase/diphenolase specificity on a structural level and have introduced an early hypothesis that states that the reason for the lack of monophenolase activity in catechol oxidases may be its structurally restricted active site. However, recent structural and biochemical studies of this enzyme class have raised doubts about this theory. Herein, the first crystal structure of a plant tyrosinase (from Juglans regia) is presented. The structure reveals that the distinction between mono- and diphenolase activity does not depend on the degree of restriction of the active site, and thus a more important role for amino acid residues located at the entrance to and in the second shell of the active site is proposed

Immobilization of Agaricus bisporus Polyphenol Oxidase 4 on mesoporous silica: Towards mimicking key enzymatic processes in peat soils

The use of immobilized enzyme-type biocatalysts to mimic specific processes in soil can be considered one of the most promising alternatives to overcome the difficulties behind the structural elucidation of riverine humic-derived iron-complexes. Herein, we propose that the immobilization of the functional mushroom tyrosinase, Agaricus bisporus Polyphenol Oxidase 4 (AbPPO4) on mesoporous SBA-15-type silica could contribute to the study of small aquatic humic ligands such as phenols.The authors thank the University of Vienna (Austria) and the Austrian Science Research Fund (FWF, P32326 to A.R.) for financial support.Peer reviewe

Purification and Characterization of Latent Polyphenol Oxidase from Apricot (<i>Prunus armeniaca</i> L.)

Polyphenol oxidase from apricot (<i>Prunus armeniaca</i>) (<i>Pa</i>PPO) was purified in its latent form (L-<i>Pa</i>PPO), and the molecular weight was determined to be 63 kDa by SDS-PAGE. L-<i>Pa</i>PPO was activated in the presence of substrate at low pH. The activity was enhanced by CuSO₄ and low concentrations (≤ 2 mM) of SDS. <i>Pa</i>PPO has its pH and temperature optimum at pH 4.5 and 45 °C for catechol as substrate. It showed diphenolase activity and highest affinity toward 4-methylcatechol (<i>K</i>_M = 2.0 mM) and chlorogenic acid (<i>K</i>_M = 2.7 mM). L-<i>Pa</i>PPO was found to be spontaneously activated during storage at 4 °C, creating a new band at 38 kDa representing the activated form (A-<i>Pa</i>PPO). The mass of A-<i>Pa</i>PPO was determined by mass spectrometry as 37 455.6 Da (Asp102 → Leu429). Both L-<i>Pa</i>PPO and A-<i>Pa</i>PPO were identified as polyphenol oxidase corresponding to the known <i>Pa</i>PPO sequence (UniProt O81103) by means of peptide mass fingerprinting

Toward Artificial Mussel‐Glue Proteins: Differentiating Sequence Modules for Adhesion and Switchable Cohesion

The synthesis of artificial mussel‐glue proteins with pH triggered cohesion control mechanism is described by extending the tyrosinase activated polymerization of peptides to sequences having specific modules for cohesion control. The high propensity of those sequence sections to adapt β‐sheets is temporarily suppressed by switch defects. This allows enzymatic activation and polymerization to proceed undisturbed. The β‐sheet formation is regained after polymerization by changing pH from 5.5 to 6.8, triggering O→N acyl transfer rearrangements that activate the cohesion mechanism. The resulting artificial mussel glue proteins exhibit rapid adsorption on alumina surfaces. The coatings resist harsh hypersaline conditions, and reach remarkable adhesive energies of 2.64 mJ/m–2 on silica at pH 6.8. In in situ switch experiments, the minor pH change increases the adhesive properties of a coating by 300% and nanoindentation confirms the cohesion mechanism to improve bulk‐stiffness by ~200%

GeV-scale electron acceleration in a gas-filled capillary discharge waveguide

We report experimental results on laser-driven electron acceleration with low divergence. the electron beam was generated by focusing 750 mJ, 42 fs laser pulses into a gas-filled capillary discharge waveguide at electron densities in the range between 1018 and 1019 cm-3/ Quasi-monoenergentic electron bunches with energies as high as 500 MeV have been detected, with features reaching up to 1 GeV, albeit with large shot-to-shot fluctuations. A more stable regime with higher bunch charge (20-45 pC) and less energy (200-300 MeV) could also be observed. The beam divergence and the pointing stability are around or below 1 mrad and 8 mrad, respectively. These findings are consistent with self-injection of electrons into a breaking plasma wave