The functionalization of synthetic polymers such as poly(ethylene terephthalate) to improve their hydrophilicity can be achieved biocatalytically using hydrolytic enzymes. A number of cutinases, ...lipases, and esterases active on polyethylene terephthalate have been identified and characterized. Enzymes from Fusarium solani, Thermomyces insolens, T. lanuginosus, Aspergillus oryzae, Pseudomonas mendocina, and Thermobifida fusca have been studied in detail. Thermostable biocatalysts hydrolyzing poly(ethylene terephthalate) are promising candidates for the further optimization of suitable biofunctionalization processes for textile finishing, technical, and biomedical applications.
Methane biogenesis in methanogens is mediated by methyl-coenzyme M reductase, an enzyme that is also responsible for the utilization of methane through anaerobic methane oxidation. The enzyme uses an ...ancillary factor called coenzyme Fsub.430, a nickel-containing modified tetrapyrrole that promotes catalysis through a methyl radical/Ni(ii)-thiolate intermediate. However, it is unclear how coenzyme Fsub.430 is synthesized from the common primogenitor uroporphyrinogen iii, incorporating 11 steric centres into the macrocycle, although the pathway must involve chelation, amidation, macrocyclic ring reduction, lactamization and carbocyclic ring formation. Here we identify the proteins that catalyse the biosynthesis of coenzyme Fsub.430 from sirohydrochlorin, termed CfbACfbE, and demonstrate their activity. The research completes our understanding of how the repertoire of tetrapyrrole-based pigments are constructed, permitting the development of recombinant systems to use these metalloprosthetic groups more widely.
We have identified a carboxylesterase produced in liquid cultures of the thermophilic actinomycete Thermobifida fusca KW3 that were supplemented with poly(ethylene terephthalate) fibers. The enzyme ...hydrolyzed highly hydrophobic, synthetic cyclic poly(ethylene terephthalate) trimers with an optimal activity at 60°C and a pH of 6. V max and K m values for the hydrolysis were 9.3 µmol⁻¹ min⁻¹ mg⁻¹ and 0.5 mM, respectively. The esterase showed high specificity towards short and middle chain-length fatty acyl esters of p-nitrophenol. The enzyme retained 37% of its activity after 96 h of incubation at 50°C and a pH of 8. Enzyme inhibition studies and analysis of substitution mutants of the carboxylesterase revealed the typical catalytic mechanism of a serine hydrolase with a catalytic triad composed of serine, glutamic acid, and histidine.
Heme d1 is a modified tetrapyrrole playing an important role in denitrification by acting as the catalytically essential cofactor in the cytochrome cd1 nitrite reductase of many denitrifying ...bacteria. In the course of heme d1 biosynthesis, the two propionate side chains on pyrrole rings A and B of the intermediate 12,18‐didecarboxysiroheme are removed from the tetrapyrrole macrocycle. In the final heme d1 molecule, the propionate groups are replaced by two keto functions. Although it was speculated that the Radical S‐adenosyl‐l‐methionine (SAM) enzyme NirJ might be responsible for the removal of the propionate groups and introduction of the keto functions, this has not been shown experimentally, so far. Here, we demonstrate that NirJ is a Radical SAM enzyme carrying two iron–sulfur clusters. While the N‐terminal 4Fe‐4S cluster is essential for the initial SAM cleavage reaction, it is not required for substrate binding. NirJ tightly binds its substrate 12,18‐didecarboxysiroheme and, thus, can be purified in complex with the substrate. By using the purified NirJ/substrate complex in an in vitro enzyme activity assay, we show that NirJ indeed catalyzes the removal of the two propionate side chains under simultaneous SAM cleavage. However, under the reaction conditions employed, no keto group formation is observed indicating that an additional cofactor or enzyme is needed for this reaction.
The heme d1 biosynthesis enzyme NirJ belongs to the Radical SAM protein family and binds two iron–sulfur clusters. One of the clusters is required for the typical SAM cleavage reaction. Overall NirJ catalyzes the removal of two propionate side chains from the substrate 12,18‐didecarboxysiroheme.
The brown alga
is one of the species successfully adapted to intertidal conditions. Inhabiting the high intertidal zone,
spends most of its life exposed to air, where it is subjected to desiccation, ...light, and temperature stresses. However, the physiological and biochemical mechanisms allowing this alga to tolerate such extreme conditions are still largely unknown. The objective of our study is to compare the biochemical composition of
during the different phases of the tidal cycle. To our knowledge, this study is the first attempt to draft a detailed biochemical network underneath the complex physiological processes, conferring the successful survival of this organism in the harsh conditions of the high intertidal zone of the polar seas. We considered the tide-induced changes in relative water content, stress markers, titratable acidity, pigment, and phlorotannin content, as well as the low molecular weight metabolite profiles (GC-MS-based approach) in
thalli. Thallus desiccation was not accompanied by considerable increase in reactive oxygen species content. Metabolic adjustment of
to emersion included accumulation of soluble carbohydrates, various phenolic compounds, including intracellular phlorotannins, and fatty acids. Changes in titratable acidity accompanied by the oscillations of citric acid content imply that some processes related to the crassulacean acid metabolism (CAM) may be involved in
adaptation to the tidal cycle.
Mass spectrometry coupled to low-temperature plasma ionization (LTPI) allows for immediate and easy analysis of compounds from the surface of a sample at ambient conditions. The efficiency of this ...process, however, strongly depends on the successful desorption of the analyte from the surface to the gas phase. Whilst conventional sample heating can improve analyte desorption, heating is not desirable with respect to the stability of thermally labile analytes. In this study using aromatic amines as model compounds, we demonstrate that (1) surface acoustic wave nebulization (SAWN) can significantly improve compound desorption for LTPI without heating the sample. Furthermore, (2) SAWN-assisted LTPI shows a response enhancement up to a factor of 8 for polar compounds such as aminophenols and phenylenediamines suggesting a paradigm shift in the ionization mechanism. Additional assets of the new technique demonstrated here are (3) a reduced analyte selectivity (the interquartile range of the response decreased by a factor of 7)-a significant benefit in non-targeted analysis of complex samples-and (4) the possibility for automated online monitoring using an autosampler. Finally, (5) the small size of the microfluidic SAWN-chip enables the implementation of the method into miniaturized, mobile LTPI probes.
Accumulation of biologically active metabolites is a specific feature of plant biochemistry, directing the use of plants in numerous applications in the pharmaceutical and food industries. Among ...these substances, the plethora of phenolic compounds has attracted particular interest among researchers. Here, we report on new findings in phlorotannin research, a large group of multifunctional phenolic substances, produced in brown algae. Comprehensive LC-MS profiling of three algal species allowed us to depict the complex pattern of this structurally diverse compound group across different tissues and subcellular compartments. We compiled more than 30 different phlorotannin series in one sample and used accurate mass spectrometry to assign tentative structures to the observed ions based on the confirmed sum formulas. From that, we found that acetylation, hydroxylation, and oxidation are likely to be the most common in vivo modifications to phlorotannins. Using an alternative data mining strategy to cope with extensive coelution and structural isomers, we quantitatively compared the intensity of different phlorotannin series in species, tissues, and subcellular compartments to learn more about their physiological functions. The structure and intra-thallus profiles of cell wall-bound phlorotannins were studied here for the first time. We suggest that one of the major dibenzodioxin-type phlorotannin series may exclusively target integration into the cell wall of fucoid algae.
A fluorimetric assay for the fast determination of the activity of polyester‐hydrolyzing enzymes in a large number of samples has been developed. Terephthalic acid (TPA) is a main product of the ...enzymatic hydrolysis of polyethylene terephthalate (PET), a synthetic polyester. Terephthalate has been quantified following its conversion to the fluorescent 2‐hydroxyterephthalate by an iron autoxidation‐mediated generation of free hydroxyl radicals. The assay proved to be robust at different buffer concentrations, reaction times, pH values, and in the presence of proteins. A validation of the assay was performed by analyzing TPA formation from PET films and nanoparticles catalyzed by a polyester hydrolase from Thermobifida fusca KW3 in a 96‐well microplate format. The results showed a close correlation (R2 = 0.99) with those obtained by a considerably more tedious and time‐consuming HPLC method, suggesting the aptness of the fluorimetric assay for a high‐throughput screening for polyester hydrolases. The method described in this paper will facilitate the detection and development of biocatalysts for the modification and degradation of synthetic polymers. The fluorimetric assay can be used to quantify the amount of TPA obtained as the final degradation product of the enzymatic hydrolysis of PET. In a microplate format, this assay can be applied for the high‐throughput screening of polyester hydrolases.
The enzymatic hydrolysis of polyethylene terephthalate (PET) can be determined by a fluorimetric assay. Terephthalate released enzymatically from PET is quantified following its conversion to the fluorescent 2‐hydroxyterephthalate by an iron autoxidation‐mediated generation of free hydroxyl radicals. The assay is robust at different buffer concentrations, reaction times, pH values, and in the presence of proteins. In a 96‐well microplate format, the method can be applied for the high‐throughput screening of polyester hydrolases.
The great research interest in the quantification of reactive carbonyl compounds (RCCs), such as methylglyoxal (MGO) in biological and environmental samples, is reflected by the fact that several ...publications have described specific strategies to perform this task. Thus, many reagents have also been reported for the derivatization of RCCs to effectively detect and quantify the resulting compounds using sensitive techniques such as liquid chromatography coupled with mass spectrometry (LC-MS). However, the choice of the derivatization protocol is not always clear, and a comparative evaluation is not feasible because detection limits from separate reports and determined with different instruments are hardly comparable. Consequently, for a systematic comparison, we tested 21 agents in one experimental setup for derivatization of RCCs prior to LC-MS analysis. This consisted of seven commonly employed reagents and 14 similar reagents, three of which were designed and synthesized by us. All reagents were probed for analytical responsiveness of the derivatives and stability of the reaction mixtures. The results showed that derivatives of 4-methoxyphenylenediamine and 3-methoxyphenylhydrazine-reported here for the first time for derivatization of RCCs-provided a particularly high responsiveness with ESI-MS detection. We applied the protocol to investigate MGO contamination of laboratory water and show successful quantification in a lipoxidation experiment. In summary, our results provide valuable information for scientists in establishing accurate analysis of RCCs.
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