Od PET lahví k dobrotám

The treatment of waste PET bottles has become a pressing global issue over the last few decades, and many scientific teams are currently working on solutions to it. There are many different approaches of how to solve this problem. The present article outlines the possibility to process terephthalic acid, which is the hydrolysis product of polyethylene terephthalate, into vanillin, a compound widely used in the food industry. The work of British scientists who have succeeded in using genetic modification to produce a strain of E. coli RARE_pVanX capable of processing polyethylene terephthalate hydrolysates to the desired vanillin is presented in a broader context.

Eradication of Myrosinase-Tethered Cancer Cells by Allyl Isothiocyanate Derived from Enzymatic Hydrolysis of Sinigrin

Sinigrin is present in significant amounts in cruciferous vegetables. Epidemiological studies suggest that the consumption of such vegetables decreases the risk of cancer, and the effect is attributed mainly to allyl isothiocyanate (AITC), a hydrolysis product of sinigrin catalyzed by myrosinase. Anticancer activity of AITC has been previously investigated for several cancer models, but less attention was paid to delivering AITC on the target site. In this study, the gene sequences of core streptavidin (coreSA) and myrosinase (MYR) were cloned in a pET-30a(+) plasmid and transformed into BL21(DE3) E. coli competent cells. The MYR-coreSA chimeric protein was expressed and purified using immobilized metal affinity chromatography and further characterized by gel electrophoresis, Western blot, and enzyme activity assay. The purified MYR-coreSA chimeric protein was tethered on the outer membrane of biotinylated adenocarcinoma A549 cells and then treated with various concentrations of sinigrin. Our results showed that 20 µM of sinigrin inhibited the growth of A549 cells tethered with myrosinase by ~60% in 48 h. Furthermore, the levels of treated cells undertaken apoptosis were determined by Caspase-3/7 activation and Annexin-V. In summary, sinigrin harnessed like a prodrug catalyzed by myrosinase to the production of AITC, which induced cell apoptosis and arrested the growth of lung cancer cells.

Molecular Dynamics of DNA Translocation by FtsK

The bacterial FtsK motor harvests energy from ATP to translocate double-stranded DNA during cell division. Here, we probe the molecular mechanisms underlying coordinated DNA translocation in FtsK by performing long timescale simulations of its hexameric assembly and individual subunits. From these simulations we predict signaling pathways that connect the ATPase active site to DNA-gripping residues, which allows the motor to coordinate its translocation activity with its ATPase activity. Additionally, we utilize well-tempered metadynamics simulations to compute free-energy landscapes that elucidate the extended-to-compact transition involved in force generation. We show that nucleotide binding promotes a compact conformation of a motor subunit, whereas the apo subunit is flexible. Together, our results support a mechanism whereby each ATP-bound subunit of the motor conforms to the helical pitch of DNA, and ATP hydrolysis/product release causes a subunit to lose grip of DNA. By ordinally engaging and disengaging with DNA, the FtsK motor unidirectionally translocates DNA.

Photolysis of the Insensitive Explosive 1,3,5-Triamino-2,4,6-trinitrobenzene (TATB)

The explosive 1,3,5-triamino-2,4,6-trinitrobenzene (TATB) is of particular interest due to its extreme insensitivity to impact, shock and heat, while providing a good detonation velocity. To determine its fate under environmental conditions, TATB powder was irradiated with simulated sunlight and, in water, under UV light at 254 nm. The hydrolysis of particles submerged in neutral and alkaline solutions was also examined. We found that, by changing experimental conditions (e.g., light source, and mass and physical state of TATB), the intermediates and final products were slightly different. Mono-benzofurazan was the major transformation product in both irradiation systems. Two minor transformation products, the aci-nitro form of TATB and 3,5-diamino-2,4,6-trinitrophenol, were detected under solar light, while 1,3,5-triamino-2-nitroso-4,6-dinitrobenzene, 1,3,5-triamino-2,4-dinitrobenzene and mono-benzofuroxan were produced under UV light. The product identified as 3,5-diamino-2,4,6-trinitrophenol was identical to the one formed in the dark under alkaline conditions (pH 13) and in water incubated at either 50 °C or aged at ambient conditions. Interestingly, when only a few milligrams of TATB were irradiated with simulated sunlight, the aci-isomer and mono-benzofurazan derivative were detected; however, the hydrolysis product 3,5-diamino-2,4,6-trinitrophenol formed only much later in the absence of light. This suggests that the water released from TATB to form mono-benzofurazan was trapped in the interstitial space between the TATB layers and slowly hydrolyzed the relatively stable aci-nitro intermediate to 3,5-diamino-2,4,6-trinitrophenol. This environmentally relevant discovery provides data on the fate of TATB in surface environments exposed to sunlight, which can transform the insoluble substrate into more soluble and corrosive derivatives, such as 3,5-diamino-2,4,6-trinitrophenol, and that some hydrolytic transformation can continue even without light.

Revealing a hidden intermediate of rotatory catalysis with X-ray crystallography and Molecular simulations

The mechanism of rotatory catalysis in ATP-hydrolyzing molecular motors remain an unresolved puzzle in biological energy transfer. Notwithstanding the wealth of available biochemical and structural information inferred from years of experiments, knowledge on how the coupling between the chemical and mechanical steps within motors enforces directional rotatory movements remains fragmentary. Even more contentious is to pinpoint the rate-limiting step of a multi-step rotation process. Here, using Vacuolar or V1-type hexameric ATPase as an exemplary rotational motor, we present a model of the complete 4-step conformational cycle involved in rotatory catalysis. First, using X-ray crystallography a new intermediate or 'dwell' is identified, which enables the release of an inorganic phosphate (or Pi) after ATP hydrolysis. Using molecular dynamics simulations, this new dwell is placed in a sequence with three other crystal structures to derive a putative cyclic rotation path. Free-energy simulations are employed to estimate the rate of the hexameric protein transfor-mations, and delineate allosteric effects that allow new reactant ATP entry only after hydrolysis product exit. An analysis of transfer entropy brings to light how the sidechain level interactions transcend into larger scale reorganizations, highlighting the role of the ubiquitous arginine-finger residues in coupling chemical and mechanical information. Inspection of all known rates encompassing the 4-step rotation mechanism implicates overcoming of the ADP interactions with V1-ATPase to be the rate-limiting step of motor action.

Biocatalytic quantification of α-glucan in particulate marine organic matter

Marine algae drive the marine carbon cycle, converting carbon dioxide into organic material. A major component of this produced biomass is a variety of glycans; and yet their chemical composition and individual involvement in production, sedimentation and bacterial uptake remain largely unknown due to a lack of analytical tools for glycan-specific quantification. Marine α-glucans include a range of storage glycans from red and green algae, bacteria, fungi and animals. Although these compounds are likely to account for a high amount of the carbon stored in the oceans they have not been quantified in marine samples so far. Here we present a method to extract and quantify α-glucans (and compare it with the β-glucan laminarin) in particulate organic matter from algal cultures and environmental samples using a sequential physicochemical extraction and enzymes as α-glucan-specific probes. This enzymatic assay is more specific and less susceptible to side reactions than chemical hydrolysis. Using HPAEC-PAD to detect the hydrolysis products allows for a glycan quantification in particulate marine samples down to a concentration of ≈ 2 μg/L. We measured glucans in three cultured microalgae as well as in marine particulate organic matter from the North Sea and western North Atlantic Ocean. While the β-glucan laminarin from diatoms and brown algae is an essential component of marine carbon turnover, our results further indicate the significant contribution of starch-like α-glucans to marine particulate organic matter. Henceforth, the combination of glycan-linkage-specific enzymes and chromatographic hydrolysis product detection can provide a powerful tool in the exploration of marine glycans and their role in the global carbon cycle.

Pyruvic acid, an efficient catalyst in SO₃ hydrolysis and effective clustering agent in sulfuric acid-based new particle formation

The role of pyruvic acid (PA), one of the most abundant α-keto carboxylic acids in the atmosphere, was investigated both in the SO3 hydrolysis reaction to form sulfuric acid (SA) and in SA-based aerosol particle formation using quantum chemical calculations and a cluster dynamics model. We found that the PA-catalyzed SO3 hydrolysis is a thermodynamically driven transformation process, proceeding with a negative Gibbs free energy barrier, ca. −1 kcal mol−1 at 298 K, ~6.50 kcal mol−1 lower than that in the water-catalyzed SO3 hydrolysis. Results indicated that the PA-catalyzed reaction can potentially compete with the water-catalyzed SO3 reaction in SA production, especially in dry and polluted areas, where it is found to be ~two orders of magnitude more efficient that the water-catalyzed reaction. Given the effective stabilization of the PA-catalyzed SO3 hydrolysis product as SA•PA cluster, we proceeded to examine the PA clustering efficiency in sulfuric acid-pyruvic acid-ammonia (SA-PA-NH3) system. Our thermodynamic data used in the Atmospheric Cluster Dynamics Code indicated that under relevant tropospheric temperatures and concentrations of SA (106 cm3), PA (1010 cm3) and NH3 (1011 and 5 × 1011 cm3), of the PA-containing clusters, only clusters with one PA molecule, namely (SA)2•PA•(NH3)2, can participate to the particle formation, contributing by ~100 % to the net flux to aerosol particle formation at 238 K, exclusively. At higher temperatures (258 K and 278 K), however, the net flux to the particle formation is dominated by pure SA-NH3 clusters, while PA would rather evaporate from the clusters at high temperatures and not contribute to the particle formation. The enhancing effect of PA of examined by evaluating the ratio of the ternary SA-PA-NH3 cluster formation rate to binary SA-NH3 cluster formation rate. Our results show that while the enhancement factor of PA to the particle formation rate is almost insensitive to investigated temperatures and concentrations, it can be as high as 4.7 × 102 at 238 K and [NH3] = 1.3 × 1011 molecule cm−3. This indicates that PA may actively participate in aerosol formation, only in cold regions of the troposphere and highly NH3-polluted environments. The inclusion of this mechanism in aerosol models may definitely reduce uncertainties that prevail in modeling the aerosol impact on climate.

A chemical screen for suppressors of dioxygenase inhibition in a yeast model of familial paraganglioma

A fascinating class of familial paraganglioma (PGL) neuroendocrine tumors is driven by loss of the tricarboxylic acid (TCA) cycle enzyme succinate dehydrogenase (SDH) resulting in succinate accumulation as an oncometabolite, and other metabolic derangements. Here we exploit a S. cerevisiae yeast model of SDH loss where accumulating succinate, and possibly reactive oxygen species, poison a dioxygenase enzyme required for sulfur scavenging. Using this model we performed a chemical suppression screen for compounds that relieve dioxygenase inhibition. After testing 1280 pharmaceutically-active compounds we identified meclofenoxate HCL, and its hydrolysis product, dimethylaminoethanol (DMAE), as suppressors of dioxygenase intoxication in SDH-loss cells. We show that DMAE acts to alter metabolism so as to normalize the succinate:2-ketoglutarate ratio, improving dioxygenase function. This work raises the possibility that oncometabolite effects might be therapeutically suppressed by drugs that rewire metabolism to reduce the flux of carbon into pathological metabolic pathways.

Use of Rapid Capillary Zone Electrophoresis to Determine Amino Acids Indicators of Herring Ripening during Salting

Currently, herring fillets are salted with acetic acid to activate muscle proteases. This causes a change in the composition of free amino acids, compared to salting of whole fish with viscera proteases. Therefore, old indicators of the ripening dynamics of salted fish based on amino acids are not current. Determination of free amino acids can be performed by many methods, but most are labor intensive and expensive. Therefore, a capillary electrophoresis method without derivatization (CZE) was used to determine the actual ripening rates of salted herring fillets. A group of hydrophobic and basic amino acids were determined in trichloroacetic acid (TCA) extracts of meat and brine to develop 16 indicators. Statistical regression analysis of the indicators (R2adj, RMSE, cluster analysis) followed by principal component analysis (PCA) correlation analysis of the indicators vs sensory evaluation parameters of texture and TPA-hardness of salted fillet meat allowed the choice of the most precise indicators. The best indicator in meat was Phe/Tyr-height, which value increased during salting. A more precise indicator of ripening was His/Tyr-height in brine, which value decreased during salting. Sensory evaluation parameters of salted herring texture correlated strongly with TPA-hardness and traditional indicators such as non-protein nitrogen and protein hydrolysis product fraction content. However, the most precise indicators were those obtained from amino acids determined by the CZE method. Results obtained in this study may be suitable for fast monitoring of the salted herring ripening process in industry.

Characterization of Conjugates between α-Lactalbumin and Benzyl Isothiocyanate—Effects on Molecular Structure and Proteolytic Stability

In complex foods, bioactive secondary plant metabolites (SPM) can bind to food proteins. Especially when being covalently bound, such modifications can alter the structure and, thus, the functional and biological properties of the proteins. Additionally, the bioactivity of the SPM can be affected as well. Consequently, knowledge of the influence of chemical modifications on these properties is particularly important for food processing, food safety, and nutritional physiology. As a model, the molecular structure of conjugates between the bioactive metabolite benzyl isothiocyanate (BITC, a hydrolysis product of the glucosinolate glucotropaeolin) and the whey protein α-lactalbumin (α-LA) was investigated using circular dichroism spectroscopy, anilino-1-naphthalenesulfonic acid fluorescence, and dynamic light scattering. Free amino groups were determined before and after the BITC conjugation. Finally, mass spectrometric analysis of the BITC-α-LA protein hydrolysates was performed. As a result of the chemical modifications, a change in the secondary structure of α-LA and an increase in surface hydrophobicity and hydrodynamic radii were documented. BITC modification at the ε-amino group of certain lysine side chains inhibited tryptic hydrolysis. Furthermore, two BITC-modified amino acids were identified, located at two lysine side chains (K32 and K113) in the amino acid sequence of α-LA.