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Industrial production of the cell protectant ectoine: protection mechanisms, processes, and products
(2014)
Bacteria, Ärchaea and Eukatya can adapt to saline environments by accumulating compatible solutes in order to maintain an osmotic equilibrium. Compatible solutes are of diverse Chemical structure (sugars, polyols, amino acid derivatives) and are beneficial for bacterial cells not only as osmoregulatory solutes, but also as protectants of proteins by mitigating detrimental effects of ffeezing, drying and high temperatures. The aspartate derivative ectoine is a wide spread compatible solute in Bacteria and possesses additional protective properties compared with other compatible solutes, and stabilizes even vvhole cells against stresses such as UV radiation or cytotoxins. The protective properties of ectoine for proteins can be explained by its strong (kosmotropic) interaction with water and subsequent exclusion ffom Protein surface, the decrease of the solubility of the peptide backbone and the strengthening of intramolecular hydrogen bonds (secondary structures). The stabilizing and UV-protective properties of ectoine attracted industry, which saw the potential to market ectoine as a novel active component in health care products and cosmetics. In joint efforts of industry and research large-scale fermentation procedures have been developed with the halophilic bacterium Halomonas elongata used as a producer strain. The two key technologies that allow for the annual production of ectoine on a scale of tons are the bacterial milking procedure and the development and application of ectoine-excreting mutants (“leaky” mutant). The details of these two procedures including the strain development and fermentation processes will be introduced and current and future applications of ectoine will be discussed.
Industrial production of the cell protectant ectoine: protection mechanisms, processes, and products
(2014)
Bacteria, Archaea and Eukarya can adapt to Saline environments by accumulating compatible solutes in order to maintain an osmotic equilibrium. Compatible solutes are of diverse chemical structure (sugars, polyols, amino acid derivatives) and are beneficial for bacterial cells not only as osmoregulatory solutes, but also as protectants of proteins by mitigating detrimental effects of freezing, drying and high temperatures. The aspartate derivative ectoine is a wide spread compatible solute in Bacteria and possesses additional protective properties compared with other compatible solutes, and stabilizes even whole cells against stresses such as UV radiation or cytotoxins. The protective properties of ectoine for proteins can be explained by its strong (kosmotropic) interaction with water and subsequent exclusion from Protein surface, the decrease of the solubility of the peptide backbone and the strengthening of intramolecular hydrogen bonds (secondary structures). The stabilizing and UV-protective properties of ectoine attracted industry, which saw the potential to market ectoine as a novel active component in health care products and cosmetics. In joint efforts of industry and research large-scale fermentation procedures have been developed with the halophilic bacterium Halomonas elongata used as a producer strain. The two key technologies that allow for the annual production of ectoine on a scale of tons are the bacterial milking procedure and the development and application of ectoine-excreting mutants (“leaky” mutant). The details of these two procedures including the strain development and Fermentation processes will be introduced and current and future applications of ectoine will be discussed.
Numerous membrane importers rely on accessory water-soluble proteins to capture their substrates. These substrate-binding Proteins (SBP) have a strong affinity for their ligands; yet, Substrate release onto the low-affinity membrane transporter must occur for uptake to proceed. It is generally accepted that release is facilitated by the association of SBP and transporter, upon which the SBP adopts a conformation similar to the unliganded state, whose affinity is sufficiently reduced. Despite the appeal of this mechanism, however, direct supporting evidence is lacking. Here, we use experimental and theoretical methods to demonstrate that an allosteric mechanism of enhanced substrate release is indeed plausible.
First, we report the atomic-resolution structure of APO TeaA, the SBP of the Na!-coupled ectoine TRAP transporter TeaBC from Halomonas elongata DSM2581T, and compare it with the substrate-bound structure previously reported. Conformational freeenergy landscape calculations based upon molecular Dynamics simulations are then used to dissect the mechanism that couples ectoine binding to structural change in TeaA. These insights allow us to design a triple mutation that biases TeaA toward apo-like conformations without directly perturbing the binding cleft, thus mimicking the influence of the membrane transporter. Calorimetric measurements demonstrate that the ectoine affinity of the conformationally biased triple mutant is 100-fold weaker than that of the wild type. By contrast, a control mutant predicted to be conformationally unbiased displays wild-type affinity. This work thus demonstrates that substrate release from SBPs onto their Membrane transporters can be facilitated by the latter through a mechanism of allosteric modulation of the former.
The halophilic bacterium Halomonas elongata takes up the compatible solute ectoine via the osmoregulated TRAP transporter TeaABC. A fourth orf (teaD) is located adjacent to the teaABC locus that encodes a putative universal stress protein (USP). By RT-PCR experiments we proved a cotranscription of teaD along with teaABC. Deletion of teaD resulted in an enhanced uptake for ectoine by the Transporter TeaABC and hence a negative activity regulation of TeaABC by TeaD. Atranscriptional regulation viaDNA binding could be excluded. ATP binding to native TeaD was shown by HPLC, and the Crystal structure of TeaD was solved in complex with ATP to a resolution of 1.9 A ° by molecular replacement. TeaD forms a dimer-dimer complex with one ATP molecule bound to each monomer, which has a Rossmann-like R/β overall fold. Our results reveal an ATP-dependent oligomerization of TeaD, which might have a functional role in the regulatory mechanism of TeaD. USP-encoding orfs, which are located adjacent to genes Encoding for TeaABC homologues, could be identified in several other organisms, and their physiological role in balancing the internal cellular ectoine pool is discussed.
Only a few myxobacteria are known to date that are classified as marine, owing to their salt dependency. In this study, the salt tolerance mechanism of these bacteria was investigated. To this end, a growth medium was designed in which the mutated Escherichia coli strain BKA13 served as sole food source for the predatory, heterotrophic myxobacteria. This enabled measurement of the osmolytes without any background and revealed that the closely related strains Enhygromyxa salina SWB007 and Plesiocystis pacifica SIR-1 developed different strategies to handle salt stress. Ple. pacifica SIR-1, which was grown between 1 and 4% NaCl, relies solely on the accumulation of amino acids, while Enh. salina SWB007, which was grown between 0.5 and 3% NaCl, employs, besides betaine, hydroxyectoine as the major compatible solute. In accordance with this analysis, only in the latter strain was a locus identified that codes for genes corresponding to the biosynthesis of betaine, ectoine and hydroxyectoine.
The compatible solute ectoine is a versatile protectant synthesized by many prokaryotes. It is used as an osmolyte helping microorganisms to maintain an osmotic equilibrium. In addition, ectoine acts as a stabilizer and protects proteins, membranes and whole cells against detrimental effects such as freezing and thawing, drying and high temperatures. Its protective effect is explained by the preferential exclusion model, which postulates that ectoine does not directly interact with biomolecules but is excluded from their surface. Interestingly, details on the interaction of ectoine with DNA are still unknown. Therefore, we studied the influence of ectoine on DNA and the mechanisms by which ectoine protects DNA against ionizing radiation. To emulate biological conditions, we used a sample holder comprising a silicon chip with a Si3N4 membrane, which allows for electron irradiation of DNA in aqueous solution. Analysis by atomic force microscopy revealed that without ectoine, DNA was damaged by irradiation with a dose of 1,7 +/-0,3 Gy. With ectoine, DNA remained undamaged, even after irradiation with 15 Gy. Simulations with dsDNA and ectoine in water revealed a preferential binding of the zwitterionic ectoine to the negatively charged DNA. According to the simulations, binding of ectoine will destabilize dsDNA. Destabilizing is probably caused by the transition of B-DNA to A-DNA and will reduce the DNA melting temperature, which was experimentally proven. The preferential binding provides a stable ectoine shell around DNA, which allows ectoine to reduce OH-radicals and electrons near the DNA and thereby mitigating the damaging effect of ionizing radiation.
The compatible solute ectoine: protection mechanisms, strain development, and industrial production
(2020)
Bacteria, Archaea, and Eukarya can adapt to saline environments by accumulating compatible solutes in order to maintain an osmotic equilibrium. Compatible solutes are of diverse chemical structure (sugars, polyols, amino acid derivatives) and are beneficial for bacterial cells not only as osmoregulatory solutes but also as
protectants of proteins by mitigating detrimental effects of freezing, drying, and high temperatures. The aspartate derivative ectoine is a widespread compatible solute in Bacteria and possesses additional protective properties compared with other compatible solutes and stabilizes even whole cells against stresses such as ultraviolet radiation or cytotoxins. Here, it is our intention to go beyond a simple description of effects, but to depict the molecular interaction of ectoine with biomolecules, such as proteins, membranes, and DNA and explain the underlying principles. The stabilizing properties of ectoine attracted industry, which saw the potential to market ectoine
as a novel active component in health care products and cosmetics. In joint efforts of industry and research, a large-scale fermentation procedure has been developed with the halophilic bacterium Halomonas elongata used as a producer strain. The development and application of ectoine-excreting mutants from H. elongata (“leaky” mutants) allow for the annual production of ectoine on a scale of tons. The details of
the strain development and fermentation processes will be introduced.
The influence of interfacial pH between AISI 4135 steel and seawater under different polarization potentials on the formation of calcareous deposits has been studied. An interfacial pH of 9.61 at −0.9 V vs. SCE using state of the art iridium oxide microelectrode was found to be the critical pH for the precipitation of magnesium hydroxide. Calcareous deposits with a double-layer structure comprising an inner-brucite layer and an outer-aragonite layer were found to form at potentials between −1.0 V and −1.2 V vs. SCE. Furthermore, the facilitation of hydrogen permeation into steel induced by the formation of calcareous deposits was verified using the Devanathan-Stachurski electrochemical test. The mechanism of calcareous deposits facilitates hydrogen permeation into steel is related to its inhibition on hydrogen recombination and escape processes.
The survey of Canadian shale sites showed a dominance of halophilic microorganisms, including Halomonas (HA). Nitrate-amended incubations of the field samples under high salinity (14.6% NaCl), revealed a dominance of HA (>72%) and an accumulation of nitrite. Nitrite accumulation directly inhibited the growth of SRB, thereby decreasing their souring and corrosion risks. However, accumulated nitrite may also contribute to iron corrosion, which will be tested by using different concentrations of nitrate as an electron acceptor to HA. Different salinities are further tested on HA strains supplemented with iron coupons to determine their effects on iron corrosion rates. HA incubated with separate cultures of corrosive methanogen and SRB were tested to determine whether a positive or adverse effect will occur between them. Lastly, analyses of iron coupons will be conducted using TOF-SIMS, FIB-SEM and EDS for corrosion product characterization
Long Term Temperature and Humidity Evolution Forecast in Near Field of Nuclear Waste Container
(2018)
The paper aims to simulate the corrosion environment of nuclear waste containers at different geological disposal periods to obtain temperature and humidity information at the interface between the container and the surrounding environment. The simulated data and calculation results of long term temperature evolution at the surface of nuclear waste (HLW) containers from some typical nuclear countries about the safety disposition were reviewed. Combining different burial patterns, this paper speculated the long term temperature evolution rule for China. According to the study about saturation variation of buffer/backfill material at home and abroad, the humidity evolution of bentonite at the surface of HLW containers was specu- lated. The study showed that the temperature of the container surface increased rapidly at the beginning, and gradually decreased after the climax. For safety reasons, the maximum temperature was designed below 100°C. The saturation of bentonite was af- fected by the mutual influences of the heat released by nuclear waste decay and the infiltration of groundwater. It was dominated by the released heat in the early stage, and later was influenced greatly by the infiltration of groundwater. It is generally believed that the water content at the surface of the container will increase obviously in about 3 years, and will be saturated in about 10 years. The prediction of long-term temperature and humidity evolution will lay a foundation for study of corrosion evolution of nuclear waste containers in China.
Surface environment changes of nuclear waste container, mainly including oxygen contents, buffer pore water components and pH under the conditions of “borehole” type and “In-floor” type with bentonite buffer/backfill and concrete buffer were summarized. This summarization provides a basic corrosion environment reference for the corrosion evolution re- search of high-level radioactive waste disposal repository in our country.
In dosimetry the determination of the effectiveness of the damaging processes is standardized and accounted for by the radiation and tissue weighting factor. For the underlying constituents of the tissue, that is the various biomolecules, such a systematic approach doesn't exist. This makes it difficult to compare results obtained under different experimental conditions. In the following work, we will describe a method to obtain comparable values for the radiation-biomolecule interaction, measured under different conditions. This approach can lead to standardization of dosedamage relationship at the molecular level. Such approach is necessary for a better understanding of the relations between the damage of the single constituents of biological tissue and the whole – finally gaining a more complete picture of irradiation damage.