Biosynthesis And The Possibility Of Using Ectoine And Hydroxyectoine In Health Care

Many authors believe that amino acids such as ectoine and hydroxyectoine provide much more effective protection against the effects of osmotic or temperature stress than other osmolytes known to date. These amino acids have a strong protective effect for enzymes and proteins. Their accumulation in the cell occurs through de novo synthesis or transportation from the external environment. Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) and its hydroxylated derivative hydroxyectoine are cyclic amino acids produced by microorganisms under stress conditions such as: salinity, high temperature, low water content environment or UV radiation. Ectoine was first isolated by Galiński et al. in 1985 [32]. This discovery took place during the study of halophilic purple bacteria Ectothiorhodospira halochloris, autotrophs which use light energy for the production of organic compounds. Ectoine and hydroxyectoine have a similar chemical structure. Hydroxyectoine differs from 1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid by the presence of a hydroxyl group at one of the carbon atoms in the aromatic ring. In all probability, microorganisms exposed to high temperature stress produce mainly hydroxyectoine [78]. In the case of salt stress, it was shown that the biosynthesis of hydroxyectoine occurs much later than the one of ectoine. This was confirmed by proving that the expression of the ectABC genes responsible for ectoine synthesis occurs earlier than the one of the ectD gene responsible for the synthesis of hydroxyectoine [91].

Ectoine belongs to the so-called compensating substances meaning that it protects against changes in the cell resulting from osmotic stress. This molecule is also referred to as a kosmotropic substance. Salt ions may have a kosmotropic effect on water, causing stabilization of the quasi-crystalline water structure or a chaotropic effect, affecting the disorganization of this structure, disturbing the polar structure of water. This is due to the interaction of a free pair of electrons with the cation and hydrogen atoms with the anion, which creates electrostatically stabilized hydration shells around the ions. Such immobilized water molecules are inaccessible to macromolecules exhibiting a hydrophobic character (e.g. proteins), because the polar solvent (water) is more strongly held by the electric field of ions with a higher charge-to-radius ratio [46]. Ectoine as a kosmotropic substance has the properties of stabilizing water molecules. The kosmotropic effect is manifested as a reduction in contact surface between water molecules and phospholipids of cell membranes. Derived from Greek κόσμος, it denotes order and refers to the ability to create orderly structures by strengthening the structure of water [31]. This phenomenon is explained by model tests carried out by Graf et al. in 2008. The dynamic simulation clearly indicates that the addition of ectoine causes a strong compensating effect consisting in the ability to re-arrange water molecules by expanding the hydrogen bond network between them [35].

Compounds having compensatory character are removed outside the hydration layer of proteins (the so-called preferential exclusion model), which explains the stabilising effect of proteins, resulting from the reduction of the surface of interaction (a phenomenon being more beneficial from the point of view of entropy) [86]. The “preferential exclusion model” is a hypothesis explaining the biophysical mechanism of the impact of ectoine on macromolecules, according to which osmoprotectants in aqueous solutions do not interact directly with macromolecules but increase the hydration of the molecule, preventing its denaturation [14, 45]. Hahn et al. showed the opposite effect of ectoine and NaCl on water molecules, using resonance Raman spectroscopy. These studies are another aspect that justifies the presence of high concentrations of osmolytes in halotolerant bacteria [38].

It turned out that the influence of ectoine and hydroxyectoine on macromolecules and cells in some aspects is mutually antagonistic [86].

The properties of ectoine cause this amino acid to be multifunctional and have a wide spectrum of applications in many industries, mainly in medicine, pharmacy, cosmetology or biotechnology. The industrial use of ectoine is based primarily on the possibility of protecting the skin and relieving inflammation (Table I), stabilizing enzymes; protection of cells and macromolecules against osmotic and temperature stress, UV radiation, desiccation (Table II, Table IV). The protective effect of ectoine described on the example of microorganisms capable of its synthesis can be also used in relation to higher organisms: human, animal and also plants.

The use of ectoine in cosmetics is based on its protective effect on the skin. It is used as a component of anti-wrinkle and moisturizing cosmetics. Human skin is a barrier between the body and the environment, so it is exposed to many external factors. The stratum corneum of the epidermis is particularly important in this regard. It has a double function in maintaining skin hydration. First, keratinized cells of the stratum corneum form a hydrophobic barrier that prevents water from entering the body through skin. Secondly, with respect to the internal environment, it maintains hydration thanks to its Natural Moisturizing Factor (NMF). It keeps water in the skin and protects it from evaporation [79]. Many environmental factors can have a destructive effect on this natural barrier, causing the skin to lose water. Skin desiccation may result, among others, from exposure to extreme temperatures, dry air, solar radiation, wind or frequent use of detergents. All this causes the skin to dry out and accelerates its aging. There are many examples in the literature confirming the anti-aging and moisturizing effect of ectoine on the skin (Table I). The first commercial use of ectoine for skin protection was related to protection against solar radiation and anti-aging activity [18]. Also, now, ectoine is widely used for this purpose [3, 4, 35]. It has been proved that ectoine protects Langerhans cells from UV radiation [10] and is responsible for blocking the release of ceramides in human epidermal keratinocytes under the influence of UVA [36]. Exposure of keratinocytes to UVA radiation, especially in humans, results in elevated levels of ceramides, and consequently activates the intracellular signalling cascade, leading to the expression of intercellular adhesion molecules. These negative effects can be effectively prevented by using ectoine, which is capable of “extinguishing” singlet oxygen [17, 36]. Büenger and Driller exposed human keratinocytes to 1 mM ectoine and UV radiation (30 J/cm²) for 24 hours. Then, they examined the release of inflammatory agents such as AP-2, ICAM-1, ceramides and showed that the initial effect of ectoine on keratinocytes leads to a decrease in the release of AP-2 inflammatory factor and increased expression of ICAM-1 adhesion molecules [17].

The cosmetics industry also uses the fact that ectoine has a stronger moisturizing effect than glycerol and ensures longer skin hydration [35]. It has been proven that the addition of 2% ectoine improves the care properties of products, causes better skin hydration, significant improvement of its elasticity and regeneration of the structure [41, 56]. Clinical studies ordered by Langsteiner LEK-Pharmaceutical company producing ectoine confirm that after 3–4 weeks of treatment, the preparation based on ectoine reduces skin dryness by up to 86% and skin desquamation by up to 70%.

The use of ectoine in pharmacy, medicine and biotechnology was influenced by its ability to protect macromolecules (Table II). Numerous studies have shown that ectoine, by forming a complex with water molecules, increases the stability of enzymes and thus reduces the susceptibility of protein to denaturation [97]. It was also shown that ectoine, like other compatible solutes, strengthens the intramolecular interactions important for protein stability [80]. Ectoine reduces the denaturation of enzymes induced by elevated temperature. It prolongs the activity of enzymes sensitive to freezing-defrosting, heating and freeze-drying, such as: lactate dehydrogenase (LDH) and phosphofructokinase [62]. In addition, it increases the stability of phytase, ribonuclease-A and DNA polymerase at elevated temperature [100]. In addition, it has been shown that an ectoine derivative – hydroxyectoine – has a better ability to protect proteins in the conditions of raised temperature [29, 90] (Table III). Ectoine can also protect macromolecules from proteolytic factors. For example, zymogen, trypsinogen and chymotrypsinogen become resistant to enteropeptidase [54]. In addition, it has been shown that ectoine and some polyols can inhibit HIV replication [58] and stabilize retrovirus vectors in gene therapy [26].

Other applications of ectoine are associated with its ability to relieve inflammation. Its protective properties have been demonstrated in the case of neutrophilic pneumonia in humans [88, 89, 93] and experimentally induced colitis in rats [1]. The administration of ectoine inhibits signalling caused by the presence of nanoparticles, which is known to be responsible for proinflammatory reactions in the epithelial cells of rat lungs. The animals which were administered the ectoine solution intrathecally prior to the introduction of carbon nanoparticles exhibited lower IL-8 expression, lower neutrophil counts in the lung, modulation of the cytokine profile, and reduced MAP kinase activation. These observations have been supported and extended by experiments on cultured human bronchial cells in which ectoine inhibited cell signalling triggered by nanoparticles and limited IL-8 induction [88, 89, 93].

Ectoine can also be used to protect the small intestine from ischaemia and reperfusion in transplantology [1]. Alleviation of the inflammatory reaction is associated with the stabilization of the intestinal barrier and the reduction of cytokine production [75]. In 2015 Bilstain et al. patented the composition of the organ storage solution with the addition of ectoine and hydroxyectoine. Further research has proved that the organ storage solution intended for transplantation – HTK (histidine-tryptophan-ketoglutarate) – modified by the addition of ectoine and hydroxyectoine exerts a beneficial effect enabling liver transplantation after cardiac arrest [84]. Cryoprotection capacity was also confirmed in umbilical cord blood cells [12] and erythrocytes [30].

Some pathological processes, such as the formation and aggregation of amyloid, trigger neurodegenerativa diseases. It was found that both ectoine and hydroxyectoine prevent the formation of amyloid (Aß42) and delay the progression of Alzheimer’s disease [48, 53, 81].

Preparations containing ectoine ensure adequate long-term moisturization of mucous membranes. Ectoine is also a natural substance performing a cell-protective function and inhibits immune reactions, including allergic reactions. Among other things, it has been investigated whether intra-tracheal administration of ectoine exerts protective effect on allergic asthma based on early allergic response (EAR), airway hyperresponsiveness (AHR) and inflammation experimentally induced in rats. The results of the study are promising, because they prove that ectoine has a significant therapeutic effect on EAR, AHR and inflammatory response in the animal model of asthma [43]. This aspect supports potential preventive and therapeutic utility of inhaling ectoine in cases of allergy and/or asthma.

It has also been demonstrated that ectoine can protect entire cells (Table IV). It increases the fluidity of cell membranes under extreme conditions [40] and increases the distance between lipid molecules, which improves membrane fluidity [28]. Other researchers report that ectoine may affect the synthesis of chaperone proteins such as heat shock proteins (Hsp), and it is also assumed that the ectoine itself may act as a chaperone molecule [7, 19]. Ectoine and some polyols make human erythrocytes more resistant to damage caused by surfactants [18]. It has been shown that this effect is stronger than in the case of lecithin (phosphatidylcholine), whose stabilizing properties are already well understood. Graf et al. studied the effect of ectoine concentration and its duration on this stabilizing effect. They proved that the higher the concentration of ectoine, the stronger the protective effect preventing damage to the membrane. A longer contact caused the membrane stability to increase by 30% after 6 hours and 60% after 24 hours. Thus, the longer cells are exposed to ectoine, the stronger the protective effect is [35]. Recently, Bownik and Stępniewska demonstrated the possibility of protecting bovine enterocytes against alpha-haemolysin of Staphylococcus aureus, which forms protein channels in the membranes of target cells [15, 16].

The current annual world production of L-amino acids significantly exceeds 2 million tons. In the industry microbiological synthesis with the participation of production strains, as well as the enzymatic and chemical synthesis method are used for production of amino acids.

The chemical synthesis of ectoine consists of the thermal cyclization of N-acetyl-benzaminobutyric acid (N-acetyl-DABA), which is formed by acetylation of L-diaminobutyric acid (L-DABA) with the participation of p-nitrophenyl acetate [59]. N-acetyl-DABA, n-butanol and triethylamine are then heated to the point of boiling for 48 hours. In this process, ectoine (60%) and N-acetyl-DABA (40%) are obtained [8]. A similar cyclization reaction takes place when the mixture of butanol and xylene (30:70, v/v) is reacted and under a reflux condenser in the presence of p-toluenesulfonic acid (20%, w/v) for 48 hours. In this case, however, less ectoine (44%) and more N-acetyl-DABA (56%) [8, 44] are obtained. The method of synthesising this compound developed and patented by Koichi et al. in 1991 is also known. This Japanese patent published under number JP-A-03031265 (Takeda Chemical Ind) concerns the chemical synthesis of ectoine from o-acetic acid trimethyl ester and various diamino carbon acids [60]. Due to the properties of ectoine and growing market interest, scientists are searching for and patenting new methods for the synthesis of ectoine. American scientists, on the basis of the method developed by Koichi et al., carried out the reaction of o-trimethyl acetate with 2,4-diaminobutyric acid [60]. Each method of chemical synthesis of amino acids is burdened with high costs resulting from expensive precursor compounds and the necessity to purify the obtained product. Moreover, the chemical synthesis method always results in a racemate, i.e. an equimolar mixture of a pair of the D and L amino acid enantiomers, with only the L-form being biologically active. Chemical synthesis therefore requires separation of the resulting enantiomeric mixture. Currently, due to the low production costs and the lack of an efficient biological alternative, the chemical method is used mainly for the production of D, L-methionine (350,000 tonnes/year).

Fig. 1.

A combination method based on the biological isomerization of a chemically obtained product may also be used for the synthesis of amino acids. Enzymatic synthesis is also possible, which has found application in industry in the production of aspartic acid, tryptophan and serine [61]. Due to the limitations of the abovementioned methods, manufacturers are searching for other, more economical methods of biotechnological production of amino acids, including ectoine [92].

The most commonly used method of obtaining amino acids is their microbiological synthesis. The largest share on the amino acid market is currently accounted for by the bioproduction of L-glutamic acid (1.5 million tonnes per year) and L-lysine (850 thousand tonnes per year) [34]. Currently, there is also a growing demand for other amino acids, including ectoine and hydroxyectoine. Biotechnological production of these amino acids is based on the use of the possibility of their synthesis by microorganisms. It is believed that ectoine is currently one of the most valuable products synthesized by microorganisms. The global consumption of ectoine is 15,000 tons per year and retail sales, only in the pharmaceutical industry, are estimated at 1,000 USD per kg [25, 87]. The mechanism of the biosynthesis of ectoine and hydroxyectoine is similar to that of other amino acids such as: L-lysine, L-methionine, L-threonine (Fig. 1). The first stage of the synthesis is the phosphorylation of L-aspartate and its conversion to 4-phospho-L-aspartate by aspartate kinase (Ask enzyme). Then, from 4-phospho-L-aspartate, L-aspartic acid 4-semialdehyde is formed. This reaction is catalysed by L-aspartate-β-semialdehyde dehydrogenase (Asd enzyme). In turn, L-aspartic acid 4-semialdehyde is converted to L-2,4-diaminobutyrate by diaminobutyric acid aminotransferase (enzyme EctB). The next stage is the transformation of the resulting acid by diaminobutyric acid acetyltransferase (EctA enzyme) to Nγ-acetyl-L-2,4-diaminobutyric acid. Another enzyme involved in the biosynthesis of ectoine is ectoine synthase (EctC), which converts Nγ-acetyl-L-2,4-diaminobutyric acid to 1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid (ectoine). Some bacteria contain ectoine hydroxylase (EctD), which allows the transformation of ectoine to hydroxyectoine (Fig. 1) [77, 92].

Biotechnological production of ectoine on an industrial scale consists of several stages. The stage of preparing the inoculum, usually it is a laboratory phase, in which the production strain is revived, and the culture is prepared in a volume of a few litres in small laboratory fermenters. The second stage is the main biosynthesis process. At each of these stages monitoring of the growth of microorganisms and control of physicochemical parameters of the culture is required. The proper biosynthesis process includes: the upstream stage, i.e. fermentation and production of the substance and the downstream stage, in which the desired substance is isolated, purified and analysed [74, 86].

The first process developed for the biotechnological production of ectoine, the so-called “bacterial milking”, was developed by Sauer and Galinski in 1998 and is currently used on an industrial scale. It allows release of synthesized osmolytes outside the cell, without degradation of the bacterial biomass. The water solubility of ectoine at 25°C is 6.5 mol/kg and it is collected in the cytoplasm of bacterial cells at a concentration up to 1 M [42]. Halophilic bacteria (e.g. Halomonas elongata), not only have adaptation mechanisms that enable them to function in an environment with a high salinity but are also adapted to a sudden drop in salinity caused by, among others, rainfall or floods. In this situation, the concentration of salt in the cytoplasm of these organisms is higher than outside the cell and there may be a sudden inflow of water from the external environment to the bacterial cell. In order to avoid lysis, the cell releases ectoine and other osmolytes outside. This is probably possible owing to mechanosensitive channels. In the case of H. elongata, three MscS channels (mechanosensitive channel of small conductance) and one MscK channel (potassium-dependent mechano-sensitive channel) has been found to be present [56]. Therefore, the main factors in the process of “bacterial milking” are the induction of osmotic shock followed by the introduction into a substrate with a higher chemical potential (lower osmolarity). Gram-negative bacteria are more sensitive to external salinity changes [63], while Gram-positive bacteria due to the multi-layered structure of murein are not so sensitive to osmotic changes and do not secrete osmoprotectants accumulated intracellularly during the “milking” process [74].

Biosynthesis of ectoine on an industrial scale is currently done mainly by the German company Bitop (Witten, Germany) which uses Halomonas elongata, isolated from the systems used for salt production on the Dutch island of Bonaire in the Caribbean Sea [94], in the production of ectoine. Bacteria are cultivated in fermenters with a capacity of 1000 and 3500 l, at a temperature of 25–40°C. They exhibit optimum growth at NaCl concentration between 3 and 6% NaCl but are able to grow on the substrate with the addition of up to 20% NaCl. Bacteria reach high cell density > 40 g dry weight per litre of culture. Ectoine is obtained in the above – -described process of “bacterial milking”, in which bacteria grown in the presence of 10% NaCl are subjected to osmotic shock by lowering the salinity to 2% NaCl. As a result, 80% of the produced ectoine is released into the medium outside the cell. In this way through the application of Halomonas elongata ectoine is produced in an amount of over 10 g/l [56]. The above-described biotechnological production of this osmolyte by H. elongata, although being a process which supplies a relatively large amount of the product, is also a long-term (120 hours) and quite an expensive process, mainly due to the high quality requirements of the substrate and costs associated with purification or final preparation of the product [25, 56, 74]. Therefore, all work related to the search for new, efficient, easy to cultivate strains for the production of ectoine is justified and much needed.

In addition to the Halomonas elongata which is used to produce ectoine on an industrial scale, many microorganisms have the ability to synthesize this osmolyte. The synthesis of ectoine and hydroxyectoine in response to high osmolarity of the environment is widespread mainly among bacteria. The analysis of 557 genomes of the representatives of Archaea domain showed that only 12 strains of the genera: Nitrosopumilus, Methanothrix and Methanobacterium have a gene cluster responsible for the synthesis of ectoine and hydroxyectoine [95]. Among the bacteria able to synthesize ectoine and/or hydroxyectoine, representatives of both Actinobacteria, Firmicutes, as well as alpha-, gamma-, delta-Proteobacteria types are listed. Most of them are microorganisms which use sugars as a source of carbon, among others, bacteria of the genera Streptomyces, Bacillus, Pseudomonas, Chromohalobacter or Halomonas.

Within the genus Halomonas, apart from H. elongata, other species are also characterized by relatively high production of ectoine, but a much lower level of its excretion outside the cell. Zhang et al. proved that H. salina produces about 923 mg/l, but the level of extracellular ectoine remains small, i.e. about 11% of the total ectoine. Moreover, it has been shown that in this case the synthesis does not proceed directly in proportion to the salinity, and the maximum synthesis was obtained at a salt concentration of 1.4% NaCl [98]. However, Halomonas boliviensis bacteria accumulate ectoine inside cells at a maximum level of 0.74 g/l in the presence of 10–15% (w/v) NaCl, while the synthesis of hydroxyectoine under these conditions is 50 mg/l [37].

In addition, as proved by Bursy et al., Streptomyces coelicolor, at the optimum growth temperature in the presence of 1.4% NaCl produces ectoine at the level of 10–55 μmol/g_dw. In turn, at elevated temperature, i.e. 39°C the synthesis of the hydroxyl derivative of ectoine predominates, reaching about 60 μmol/g_dw [20]. Also the bacteria of the genus Bacillus [21] are known as producers of ectoine. Kuhlmann and Bremer demonstrated that B. pasteurii strain (DSM 33T) synthesizes ectoine at the level of 0.36 to 0.59 mmol/g_dw [2, 55]. Other bacteria commonly occurring in the environment, in which the ability to synthesize ectoine was confirmed, are bacteria of the genus Pseudomonas. Seip et al. demonstrated that they synthesize ectoine at a maximum level of about 50 μmol/g_dw and that its concentration increases along with an increase in salinity in the range of 2–7.5% NaCl [82]. Another example are bacteria of the genus Chromohalobacter [22, 33, 73, 83]. It has been shown that these bacteria produce a maximum of 1.2 mmol of ectoine per gram of dry weight of bacteria, which makes them more efficient in the production of ectoine than the above-described Streptomyces sp., Bacillus sp. and Pseudomonas sp.

Halophiles are microorganisms with a high biotechnological potential associated with the production of enzymes, β-carotene or osmolytes. In order to maintain the osmotic balance between the cytoplasm and the substrate in high salinity conditions, these microorganisms developed two basic strategies. The first mechanism concerns the maintenance of high concentration of potassium ions inside the cell. The second strategy involves the biosynthesis of organic osmotic solutes such as sugars (e.g. trehalose), amino acids (e.g. glycine, betaine, glutamic acid, L-proline, ectoine, hydroxyectoine) and polyols (e.g., glycerol). The protective effect of compatible compounds described on the example of microorganisms capable of synthesizing them may also be used in relation to other organisms: human, animals or plants. In recent years, a rapid increase in the demand for ectoine has been observed, mainly due to its properties related to the health protection, and especially that of skin and mucous membranes. Pharmaceutical companies are increasingly willing to test and introduce products in which ectoine is the active substance.