Appl Microbiol Biotechnol (2006) 72:623–634

DOI 10.1007/s00253-006-0553-9

MINI-REVIEW

Extremolytes: natural compounds from extremophiles

for versatile applications
Georg Lentzen & Thomas Schwarz

Received: 2 May 2006 / Revised: 20 June 2006 / Accepted: 20 June 2006 / Published online: 7 September 2006
# Springer-Verlag 2006

Abstract Extremophilic microorganisms have adopted a Introduction

variety of ingenious strategies for survival under high or
low temperature, extreme pressure, and drastic salt concen- The effectiveness of salt treatment against the growth of
trations. A novel application area for extremophiles is the harmful microorganisms has been used for food
use of “extremolytes,” organic osmolytes from extremo- preservation for thousands of years and it was therefore
philic microorganisms, to protect biological macromole- long believed that at a salt concentration in access of
cules and cells from damage by external stresses. In 100 g NaCl/l, microbial growth is impossible. The
extremophiles, these low molecular weight compounds are discovery of extremophiles, organisms that thrive under
accumulated in response to increased extracellular salt high salt concentration, high temperature, and other
concentrations, but also as a response to other environmen- stresses has changed this picture (for review, see
tal changes, e.g., increased temperature. Extremolytes Rothschild and Mancinelli 2001). Organisms that are
minimize the denaturation of biopolymers that usually capable of growing at high salt concentration achieve
occurs under conditions of water stress and are compatible osmotic balance across the cell membrane by two different
with the intracellular machinery at high (>1 M) concen- strategies. The first mechanism, which is typical for the
trations. The ectoines, as the first extremolytes that are archaeal Halobacteriaceae, uses inorganic solutes (potas-
produced in a large scale, have already found application as sium and chloride ions) to achieve osmotic equilibrium.
cell protectants in skin care and as protein-free stabilizers of This method leads to a relatively narrow adaptation for a
proteins and cells in life sciences. In addition to ectoines, a specific high-salt environment. The second mechanism is
range of extremolytes with heterogenous chemical struc- widely used in eubacterial halophiles and employs the
tures like the polyol phosphates di-myoinositol-1,1′-phos- accumulation of small organic molecules of low molecular
phate, cyclic 2,3-diphosphoglycerate, and α-diglycerol weight to counterbalance the salt concentration in the
phosphate and the mannose derivatives mannosylglycerate environment. These small molecules are chemically
(firoin) and mannosylglyceramide (firoin-A) were charac- diverse and can be zwitterionic, uncharged, or anionic
terized and were shown to have protective properties (K+ is often used as a counterion). In mesophilic micro-
toward proteins and cells. A range of new applications, all organisms, a variety of chemically diverse osmolytes is
based on the adaptation to stress conditions conferred by found. These osmolytes can be amino acids like proline,
extremolytes, is in development. amino sulfone acids (taurine), sugars (trehalose), inositols
(myo-inositol), and betaines (glycine betaine). All these
Keywords Ectoine . Compatible solutes . Osmolytes . compounds have a relatively low molecular weight and
Extremophiles . Extremolytes . Cytoprotection are very polar. Some osmolytes, like proline, are also
components of primary metabolic pathways.
G. Lentzen (*) : T. Schwarz The mechanism of using osmolytes has the advantage of
bitop AG,
being more flexible and suitable over a wider range of
Stockumer Strasse 28,
58453 Witten, Germany salinity. Organic osmolytes play an essential role in
e-mail: lentzen@bitop.de maintaining and protecting intracellular homeostasis in
624 Appl Microbiol Biotechnol (2006) 72:623–634

organisms ranging from bacteria to humans. Their primary 1998). Molecular dynamics simulation performed for a
function is the adaptation to changing extracellular osmo- model ectoine–water mixture using chymotrypsin inhibitor
larity. Osmolytes are accumulated as a consequence of 2 as a target protein are consistent with the idea that the
increased extracellular salinity and their intracellular con- ectoine does not interact with the protein surface or change
centration is reduced. The concentration of osmolytes in the the hydration structure (Yu and Nagaoka 2004). Instead, the
cell can range from millimolars to 1–2 M in response to the solvent (water) property is altered leading to a large
extracellular osmolarity. This means that some osmolytes slowdown of water diffusion and thus stabilization of
are tolerated by the macromolecular machinery of the cell protein conformation. Therefore, in line with the compat-
over a wide concentration range, hence, these compounds ibility with the cellular machinery at high molar concen-
were also termed compatible solutes (Brown 1976). The trations, the general mode of action of extremolytes is a
biosynthesis and release of osmolytes is tightly regulated. physical effect on the water properties.
In general, the microorganism can accumulate osmolytes Extremolytes exhibit protective properties not only
either by de novo synthesis or by uptake from the toward globular proteins but also toward whole cells and
environment. Specific uptake systems exist, e.g., for ectoine nucleic acids (see Table 1). These protective effects may
in Marinococcus marinus, where the BCCT type ectoine partially be secondary effects of protein stabilization (e.g.,
transporter EctM serves as a high-affinity uptake system for stabilization of membrane proteins) or are due to, e.g., the
ectoine (Vermeulen and Kunte 2004). Often, a mixture of replacement of water by hydroxyl group bearing extrem-
several osmolytes is produced, and the composition of the olytes like hydroxyectoine upon drying (Lippert and
osmolyte pool changes with environmental conditions: Galinski 1992).
Halomonas elongata predominantly accumulates potassium Ectoine ((4S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-
glutamate at intermediate salinity (0.5 M NaCl) and ectoine carboxylic acid) was originally discovered by Galinski et
at higher salinity (Kraegeloh and Kunte 2002). al. (1985) in Halorhodospira (formerly Ectothiorhodo-
spira) halochloris, an extremely halophilic phototrophic
eubacterium, isolated from Wadi Natrun, Egypt. Later the
Extremolytes: compatible solutes from extremophilic substance was found in a wide range of halophilic and
microorganisms halotolerant bacteria. The ability to accumulate ectoine is
also widespread among organisms that are unable to
Cells exposed to changing and elevated osmolarities have synthesize ectoines like Escherichia coli (Jebbar et al.
developed an osmolyte strategy. In the case of extrem- 1992), Bacillus subtilis (Jebbar et al. 1997), Corynebacte-
ophilic microorganisms, which are adapted to extremely rium glutamicum (Steger et al. 2004), and Sinorhizobium
high salinities and other environmental extremes like high melilotii (Jebbar et al. 2005). The hydroxy derivative
(or low) temperature, a specific set of osmolytes is found. hydroxyectoine ((4S,5S)-2-methyl-5-hydroxy-1,4,5,6-tetra-
We have termed these compounds extremolytes (osmolytes hydropyrimidine-4-carboxylic acid) was first discovered in
from extremophiles, Fig. 1). an actinomycin-D-producing Streptomyces strain (Inbar and
The ability of extremolytes to compensate osmotic Lapidot 1988) and is present in many strains that also
pressure and to stabilize macromolecules was studied produce ectoine. The relative amount of hydroxyectoine
extensively and a model for their mode of action of often increases with growth temperature, e.g., in
macromolecule stabilization was proposed. The preferential Streptomyces.
exclusion model postulates that the extremolytes are less Mannosylglycerate (firoin) and mannosylglyceramide
likely to be found at the surface of proteins, thus leading to (firoin-A) are examples for carbohydrate extremolytes. As
an increased (preferential) hydration of the protein typical for this group of extremolytes, the chemically
(Arakawa and Timasheff 1985). Thus preferential hydration reactive end of the sugar forms a glycosidic bond with a
favors the native state of the protein by making the status of hydroxyl group of glyceric acid or glyceramide. Rhodo-
unfolding less favorable in the presence of extremolytes thermus marinus synthesizes both mannosylglycerate and
(Fig. 2). Because osmolytes do not interact directly with mannosylglyceramide. Whereas the anionic mannosyl-
proteins, the catalytic activity of enzymes is largely glycerate is accumulated in response to heat stress, the
undisturbed. The effect of the osmolyte glycine on the uncharged mannosylglyceramide increases with elevated
stabilization of chymotrypsin inhibitor 2 was studied by NaCl levels (Silva et al. 1999). It is interesting to note that
NMR and it was found that the presence of a high mannosylglycerate is also found in eukaryotic red algae
concentration of glycine has a stabilizing effect on (Karsten et al. 1993).
chymotrypsin inhibitor 2, which is accompanied by a large Several archaea accumulate negatively charged deriva-
reduction of the exchange rate constants of most slowly tives of inositol and glycerol. Di-myoinositol-1,1′-phos-
exchanging amide protons (Foord and Leatherbarrow phate (DIP), a phosphodiester derivative of the uncharged
Appl Microbiol Biotechnol (2006) 72:623–634 625

O O OH OH
HO OK HO OH HO OH
HO NH2 O OK
O P
O
HO HO O O OH
O HO
O OH OH
OH
HO OH DIP
HO (L,L-Di-myo-1,1´(3,3´)-Inositolphosphate)
OH
OH
Firoin-A OH
Firoin
(2-O- α-Mannosylglyceramide)
(2-O-α-Mannosylglycerate) HO O OK
P
HN HO O O
+ O OH
N
DGP
H (α-Diglycerol phosphate)
O
Ectoine
((4S)-2-Methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid)
OK
OH O P
HN
O
+ O OK
N O P
H OK O
O
Hydroxyectoine cDPG
((4S,5S)-2-Methyl-5-hydroxy-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid) (cyclic 2,3-Diphosphoglycerate)
Fig. 1 The chemical diversity of extremolytes (extremophilic osmolytes). The anionic extremolytes are depicted as potassium salts, K+ being the
main counterion in the organisms from which they originate

osmolyte myo-inositol found in eukaryotes, is typical for and was first detected in the thermophilic methanogen
halotolerant hyperthermophilic archaea like Pyrococcus Methanothermobacter thermoautotrophicus (Kanodia and
furiosus and Thermotoga maritima (Scholz et al. 1992; Roberts 1983). cDPG exhibits a somewhat unusual behav-
Ciulla et al. 1997). In Thermotogales, the concentration of ior because its intracellular concentration is not modulated
DIP increases at NaCl levels above the optimum for growth in response to high NaCl concentrations (Ciulla et al.
(Martins et al. 1996). In many cases, however, an even 1997). The primary role in Methanothermobacter may
stronger accumulation of DIP is observed in response to therefore be as a phosphate storage compound. A role in
temperatures above the optimum for growth (Santos and da thermoadaptation was proposed for Methanothermus fervi-
Costa 2002) reaching 20-fold in P. furiosus at 101 °C dus, which accumulates high levels of cDPG (Matussek et
(Martins and Santos 1995). al. 1998). In line with this, cDPG was shown to stabilize
α-Diglycerol phosphate (DGP) also belongs to the archaeal glyceraldehyde-3-phosphate dehydrogenases at
anionic phosphodiesters found in halotolerant archaea. high temperature (Hensel and Jakob 1994).
DGP was identified as a new extremolyte in Archaeoglobus
fulgidus and shown to be an effective protein stabilizer in Production of extremolytes
vitro (Lamosa et al. 2000, Pais et al. 2005). A. fulgidus also
synthesizes DIP and while DGP is accumulated in response The high level of accumulation (up to 25% of cell dry mass)
to elevated external NaCl, increase in temperature leads to of extremolytes makes the development of bioprocesses for
enhanced DIP accumulation (Lamosa et al. 2000). large-scale production feasible. A prerequisite for this
Cyclic 2,3-diphosphoglycerate (cDPG), a cyclic approach is the ability to cultivate the corresponding
trianionic pyrophosphate, is accumulated in some archaea microorganism in fermenter scale and high cell densities at
626 Appl Microbiol Biotechnol (2006) 72:623–634

nonstandard conditions, e.g., >10% (w/v) NaCl and high with a gradual increase of salinity due the evaporation and
temperature. Alternatively, extremolytes can be produced in fast dilution due to rainfall or flooding of evaporation
recombinant hosts, e.g., E. coli or yeast, which are more ponds by tidal wave. This robustness of H. elongata,
common in bioprocesses. A prerequisite for this approach is together with the ability to achieve high cell densities
the availability of the synthetic pathway genes for cloning (>40 g dry weight/l corresponding to >10 g/l ectoine)
and the construction of a corresponding recombinant and the safety of the microorganism indicated by its use in
organism. This approach was chosen for mannosylglycerate food processing (Hinrichsen et al. 1994), have led to its
where the bifunctional mannosylglycerate synthase from use in the manufacturing process for ectoines. Strains of
Dehalococcoides ethenogenes was overproduced in E. coli H. elongata, which continuously release ectoine, can also
and Saccharomyces cerevisiae (Empadinhas et 2al. 2004). accumulate ectoine in the culture broth at concentrations
In S. cerevisiae, a moderate NaCl-dependent accumulation >20 g/l (Grammann et al. 2002).
of mannosylglycerate was observed. Another approach was A proprietary industrial bioprocess, termed “bacterial
chosen for DGP where chemical synthesis is the most milking” was established for the industrial-scale production
feasible way of production (Santos et al. 1998). of the extremolyte ectoine (Sauer and Galinski 1998). In
For cDPG, a production process using a recombinant this process H. elongata, a moderately halophilic ectoine
E. coli harboring the genes for 2-phosphoglycerate kinase producer, is grown under high-salt conditions (15–20% w/v
and cDPG synthetase was outlined (Moritz 2003). NaCl) and the intracellularly accumulated ectoines are
zDIP can be produced by fermentation of P. furiosus released by applying an osmotic down-shock, leading to
at 98 °C under anaerobic conditions, the productivity of the opening of mechanosensitive channels in the inner
this continuous process, however, is comparably low. A membrane of H. elongata. The biomass is returned to the
more economically feasible, recombinant approach to DIP fermenter for the next round of fermentation while the
production has to rely on the more complete characteriza- product solution is further purified by electrodialysis,
tion of the biosynthetic pathway, which is under way chromatography, filtration, evaporation, and crystallization.
(Chen and Roberts 1998; Chen et al. 1998). An ectoine bioprocess with an even higher productivity,
H. elongata was originally isolated from a solar saltern based on the continuous fermentation of H. elongata, was
(Vreeland et al. 1980) and can adapt to a wide range of developed recently (Lentzen and Schwarz 2005). This
salinities. This is due to the adaptation to an environment continuous “permanent milking” process is now used by

Fig. 2 Mode of action of

extremolytes. The hydrated ter-
tiary structure of globular pro-
teins in aqueous solution (a) is
stabilized in the presence of
extremolytes (b), which build
solute hydrate clusters (Galinski
et al. 1997) that are preferen-
tially excluded from the hydrate
shell of the protein (Arakawa
and Timasheff 1985). This leads
to a more compact tertiary
structure with reduced surface
area (c). Thus, the solute does
not interact directly with the
protein, the stabilizing effects
are rather due to modification of
the solvent (water) properties
Appl Microbiol Biotechnol (2006) 72:623–634 627

Table 1 Extremolyte applications

Application Extremolyte(s) Reference

Protection of biological macromolecules

Protection of oxidative protein damage (LDH) Hydroxyectoine Andersson et al. 2000
Stabilization of enzymes against thermal stress and freeze drying Mannosylglycerate Borges et al. 2002; Ramos et al. 1997
Stabilization of recombinant nuclease Mannosylglycerate Faria et al. 2004
Enzyme stabilization against heating, freezing, and drying Ectoine Lippert and Galinski 1992
Protection of LDH against heat and freeze-thawing Ectoine Göller and Galinski 1999
Protection against proteolytic cleavage of antibodies Ectoine Bersch et al. 2000
Thermostabilization of proteins DGP Lamosa et al. 2000
Thermostabilization of rubredoxin DGP Lamosa et al. 2003
Cutinase unfolding and stabilization Mannosylglycerate Melo et al. 2001
Inhibition of insulin amyloid formation Ectoine Arora et al. 2004
Protection against freeze-thaw stress of immunotoxins Hydroxyectoine Barth et al. 2000
Reduction of VLS in immunotoxin therapy Hydroxyectoine Barth 2000
Expression of functional recombinant proteins Hydroxyectoine Barth et al. 2000
Stabilization of retroviral vaccines Mannosylglycerate; Cruz et al. 2006
Hydroxyectoine
Reduction of apoptotic cell death induced by MJD gene product Ectoine Furusho et al. 2005
Inhibition of aggregation and neurotoxicity of Alzheimer’s Ectoine, Hydroxyectoine Kanapathipillai et al. 2005
beta-amyloid
Protection of cells
Stabilization of E. coli during drying and storage Ectoine; Hydroxyectoine Louis et al. 1994;
Manzanera et al. 2004
Induction of thermotolerance in E. coli Hydroxyectoine Malin and Lapidot 1996
Protection of P. putida against anhydrobiotic stress Hydroxyectoine Manzanera et al. 2002
Osmoprotection of lactic acid bacteria Ectoine Baliarda et al. 2002
Stabilization of tobacco cells against hyperosmotic stress Ectoine Nakayama et al. 2000
Block of UVA-induced ceramide release in human keratinocytes Ectoine Grether-Beck et al. 2005
Protection of human RBC membranes Ectoine Bünger et al. 2001
Protection of mitochondrial DNA in human dermal fibroblasts Ectoine Bünger and Driller 2004
Protection of skin
Protection of the skin barrier against water loss and drying out Ectoine Bünger 1999
Protection of skin immune cells against UV radiation Ectoine Beyer et al. 2000
Reduction of UV-induced SBCs Ectoine Bünger et al. 2001
Prevention of UVA-induced photoaging Ectoine Bünger and Driller 2004
Cytoprotection of keratinocytes Ectoine Buommino et al. 2005

bitop AG for the production of ectoines in metric ton scale accumulated in their natural producers upon increase of
(Fig. 3). temperature, e.g., hydroxyectoine in Streptomyces (Malin
For the production of hydroxyectoine, a bioprocess using and Lapidot 1996), it was of great interest to study heat
the Marinococcus strain M52 was described (Frings et al. protection effects in vitro. In a study involving a variety of
1995). Alternatively, hydroxyectoine can be produced with solutes, 1 M of hydroxyectoine showed the best perfor-
H. elongata by changing the fermentation conditions in the mance in heat stabilization, increasing the midpoint of
bacterial milking process. thermal inactivation by 14 °C for the model enzymes
phosphofructokinase and lactate dehydrogenase (LDH)
Protection of proteins by extremolytes (Lippert and Galinski 1992). In a study comparing
mannosylglycerate (firoin) with trehalose, mannosylglycerate
The physicochemical mode of action of extremolytes was the superior thermoprotectant for model enzymes from
(Fig. 2) by modifying the water properties is in line with both hyperthermophilic and mesophilic origin, in line with its
the many experimental data for protein stabilization by role as thermostabilizer under physiological conditions
extremolytes, which at the same time do not interfere with (Ramos et al. 1997). A comparative study of extremolytes,
the enzymatic or binding activity. The stabilization against together with some other more common osmolytes like
heat denaturation is a direct measure for increased protein glycerol, showed for the model enzyme LDH that manno-
stability in solution. Because some extremolytes are sylglycerate and hydroxyectoine were the best protectants
628 Appl Microbiol Biotechnol (2006) 72:623–634

against heat stress, while other compounds like ectoine or nase, mannosylglycerate was superior to trehalose in both
glycerol showed a much lower degree for stabilization cases (Ramos et al. 1997).
(Borges et al. 2002). At least for mannosylglycerate the Probably as a consequence of the stabilization of the
stabilization does not depend on the basic net charge of LDH tertiary structure of proteins, ectoine was also shown to
because it was also a good thermoprotectant of the acidic protect proteins from proteolysis by trypsin and trypsinogen
glucose oxidase from Aspergillus niger. DGP, which (Kolp et al. 2003) and proteolytic cleavage of antibodies
originates from a thermophilic archaeon, was found to (Bersch et al. 2000). Hydroxyectoine is able to protect LDH
protect a range of enzymes (LDH, alcohol dehydrogenase, from metal-catalyzed oxidation and against oxidation by
glutamate dehydrogenase, and rubredoxin) against heat hydrogen peroxide (Andersson et al. 2000), indicating that
inactivation (Lamosa et al. 2000). An NMR study indicates the protection is independent of the mechanism of
that the stabilization of rubredoxin by DGP is mainly due to oxidation. Also, ectoine is able to protect against metal-
a stabilization of the closed, more compact form of the catalyzed oxidation of LDH. In addition to this potentially
protein (Lamosa et al. 2003). Staphylococcus aureus indirect antioxidative effect (due to the protection of
nuclease A was used as a model enzyme to test the effect oxidizable groups in the protein), we have also assessed
of the extremolyte mannosylglycerate (Faria et al. 2004). the antioxidative properties of extremolytes (Table 2).
Mannosylglycerate at 0.5 M increases the thermal denatur- cDPG, DGP, and DIP strongly protect plasmid DNA from
ation transition point (Tm) by 7 °C and increases the heat damage by hydroxyl radicals, whereas the control com-
capacity by a factor of 2, but does not influence the (un) pounds proline and trehalose are inactive even at a 20-fold
folding pathway of the nuclease. higher concentration. Using the superoxide generating
Hydroxyectoine was shown to be an especially effective phenazine methosulfate/NADH system (Valentao et al.
cryoprotectant of antibody conjugates (Barth et al. 2000). 2002), we could show that cDGP is also a very efficient
When stored in a 1 M solution of hydroxyectoine, an scavenger of superoxide radicals with one third of the
antibody conjugate was shown to maintain 90% of binding activity of the strong antioxidant ascorbic acid.
activity even after four freeze–thaw cycles, whereas binding
activity was completely lost after only two freeze–thaw Use of extremolytes in protein chemistry and protein
cycles in the absence of hydroxyectoine. expression
Many proteins are inactivated by freeze-drying, which
combines the freezing stress with desiccation stress. When Due to their stabilization of the native structure of
comparing the remaining activity after freeze-drying for proteins, some extremolytes can inhibit protein aggrega-
betaine, ectoine, hydroxyectoine, trehalose, maltose, and tion seen in different aggregation pathways. One such
sucrose, hydroxyectoine and trehalose turned out as the best pathway is the formation of inclusion bodies that occurs
protectants against dehydration, whereas ectoine resulted in upon overexpression of recombinant proteins. Often, the
a lower remaining activity (Lippert and Galinski 1992). formation of inclusion bodies is desired because it
Here, the presence of hydroxyl groups, which are able to simplifies the extraction of pure protein from the biomass.
replace water upon desiccation, seems to be essential. It is then critical to develop a protocol for efficient
When comparing residual activities after freeze-drying of a refolding of the inclusion body aggregates. Using a
mesophilic LDH and a thermophilic glutamate dehydroge- standard refolding protocol based on dissolving the

Fig. 3 Ectoine bioprocess Medium feed

scheme: H. elongata is cultivat-
ed in a continuous fermentation
and the culture broth is separat-
ed from the biomass by micro- Electrodialysis
filtration. The concentrated Microfiltration II
biomass is exposed to an os-
motic down-shock and again
concentrated by microfiltration.
Chromatography
The ectoine containing filtrates Bioreactor
are the starting material for the
purification process with the key Crystallisation & drying
steps electrodialysis, chroma-
tography, and crystallization
Biomass
yielding a highly pure, crystal-
line product
Final product
Osmotic down shock
Microfiltration I
Appl Microbiol Biotechnol (2006) 72:623–634 629

Table 2 Antioxidative activity of extremolytes in a plasmid relaxation animal protein, extremolytes offer an attractive novel
assay approach to the protein-free and animal-free stabilization
Protection of plasmid DNA from hydroxyl and storage of sensitive proteins.
radicals
Amyloid inhibition by ectoines
Mannosylglycerate +++
Mannosylglyceramide +
The number of diseases associated with the misfolding of
Hydroxyectoine ++
cDPG +++ proteins is growing fast (Dobson 2003). In an important
DGP +++ subgroup of misfolding diseases, protein aggregation
DIP +++ leading to the formation of highly regular aggregates
Proline (1 M) − termed amyloids plays a key role and includes diseases
Trehalose (1 M) − like Alzheimer’s disease and spongiform encephalopathies.
In a study that used the formation of insulin amyloid in
Extremolytes (50 mM) were incubated with 20 ng of plasmid DNA in
an OH radical-producing system (1 mM H2O2, 0.1 mM FeCl3, and vitro as a model system, it was shown that ectoine is a very
0.1 mM ascorbic acid) for 30 min at 37°C, the reaction stopped by the effective inhibitor of amyloid formation decreasing both the
addition of EDTA and damage to the circular plasmid DNA was initiation and elongation phase of amyloid formation (Arora
assessed by agarose gel electrophoresis.
et al. 2004). In a further study, ectoine and hydroxyectoine
–No protection of circular plasmid DNA, + weak, ++ intermediate,
and +++ strong protection of supercoiled circular DNA from were investigated for their effect on Aβ peptide amyloid
hydroxyl radical cleavage formation (Kanapathipillai et al. 2005). Aβ peptide is the
major constituent of senile plaques, the key pathological
feature of Alzheimer’s disease. Ectoine and hydroxyectoine
were both effective inhibitors on the inhibition of Aβ42
inclusion bodies in the chaotropic reagent guanidinium aggregation and toxicity to human neuroblastoma cells.
chloride and diluting the dissolved protein to achieve Protein misfolding is also a key event in the pathogenesis of
refolding, we could show that ectoine improves the polyglutamine diseases such as Machado–Joseph disease
refolding yield of a model protein (Fig. 4). Addition of (MJD). Looking at apoptotic cell death produced by the
ectoine, hydroxyectoine, or firoin-A (mannosylglyceramide) truncated MJD gene product with an expanded polygluta-
to the refolding reaction increases the refolding yield by mine tract in cultured neuro2a cells, Furusho et al. (2005)
several folds, while addition of betaine, trimethylamine- showed that ectoine reduces the total amount of aggregates
N-oxide, or firoin is not beneficial. The effect of ectoine is and changes its intracellular distribution. In consequence,
concentration-dependent up to 1 M and additive to the apoptotis is decreased after ectoine application.
effect of arginine, an amino acid sometimes used in
refolding protocols. The data show that the use of
extremolytes in refolding protocols can improve yields of Stabilization of nucleic acids
active protein.
The use of extremolytes may be especially useful in the Although most research on stabilization of macromolecules
expression and purification of problematic, aggregation- by extremolytes has focused on proteins, there is also
prone proteins. As shown with immunotoxins (Barth et al. evidence for the stabilization of other cellular macro-
2000), osmolytes allow increased yields when used in the molecules, especially DNA. In general, high concentrations
periplasmatic expression of proteins in E. coli (Barth et al. of zwitterionic solutes increase the dielectric constant of the
2000) and furthermore stabilize the protein during the solution, thus decreasing ionic interactions and affecting the
purification process. DNA duplex (Flock et al. 1996). For ectoine it was
Ectoine was also successfully used for the optimization experimentally shown that it lowers the melting tempera-
of protein crystallization conditions for X-ray crystallogra- ture of double-stranded DNA (Lapidot et al. 1999).
phy, resulting in larger and more regular protein crystals Furthermore, ectoine and hydroxyectoine increase the
(Harjes et al. 2004). thermal stability of DNA polymerases at elevated temper-
Taken together the data on protein stabilization by atures and can thus be used to improve primer extension,
extremolytes in vitro and in vivo, these low molecular sequencing and polymerase chain reactions (Lapidot et al.
weight compounds do combine many properties of ideal 1999). Ectoine and hydroxyectoine were also implicated in
protein protectants, i.e., high stability and no interference the self-defense mechanism of antitumor antibiotic-produc-
with binding and enzymatic activity. Due to the increasing ing Streptomyces strains: Here, the ectoines may protect
sensitivity for the potential propagation of transmissible Streptomyces DNA from intercalation by actinomycin D
spongiform encephalopathies by stabilizers containing (Inbar and Lapidot 1988).
630 Appl Microbiol Biotechnol (2006) 72:623–634

Fig. 4 Inclusion of body

refolding yield for a galactosi-
dase enzyme. Relative enzyme
activity of the refolded protein is
shown. Activity in the sample
refolded in the absence of addi-
tives was taken as one

Another DNA-protective effect is seen in the inhibition The positive effects of extremolytes on the expression of
of hydroxyl radical cleavage of DNA by extremolytes recombinant proteins and the fermentative yield of amino
(Table 2) in vitro and in the protection of mitochondrial acids indicate a more general potential for the use of
DNA in human dermal fibroblast against UVA-irradiation- extremolytes as fermentation additives. In particular, bio-
induced damage (Bünger and Driller 2004). processes that involve high salt concentrations, like the
production of fermented food, could be improved by the
use of extremolytes.
Protection of cells by extremolytes Cytoprotection by ectoines, however, is not limited to
bacteria but is also seen with eukaryotic cells, leading to
Extremolytes do not only stabilize proteins and other some of the most interesting applications. The effect of
macromolecules; they are also potent cell protectants. The ectoine on membranes was tested with red blood cell
protective effect is obvious in the extremolyte-producing (RBC) assay. This test is a biological in vitro test for the
strains, but is also seen with other prokaryotes and even rapid estimation of membrane and protein denaturing
eukaryotic cells (Table 1). Louis et al. (1994) showed that properties of surfactants. The standard protocol uses
ectoine and hydroxyectoine do stabilize air-dried and erythrocytes, nonnucleated blood cells containing hemo-
freeze-dried E. coli during drying. Ectoines added to the globin. In the event that the cell membrane is damaged,
culture medium reversed the inhibition of E. coli growth hemoglobin is released from the cell and can be quantified
caused by osmotic stress and increased temperature (Malin by a standard photometric determination. RBCs were
and Lapidot 1996). Manzanera et al. (2004) showed that E. incubated with ectoine before the addition of surfactant as
coli dried in hydroxyectoine exhibited a high degree of the lytic agent. The action of ectoine was compared to the
desiccation tolerance, similar to that achieved using the well-known membrane stabilizer phosphatidylcholine. Five
more common trehalose as an extracellular protectant. different surfactants were used and ectoine showed mem-
Ectoine also serve as a specific osmoprotectant for the brane stabilization for all detergents tested, and for four out
lactic acid bacterium Tetragenococcus halophila, which is of five detergents, the effect was stronger than with
used in soy sauce fermentation (Baliarda et al. 2002). In the phosphatidylcholine (Bünger et al. 2001). When applying
gram-negative organism, Pseudomonas putida, which is UVA irradiation as a stress to human keratinocytes pre-
used for bioremediation due to its ability to degrade organic treated with ectoine, cells are protected from damage. The
solvents, hydroxyectoine was superior to trehalose as a effect seems to be exerted via a modulation of the
drying excipient for storage (Manzanera et al. 2002). keratinocyte cell membrane because the release of
Hydroxyectoine is therefore well-suited for the improve- ceramides from sphingomyelin is decreased in skin cells
ment of desiccation tolerance and as a useful excipient for pretreated with ectoine (Bünger and Driller 2004). This
dry storage and shipment of sensitive microorganisms. leads to the inhibition of the release of proinflammatory
Ectoine was also used in fermentation technology to cytokines like intercellular adhesion molecule-1 and pro-
increase the osmotolerance and yield in the production of cesses that are involved in UVA-induced accelerated skin
amino acids by coryneform bacteria (Yasuhiko et al. 1997). aging. In the same study it was also shown that ectoine
Appl Microbiol Biotechnol (2006) 72:623–634 631

protects mitochondrial DNA from UVA-induced mutation effect, protecting skin from potentially damaging UV light.
in human cell culture. A possible mechanism for the ectoine A recent study (Buommino et al. 2005) shows the
effects could be the stabilization of membrane structures, cytoprotective effects of ectoine also when bacterial lipo-
leading to an increased resistance to potentially damaging polysaccharides are used as a stressor because pretreatment
environmental influences like UVA. The cytoprotective with ectoine prevents cell damage by maintaining an
effect of ectoine even extend to plant cells: Transgenic elevated level of the Hsp70.
tobacco (Nicotiana tabacum) cells expressing the genes for
ectoine biosynthesis ectA, ectB, and ectC accumulated
small levels of ectoine, showed increased tolerance to Outlook: novel areas of application
hyperosmotic shock, and could grow at elevated salt levels
(Nakayama et al. 2000). A major application area for extremolytes established today
is in cosmetics where ectoine (Ectoin™) is now used in a
growing range of skin care products. Another established
Protection of skin by ectoine use of extremolytes is in stabilization of proteins and
nucleic acids in life science and protein chemistry. The high
Because of it’s origin from water-stressed organisms and compatibility with biological systems together with the
the accessibility of bulk amounts due to an economical macromolecule-stabilizing and cytoprotective properties of
biotechnological production process, ectoine raised com- extremolytes raise possibilities for a much wider range of
mercial interest as a moisturizer for skin care products. applications, for which we give a few examples below.
Initial studies did focus on the protection of the skin by of Many immunotoxins and immunomodulators do have a
ectoine against water loss and desiccation (Bünger 1999). particularly severe side effect, termed “vascular leak
The influence of ectoine on the transepidermal water loss syndrome” (VLS), which reduces the suitability of other-
was measured in a test with human volunteers where skin wise effective therapies because it strongly limits the
was pretreated with an emulsion containing 0, 2, or 5% applicable therapeutic dose (Baluna and Vitetta 1997).
ectoine. With increasing amounts of ectoine, the skin Hydroxyectoine, which is able to stabilize immunotoxins
becomes less susceptible to damage by the detergent in vitro (Barth et al. 2000), also ameliorates the highly toxic
sodium dodecyl sulfate and the subsequent water loss. side effects of immunotoxins in an animal model when
Furthermore, ectoine treatment in a cosmetic formulation administered in combination with the immunotoxin (Barth
protects the skin from drying out subsequent to application 2000). Hydroxyectoine could therefore be a powerful
of a hygroscopic silica gel and thus has a prophylaxic effect functional excipient for cancer drugs with VLS toxic side
against dry skin. The ideal formulation properties of ectoine effect. The high compatibility, lack of toxicity, and
as a stable, crystalline white powder taken together with the macromolecule-stabilizing properties of extremolytes make
lack of interference with cell metabolism and lack of them promising candidates for the use as pharmaceutical
irritative potential are further factors for its suitability as a excipients. Mannosylglycerate (firoin) allowed long-term
cosmetic ingredient. storage (half-life >1 year) of adenoviral vectors, which are
Ectoine was also tested for its ability to reduce the UV- promising tools for gene therapy, but are hampered by loss
induced damage to keratinocytes, also termed sunburn cells of vector infectivity during storage and transport (Cruz et
(SBCs), which are formed as a consequence of UV al. 2006).
irradiation. Using a skin model system it was shown that The chiral ectoines, which are now available in bulk
preincubation of the skin equivalent for 24 h with ectoine quantities, could also serve as interesting starting points for
significantly reduced the number of SBCs (Bünger et al. the design of natural product chemical libraries. In
2001). The photoprotective properties of ectoines seen in a particular, hydroxyectoine, with its two chiral centers,
skin model could also be shown in vivo. The effect is provides an interesting starting point for the synthesis of
especially pronounced with the Langerhans cells (LC), chiral compounds.
which present antigens crossing the skin barrier and induce It was recently shown that ectoine is accumulated in
T cell immune response. LC are particularly sensitive to certain cheeses up to 89 mg/100 g of cheese (J. Klein et
UV-induced stress and as a consequence of UV irradiation, al., submitted for publication). The occurrence in cheese is
the number of cells visible by ATPase staining is reduced, due to the growth of Brevibacterium linens, an ectoine
correlating with a loss of their antigen-presenting proper- producer traditionally used in surface ripening of red-
ties. Pretreatment with an emulsion containing 1% ectoine smear cheeses such as Limburger, Munster, French cheese,
led, however, to a highly significant protective effect of the Swiss cheese, and Tilsiter (Rattray and Fox 1999). The
LC against UV-induced damage and destruction (Beyer et occurrence of ectoine in food is probably not limited to
al. 2000). Thereby, ectoine exerts an immunoprotective cheese, but could extend to other food where the
632 Appl Microbiol Biotechnol (2006) 72:623–634

production involves the fermentation with ectoine-produc- keratinocytes modulating the proinflammatory response. Cell
ing microorganisms under high salt conditions like in soy Stress Chaperones 10:197–203
Chen L, Roberts MF (1998) Cloning and expression of the inositol
sauces, Natto (a Japanese soy fermentation product monophosphatase gene from Methanococcus jannaschii and
produced by a Bacillus species), fermented fish sauces, characterization of the enzyme. Appl Environ Microbiol
and cured meat. The occurrence in food, taken together 64:2609–2615
with the known protein and cell protection properties of Chen L, Spiliotis ET, Roberts MF (1998) Biosynthesis of di-myo-
inositol-1,1′-phosphate, a novel osmolyte in hyperthermophilic
ectoine, open a new area of application for extremolytes archaea. J Bacteriol 180:3785–3792
as functional food ingredients. One application could be Ciulla RA, Diaz MR, Taylor BF, Roberts MF (1997) Organic
the use of ectoine to increase the stability and freshness Osmolytes in Aerobic Bacteria from Mono Lake, an Alkaline,
of foods by stabilizing food components with additional Moderately Hypersaline Environment. Appl Environ Microbiol
63:220–226
potential health benefits due to the cytoprotective Cruz PE, Silva AC, Roldao A, Carmo M, Carrondo MJ, Alves PM
properties of ectoines. (2006) Screening of novel excipients for improving the stability
of retroviral and adenoviral vectors. Biotechnol Prog 22:568–576
Dobson CM (2003) Protein folding and disease: a view from the first
Horizon Symposium. Nat Rev Drug Discov 2:154–160
Empadinhas N, Albuquerque L, Costa J, Zinder SH, Santos MA,
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