macromolecules such as proteins and membrane, and via production of anti- oxidants that counter damages resulted from reactive oxygen species. These
protections are often induced by drying, and some of the genes involved may be
homologous in microbes, plants, and animals. As mentioned above that trehalose is also involve in preserving biomolecules
from cold, hence it is well used as cryopreservation agents. Additional of sugars or salts to the solution distort the water structure where which these compounds
may enhance the tetrahedral coordinated hydrogen bond structure or reduced it that respectively called as water structure maker and water structure breaker.
Preservation of biological membrane would be effective with additional of water solute act as water structure breaker that reduced freezable water Branca et al
1999. Including in water structure breaker are ClO
4 -
, MnO
4 -
, Br
-
, Cl
-
, K
+
, Cs
+
, sugars and I
-
. Those belong to water structure maker are Li
+
, Cu
+
, Al
+
, Mg
+
, and OH
-
. While Na
+
, Ag
+
and Ba
+
are in borderline Bryant et al. 2001. Miller and Pablo 2000 suggested heat solution value of trehalose, maltose and sucrose as
water structure breakers. The conformation of sugars in the solution release heat enthalpy, where 19.1, 15.6 and 5.95 kJmol of trehalose, maltose and sucrose
respectively that in turn reduce freezable water. Trehalose is also associated with resistance of organism to oxidative stress,
such to super oxide. Yeast cell that exposed to mild heat stress induced accumulation of trehalose and its resistance to hydrogen peroxide. Conversely,
those lacking of trehalose synthase were sensitive to hydrogen peroxide as its protein oxidation run faster Benarouj et al. 2001. Trehalose is also found to be
essential for mycolic acid biosynthesis in Corrynebacterium glutamicum. The absence of mycolic acid in defective trehalose synthase mutant caused cell wall
disorder, excretion of amino acids and impairment with bacterial growth Wolf 2002.
There is no discussion about trehalose in combating the deleterious effect of high salinity has been reported. However, Garg et al. 2002 and Jang et al.
2003 found that transgenic rice bearing gene for trehalose synthase fusion of TPS1-TPP
were also tolerant to salt and cold, despite to drought.
2.4 Other compatible solutes
Organism has seceral strategies to copewith stresses, such as by accumulating compatible solutes. Osmolytes usually do not affect cellular
processes; hence, they are called as compatible solutes. The osmolytes can be synthesized or taken from medium. They interact to biological compound
accordingly, such as via making hydrogen bonding or electrical interaction Bryant et al. 2001. Beside their function for osmolytes these compound also
stabilize biological compounds. Ion channel, membrane protein involves in sensing external and internal differences, such as salt concentration, cell volume
and turgor pressure changes. They may not act exclusively in particular organism and particular stress, yet they may be accumulated at different stresses, and act
together that called as cocktail solutes. There are many compatible solutes that can be categories into in several
ways. Anonyl Anonim, www.sbu.ac.uk suggested that it can be devided into inoprganic and organis compatible solutes. Including in inorganic solutes are
such as: K, Mg, SO
4
, HPO
4
, Ca, Li, Na, H, and OH. While organic osmolytes include polyol and derivates, amino acid and derivates, betaine, ectoin and
occasionally peptides Robert MF for review.that can be sub categories into three groups: zwitterionic solutes, ii noncharged solutes, and iii anionic
solutes. The first group includes glycine-betain, ectopin and Na-acetyl b-lysine and b-glutamin. The second group includes carbohydrates glycerol, myo-inositol,
trehalose, sucrose etc and uncharged amino acid and peptides such as carboxamine and acetylated neutral glutamine dipeptides, ß-glutamate, ß-
hydroxybutyrate and derivatives. The unionic solutes are anionic polyols and carbohydrates. Neutral osmolytes usually accumulated by bacteria, while archea
tend to accumulate negative charged osmolytes.
2.5 Trehalose biosynthesis
There are three pathways of trehalose synthesis, first known as OtsA-OtsB pathway. This pathway involves two steps of enzymatic reactions, using glucose
and glucose 6 P to be condensed into trehalose 6 Phosphate. This reaction is catalyzed by trehalose Synthase I TPS1 on plant or OtsA within E. coli. The
second step of this pathway is catalyzed by TPP or OtsB that remove phosphate from trehalose 6 phosphate. The second pathway of trehalose synthase is known
as TreY-TreZ. This pathway convert oligomere of glucose such as oligocyl maltose or glycogen to trehalose by converting the most end of glucose from 1-4
linkage to 1-1 linkage, catalyzed by enzyme TreY by which trehalose is produced but still attached at the oligomere. TreZ enzyme then removes trehalose from
Trehalose G6P
UDP-G
trehalose 6-phosphate
OtsA
OtsB
1-O- α
-D-glucopyranosyl- α
-D-glucopyranoside
inhibites
1. OtsA–OtsB pathway
Trehalose 6 P T6P
Figure 2: Trehalose biosynthesis. Three pathways Smet et al. 2000. 1. OtsA- OtsB
pathway, 2. TreY-TreZ pathway and 3. TreS pathway. Avonce et al 2006 proved the presence of 2 other pathways beside the 3
mentioned, distributed in diverse organisms called as TreP 4
th
pathway and TreT the 5
th
pathways. oligomere. Third pathway called TreS pathway, one enzymatic reaction involves
in the pathway, by which enzyme trehalose synthase TRES convert maltose to trehalose. Similarly to the second pathway, this enzyme also rearrange
intermolecular of maltose from α
1-4 glycosidic linkage to α
- α
, 1-1 glycosidic linkage of trehalose, hence maltose is converted to trehalose. This enzyme is
also capable to convert trehalose to maltose Smet et al. 2000 with equilibrium state at 60 trehalose and 40 maltose Wei et al. 2004. Avonve et al 2006
proved 2 others pathways beside these pathways, they are called as TreP and TreT pathways Figure 2. TreP pathway, converts glucose-1P and glucose to
trehalose and release Pi catalysed by trehalose phosphorylase TreP. This pathway mainly occurs in eubacteria and small portion occurs in archaea. TreT
pathway converts ADP-glucose and glucose to produce trehalose and ADP is released. This conversion is catalysed by trehalose glycolsiltransfering synthase,
4. TreP Pathway
TreP
OP ADP
TreT
5. TreT Pathway
G-1P Glucose
ADP-G
and is found mainly in archaea with small event in eubacteria Figure 3. The first pathway is widely spread in many organisms, E. coli Murata et al.
1971 and S. cerevisiae Smet et al. 2001; S. pombe Franco et al. 2000; Aspergillus nidulan
Fillinger et al. 2001; A. niger Wolschek and Kubicek, 1997; Soto et al. 1999 and also in Arabidopsis thaliana Leymann et al. 2001. The
second pathway can be found such as in Brevibacterium helvolvum Kim et al. 2000, Arthrobacter Nakada et al. 1995, Rizobium Murata et al. 1996 and
Archeon Sulfolobus acidocaldarius Murata et al. 1996. While the third pathway
can be found in Thermus aquaticus Tsusaki et al. 1997, Agaricus bisphorus Wannet et al. 1998, Pimelobacter Nishimoto et al. 1995, Thermobifida fusca
Wei et al. 2004, Thermus acidophyllus and Grifola frondosa Saito et al. 1998. Some organism has more than one pathways, it may be two or three pathways.
Mycobacterium tuberculosis Murphy et al. 2005, M. smegmatis, Mycobacterium
bovis and M. leprae have those three pathways in trehalose synthesis Pan et al.
2004. Figure 3. Distribution of trehalose pathways in organisms adopted from Avonce
et al 2006. Diverse pathways found in eubacteria and archaea, while in
eukaryotic organisms the first pathway only is observed and in vertebrates degrading pathway only is available.
2.6 Characterization of Trehalose Synthase TRESTSASE
Enzyme with optimum condition similar to that plant cellular is favorable for generating transgenic plant. Information was gathered regarding to enzyme
characters pH, temperature, K
m
of trehalose synthase from few organisms that has been reported, summarized at appendix1. It is shown that among he reported
trehalose synthase, TSase with optimum temperature of 20
o
C, pH 6.5 with low K
m
and high maximum conversion, seems to be most possible to be active in plant cellular. Saito et al. 1998 found trehalose synthase from Grifola frondosa that is
annotated as TSase has a molecular mass 120 kDa consisted of a dimmer protein 60 kDa. This enzyme is very interesting, since it converses a-D glucose
and a-D glucose 1P. While other TSae used maltose as substrate. TSase from G. frondosa also catalyses trehalose phosphorylation into its
substrate; however its equilibrium is favor to trehalose synthesis. The temperature optimums of trehalose synthesis and trehalose phosphorylation were
32.5-35
o
C and 35-37.5
o
C respectively. The rate conversion of maltose to trehalose by trehalose synthase is in optimal rate when it occurs at pH of 6.5,
while the opposite direction is optimal at pH of 6.5-6.8. In the optimal condition, the conversion rate of trehalose synthesis was 18mM h
-1
with maximum conversion rate was 90 of the substrate added. Interestingly, this enzyme also
can produce trehalose from sucrose when 1.05 U of glucose isomerase and 20 mM MgSO
4
is added to the reaction mixture. The rate production of the second way of synthesis was 4.5 mM h
-1
. The same enzyme from Pimelobacter, is annotated as TreS. TRES from
Pimelobacter sp. was shown to catalyze an intramolecular rearrangement of
maltose to convert the α
-1,4-glycosidic linkage of this disaccharide to the
α ,?
α 1,1-
glycosidic linkage of trehalose Nishimoto et al. 1995. TreS of Pimelobacter
consisted of a 1719-bp gene, coding a 573-residue amino acid sequence. It has
homologues area with maltases from Saccharomyces carlsbergenesis and Aedes
aegypti at 220 N-terminal residues. The same enzyme from Thermus
acidophyllus annotated as TS has a molecular mass of 110 kDa. At pH of 7.0
this enzyme has optimum temperature at 40
o
C with K
m
of 0.9 mM. While at pH 6.5, it has optimum temperature at 45
o
C with K
m
of 4.2 mM. TRES from Mycobacterium smegmatis reported by Pan et al. 2004. This
enzyme that was isolated from cytosol of M.smegmatis has a molecular weight of
about 68 kDa, however in the active form it has 390 kDa. Hence it is an hexamere of 6 identical monomers. This enzyme catalyzes maltose to trehalose
and capable of interconversion with equilibrium state at 42-45 of each that can be reached at incubation for 6h with 0.5 mM maltose as the substrate. Higher
concentration reduced the conversion rate. When trehalose was used as substrate, 30 maltose was obtained after et least 12h incubation with 8-10 of
glucose was obtained. The optimum pH was 7 and experiment was conducted at 37
o
C. When maltose is used as substrate it has K
m
was 10 mM but he K
m
for trehalose was 90 mM.
The character of the enzyme TRES from Thermobifida fusca was also reported Wei et al. 2004 reported. It has a molecular weight of 66 kDa with
optimum condition of pH at 6.5 and temperature at 25
o
C. This enzyme converts matose into trehalose, and has capability to reverse back trehalose into maltose
at equilibrium state, about 60 trehalose concentration. At higher temperature 37
o
C, or when the enzyme concentration is high, the enzyme also diverts matose into glucose up to 15 of the substrate. Reaction mixture with15
maltose, it converts substrate about 55-60 at optimal condition. Heavy metal Cu
2-
, Mn
2-
and Zn
2-
at 5 mM did not inhibite its activity. The catalytic sites of this enzyme laid at 223-257 amino acids and 400-439 amino acids.
TreS is also found in Mycobacterium tuberculosis Murphy et al. 2005. In
this bacteria, there 3 pathways are present, and TreS is not the used major pathway compared to the two others. While Cardoso and Castro 2007 reported
2 pathways OtsA-OtsB and TreS found in Propionibacterium freudenreichii. This bacteria accumulated high level of trehalose especially in response to stresses
osmotic, oxidative and acid stresses. The first pathway is suggested to function in trehalose synthesis while the second is functioning as trehalose degradation.
2.7 Foreign genes encoding for Trehalose Synthase on plants