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Starch-hydrolyzing enzymes from thermophilic archaea and bacteria

Costanzo Bertoldo and Garabed Antranikian*
Extremophlic microorganisms have developed a variety of
molecular strategies in order to survive in harsh conditions.
For the utilization of natural polymeric substrates such as
starch, a number of extremophiles, belonging to different
taxonomic groups, produce amylolytic enzymes. This class of
enzyme is important not only for the study of biocatalysis and
protein stability at extreme conditions but also for the many
biotechnological opportunities they offer. In this review, we
report on the different molecular properties of thermostable
archaeal and bacterial enzymes including -amylase,
-glucosidase, glucoamylase, pullulanase, and cyclodextrin
glycosyltransferase. Comparison of the primary sequence of
the pyrococcal pullulanase with other members of the glucosyl
hydrolase family revealed that significant differences are
responsible for the mode of action of these enzymes.
Technical University Hamburg-Harburg, Institute of Technical
Microbiology, Kasernenstrasse 12, 21073 Hamburg, Germany
Current Opinion in Chemical Biology 2002, 6:151160
1367-5931/02/$ see front matter
2002 Elsevier Science Ltd. All rights reserved.
CGTase cyclodextrin glycosyltransferase

Microorganisms that are adapted to grow optimally at high
temperatures (60108C) have been isolated from hightemperature terrestrial and marine habitats. In recent years,
many heterotrophic extreme thermophilic (6080C) and
hyperthermophilic (80110C) microorganisms have been
discovered that utilize natural polymeric substrates as their
carbon and energy source. These extremophilic microorganisms, which belong to the Archaea and Bacteria, can
facilitate the enzymatic degradation of polymeric substrates
such as starch, cellulose, xylan, pectin and chitin [1,2,3].
Because starch is rare in deep-sea hydrothermal vents, it
can be assumed that the presence of starch-hydrolyzing
enzymes in microorganims living in these ecological niches
is required to degrade glycogen. Glycogen can be provided
from animal cells and microorganisms. In a number of
archaea, glycogen serves as a storage material in the cell
[4]. The fact that starch-hydrolyzing enzymes are abundant
in extremophiles suggests that they play an important role
in their metabolism. The enzymatic hydrolysis and modification of polysaccharides is of great interest in the field of
biotechnology. Thermostable starch-hydrolyzing enzymes
such as amylases, pullulanases and glucoamylases play an
important role in food, chemical, and pharmaceutical
industries. The potential exploitation of this natural source
of sugars is not only useful for glucose/fructose syrup

production, but also for the synthesis of non-fermentable

carbohydrates, anti-cariogenic and anti-staling agents in
baking [5]. It has been shown that in most hyperthermophilic microorganisms, the level of starch-hydrolyzing
enzymes appears to be too low for biotechnological applications. The molecular cloning of the corresponding genes
and their expression in heterologous hosts, however,
circumvents the problem of insufficient expression in the
natural host. The number of genes from thermophiles
encoding amylolytic enzymes that have been cloned and
expressed in mesophiles has been increasing significantly
[3,6,7]. In most cases, the thermostable proteins expressed
in mesophilic hosts maintain their thermostability, are
correctly folded at low temperature, are resistant to host
proteolysis and can be easily purified by using thermal
denaturation of the mesophilic host proteins. The degree
of enzyme purity obtained is generally suitable for most
industrial applications. In this review, we focus on
amylolytic enzymes, which have been isolated and characterized from extreme thermophilic and hyperthermophilic
archaea and bacteria. Some of these aspects have been
already presented in recent reviews [3,4,59,10].

Microorganisms growing at high temperatures

Many parts of the world are considered extreme, such as
geothermal environments, polar regions, acid and alkaline
springs and the cold pressurized depths of the oceans. As
conditions become increasingly demanding, extreme environments become exclusively populated by microorganisms
belonging to the bacterial and archaeal domains (prokaryotes).
It is very likely that higher organisms are unable to survive
under extreme conditions because of their cellular complexity
and compartmentalization. The realization that extreme
environments harbor different kinds of prokaryote lineage
has resulted in a complete reassessment of our concept of
microbial evolution and has given considerable impetus to
extremophile research [11,12].
Microorganisms that are adapted to grow optimally at high
temperatures (60108C) have been isolated from hightemperature terrestrial and marine habitats. The most
common biotopes are volcanically and geothermally heated
hydrothermal vent systems such as solfataric fields, neutral
hot springs, and submarine hot vents. Submarine hydrothermal
systems are situated in shallow and abyssal depth. They
consist of hot fumaroles, springs, sediments and deep-sea
vents with temperatures up to 400C (black smokers)
[2,13]. Shallow marine hydrothermal systems are located
at the beaches of Vulcano, Naples and Ischia (Italy);
Sao Miguel (Azores); and Djibouti (Africa). Examples of
deep-sea hydrothermal systems are the Guaymas Basin
(depth 1500 m) and the East Pacific Rise (depth 2500 m),
both off the coast of Mexico; the Mid-Atlantic Ridge (depth
3700 m); and the Okinawa Trough (depth 1400 m) [10].


Biocatalysis and biotransformation

Figure 1





-Limit dextrin

Pullulanase II
Pullulanase I

Linear oligosacch.





Maltose and




Linear oligosaccharides

type I and II

hydrolase type I


hydrolase type II


hydrolase type III



Current Opinion in Chemical Biology

Starch-hydrolyzing enzymes from thermophilic archaea and bacteria Bertoldo and Antranikian


Figure 1 legend
Schematic presentation of the action of amylolytic and pullulytic enzymes. Pullulanase type I also attacks -1,6-glycosidic linkages in
oligosaccharides and polysaccharides. Pullulanase type II also attacks -1,4-linkages in various oligosaccharides and polysaccharides [3]. Black
circles indicate reducing sugars.

Microorganisms capable of growing optimally at temperatures between 50C and 60C are designated as moderate
thermophiles. Most of these microorganisms belong to
many different taxonomic groups of eukaryotic and
prokaryotic microorganisms such as protozoa, fungi, algae,
streptomycetes and cyanobacteria, which comprise mainly
mesophilic species. It can be assumed that moderate
thermophiles, which are closely related phylogenetically to
mesophilic organisms, may be secondarily adapted to life
in hot environments.
Extreme thermophiles, which grow optimally between 60C
and 80C, are widely distributed among the genera Bacillus,
Clostridium, Thermoanaerobacter, Thermus, Fervidobacterium,
Thermotoga and Aquifex. Most of the hyperthermophiles, on
the other hand, grow optimally between 80C and 110C
[11].The Archaea consists of two major kingdoms: the
Crenarchaeota (some genera are Sulfolobus, Picrophilus,
Pyrodictium, Pyrolobus, Pyrobaculum and Thermoproteus) and
the Euryarchaeota, which include hyperthermophiles (some
genera are Thermococcus and Pyrococcus), methanogenes
(e.g. Methanococcus, Methanobacterium and Methanosarcina),
sulfate-reducer (Archaeoglobus) and halophiles (including
genera such as Halobacterium and Halococcus). Short phylogenetic branches indicate a rather slow clock of evolution.
Deep branching-points are evidence for early separation of
the two groups. The separation of the Bacteria from the
EukaryaArchaea lineage is the deepest and earliest branching point known so far. Hyperthermophiles are represented
among all the deepest and shortest lineages, including the
genera Aquifex and Thermotoga within the Bacteria and
Pyrodictium, Pyrobaculum, Thermoproteus, Desulfurococcus,
Sulfolobus, Methanopyrus, Pyrococcus, Thermococcus, Methanococcus
and Archaeoglobus within the Archaea [13].
Solfataric fields are the most important biotopes of
microorgansms that prefer to live under both thermophilic
and acidic conditions. Thermoacidophiles belonging to the
genera Sulfolobus, Acidianus, Thermoplasma and Picrophilus,
with growth optima between 60C and 90C and pH 0.7 to
5.0 are commonly found in the aerobic upper layer, whereas
slightly acidophilic or neutrophilc anaerobes such as
Thermoproteus tenax or Methanothermus fervidus can be
isolated from the lower layer. Species of Thermoplasma
(growth optimum: pH 2 and 60C) have been found in hot
springs, solfataras and coal refuse piles [14]. Their closest
known phylogenetic relatives, also found in solfataras, are
species of the genus Picrophilus, which are so far the most
extreme acidophiles with growth close to pH 0. Picrophilus
oshimae and P. torridus are both aerobic, heterotrophic
archaea that grow optimally at 60C and pH 0.7 and utilize

various polymers such as starch and proteins. On the other

hand, the alkaliphiles have been found in carbonate-rich
springs and alkaline soils, where the pH can be around 10.0
or even higher, although the internal pH is maintained
around 8.0. The soda lakes in the Rift Valley of Kenya and
similar lakes found in a few other places on earth are highly
alkaline with pH values between 11.0 or 12.0 and represent
a typical habitat where alkaliphilic microorganisms can be
isolated. Very recently, two thermoalkaliphilic bacteria,
Anaerobranca gottschalkii and Anaerobranca horikoshii have
been isolated from Lake Bogoria in Kenya and from
Yellowstone National Park, respectively. The new isolates
represent a new line within the Clostridium/Bacillus
subphylum. The two archaeal thermoalkaliphiles identified
to date are Thermococcus alcaliphilus and Thermococcus
acidoaminivorans, both growing at 85C and pH 9.0 [13,15].

Starch-processing enzymes
Starch is a ubiquitous and easily accessible source of energy.
In plant cells or seeds, starch is usually deposited as large
granules in the cytoplasm. Starch is composed exclusively
of -glucose units that are linked by -1,4- or -1,6-glycosidic bonds. The two high-molecular-weight components
of starch are amylose (1525%), a linear polymer consisting
of -1,4-linked glucopyranose residues, and amylopectin
(7585%), a branched polymer containing, in addition to
-1,4 glycosidic linkages, -1,6-linked branch points
occurring every 1726 glucose units. -Amylose chains,
which are not soluble in water but form hydrated
micelles, are polydisperse and their molecular weights vary
from hundreds to thousands. The molecular weight of
amylopectin may be as high as 100 million and in solution
such a polymer has colloidal or micellar forms [3].
Because of the complex structure of starch, cells require an
appropriate combination of enzymes for its depolymerization to oligosaccharides and smaller sugars, such as glucose
and maltose. They can be simply classified into two
groups: endo-acting or endo-hydrolases, and exo-acting
enzymes or exo-hydrolases. Endo-acting enzymes, such as
-amylase (-1,4-glucan-4-glucanohydrolase; EC,
hydrolyze linkages in the interior of the starch polymer in
a random fashion, which leads to the formation of linear
and branched oligosaccharides [3].
Exo-acting starch hydrolases include -amylase, glucoamylase, and -glucosidase. These enzymes attack the
substrate from the non-reducing end, producing small
and well-defined oligosaccharides such as maltose by
-amylase (EC and glucose by glucoamylase
( [3] (Figure 1).


Biocatalysis and biotransformation

-Glucosidase (EC, or -D-glucoside glucohydrolase,

attacks the -1,4 linkages of oligosaccharides. Unlike
glucoamylase, which produces glucose with -anomeric
configuration, -glucosidase liberates glucose with an
-anomeric configuration [3].
Enzymes capable of hydrolyzing -1,6 glycosidic bonds in
pullulan are defined as pullulanases. On the basis of
substrate specificity and product pattern, pullulanases
have been classified into two groups: pullulanase type I
and pullulanase type II. Pullulanase type I (EC
specifically hydrolyzes the -1,6-linkages in pullulan as
well as in branched oligosaccharides (also called debranching enzyme); its degradation products are maltotriose and
linear oligosaccharides, respectively. Pullulanase type I is
unable to attack -1,4-linkages in -glucans (e.g. amylose).
Pullulanase type II, or amylopullulanase, attacks -1,6-glycosidic linkages in pullulan and branched polysaccharides.
Unlike pullulanase type I, this enzyme also attacks -1,4glycosidic linkages in branched and linear polysaccharides.
The action of pullulanase type II on amylopectin leads to
the formation of small sugars such as glucose, maltose and
maltotriose [3] (Figure 1).
In contrast to the previously described pullulanases, pullulan
hydrolases type I and type II are unable to hydrolyze
-1,6-glycosidic linkages in pullulan or in branched substrates. They cleave -1,4-glycosidic linkages in pullulan
leading to the formation of panose or isopanose. Pullulan
hydrolase type I or neopullulanase (EC
hydrolyzes pullulan to panose (-6-D-glucosylmaltose).
Pullulan hydrolase type II or isopullulanase (EC
hydrolyzes pullulan to isopanose (-6-maltosylglucose).
Recently, pullulan-hydrolase type III was described. This
archael enzyme attacks -1,4- as well as -1,6-glycosidic
linkages in pullulan-forming maltotriose, panose and
maltose (Figure 1) [3].
The branching enzyme -1,4-glucan (-1,4-glucan
6--glycosyltransferase; EC is one of the key
enzymes in the biosynthesis of starch and glycogen. The
enzyme forms -1,6 branching linkages by a transglycosylation reaction of -1,4 glucan. Intermolecular as well as
intramolecular reactions have been demonstrated for the
branching enzyme. In the first case, the enzyme cleaves
-1,4-glycosidic linkages of -1,4 glucan in one polysaccharide chain and then transfers the fragment to another
-1,4 glucan to create -1,6-branching linkages. In the
intramolecolar mode of action, the hydrolysis and the
transglycosylation reaction occur in the same polysaccharide chain. Such an intramolecolar branching reaction also
produces cyclic glucans from amylose and amylopectin .
Cyclodextrin glycosyltransferase (CGTase, EC, or
-1,4-D-glucan -4-D-(1,4-D-glucano)-transferase, is an
enzyme that is generally found in bacteria and was recently
discovered in archaea. This enzyme produces a series of
non-reducing cyclic dextrins (-, - and -cyclodextrins)

from starch, amylose, and other polysaccharides. -, - and

-cyclodextrins contain six, seven and eight glucose units,
respectively, that are linked by -1,4-bonds (Figure 1) [3].
In general, all these enzymes, which share four conserved catalytic regions (I, II, III, IV) and adopt a common (/)8barrel
structure [14], belong to the -amylase superfamily.

Thermoactive amylolytic enzymes: heat-stable

amylases and glucoamylases
Thermoactive amylolytic enzymes have been detected in
hyperthermophilic archaea of the genera Sulfolobus,
Thermophilum, Desulfurococcus and Staphylothermus [16].
The most thermoactive -amylases have been characterized from the hyperthermophilic archaea Pyrococcus woesei,
Pyrococcus furiosus, Thermococcus profundus and Thermococcus
hydrothermalis [1720]. The optimal temperatures for the
activity of these enzymes is between 80C and 100C. The
gene encoding an extracellular -amylase from P. furiosus
has been cloned and the recombinant enzyme has been
expressed in Bascillus subtilis and Escherichia coli [20,21].
This is the first report on the expression of an archaeal
gene derived from an extremophile in a Bacillus strain [21].
In addition, an intracellular -amylase gene from P. furiosus
has been cloned and sequenced [22]. It was interesting to
note that the four highly conserved regions usually
identified in -amylases are not found in this enzyme. The
-amylase from P. woesei is a dimeric enzyme with a subunit molecular mass of 50 kDa [23,24]. The enzyme (80%
activity at 120C) is inhibited by Zn2+ and does not require
the addition of calcium ions for its activity or stability. The
analysis of three-dimensional structure, however, reveals
that Ca2+ is tightly bound to the enzyme by a high-affinity
calcium-binding site (M Wilmanns, EMBL, Hamburg,
personal communication). -Amylases with lower thermostability have been isolated from the archaea Thermococcus
profundus, Pyrococcus sp. KOD1 and the bacteria Thermotoga
maritima and Dictioglomus thermophilum [2528]. The genes
encoding these enzymes were successfully expressed in
E. coli. Similar to the amylase from Bacillus licheniformis,
which is commonly used in liquefaction, the enzyme from
T. maritima requires Ca2+ for activity [27]. Further investigations have shown that the extreme hyperthermophilic
archaeon Pyrodictium abyssi can grow on various polysaccharides
and also secretes a heat-stable amylase (C Andrade,
G Antranikian, unpublished data).
Unlike -amylase, the production of glucoamylase seems to
be very rare in extremely thermophilic and hyperthermophilic
bacteria and archaea. Among the thermophilic anaerobic
bacteria, glucoamylases have been purified and characterized
from Clostridium thermohydrosulfuricum 39E [29], Clostridium
thermosaccharolyticum [30] and Thermoanaerobacterium thermosaccharolyticum DSM 571 [31]. Recently, it has been shown that
the thermoacidophilic archaea Thermoplasma acidophilum,
Picrophilus torridus and Picrophilus oshimae produce heat-stable
and acid-stable glucoamylases. These archaeal enzymes are
optimally active at pH 2.0 and 90C. The enzyme does not

Starch-hydrolyzing enzymes from thermophilic archaea and bacteria Bertoldo and Antranikian

lose activity at pH 1.04.0 after 6 h of incubation at 70C,

whereas it is rapidly inactivated at pH 8.0. No loss of activity
was observed in the presence of 1 mM sodium dodecyl sulfate,
3 M urea, 1 M guanidine HCl and 1% Triton X-100. Catalytic
activity is still detectable at pH 0.5 and 100C. This is the first
report on the production of glucoamylases in thermophilic
archaea (C Andrade, G Antranikian, unpublished data).


-Glucosidases are present in thermophilic archaea and

bacteria. -Glucosidases are generally involved in the last
step of starch degradation. These enzymes are inactive
against high-molecular-weight substrates such as starch or
pullulan. Only few thermoactive bacterial -glucosidase
have been described: from Thermoanaerobacter ethanolicus
and T. maritima [3,32]. The enzyme from T. maritima has
the unusual property of requiring NAD+ and Mn2+ for its
activity. This is the first example of a maltodextrin-degrading enzyme with these unusual characteristics. Among the
archaea, -glucosidase has been detected in P. furiosus,
P. woesei, Sulfolobus shibatae [4], Sulfolobus solfataricus [33],
Thermococcus strain AN1, and T. hydrothermalis [34,35]. An
intracellular -glucosidase has also been purified from
P. furiosus [36]. The enzyme exhibits optimal activity at
pH 5.0 to 6.0 over a temperature range of 105C to 115C;
the half-life at 98C is 48 h. An extracellular -glucosidase
from the thermophilic archaeon Thermococcus strain AN1
was purified and its molecular characteristics determined
[34]. The monomeric enzyme (60 kDa) is optimally active
at 98C. The purified enzyme has a half-life around
35 min, which is increased to around 215 min in the
presence of 1% (w/v) dithiothreitol and 1% (w/v) bovine
serum albumin. The substrate preference of the enzyme
is: para-nitrophenyl--D-glucoside > nigerose > panose >
palatinose > isomaltose > maltose and turanose. No activity
was found with starch, pullulan, amylose, maltotriose,
maltotetraose, isomaltotriose, cellobiose and -gentiobiose.
The gene encoding -glucosidase from T. hydrothermalis
was cloned by complementation of a Saccharomyces cerevisiae
maltase-deficient mutant strain [35]. The cDNA clone
isolated encodes an open reading frame corresponding to a
protein of 242 amino acids. The protein shows 42% identity to
a P. horikoshii unknown open reading frame but no similarities were obtained with other polysaccharidase sequences.

Thermoactive pullulanase, branching enzyme

and CGTase
Thermostable and thermoactive pullulanases from
extremophilic archaea have been detected in Thermococcus
celer, Desulfurococcus mucosus, Staphylothermus marinus and
Thermococcus aggregans [6,36]. Temperature optima between
90C and 105C, as well as remarkable thermostability even in
the absence of substrate and calcium ions, have been observed.
Most thermoactive pullulanases identified to date belong to
the type II group, which attack -1,4- and -1,6-glycosidic
linkages in polysaccharides. They have been purified from
P. furiosus, Thermococcus litoralis, T. hydrothermalis and Pyrococcus
strain ES4 [3638]. Pullulanase type II from P. furiosus, P. woesei


and T. hydrothermalis have been expressed in E. coli [3941].

The unfolding and refolding of the pullulanase from P. woesei
has been investigated using guanidinium chloride as denaturant. The monomeric enzyme (90 kDa) was found to be very
resistant to chemical denaturation and the transition midpoint
for guanidinium-chloride-induced unfolding was determined
to be 4.86 +/ 0.29 M for intrinsic fluorescence and
4.90 +/ 0.31 M for far-UV CD changes. The unfolding process
was reversible. Reactivation of the completely denatured
enzyme (in 7.8 M guanidinium chloride) was obtained upon
removal of the denaturant by stepwise dilution. 100% reactivation was observed when refolding was carried out with a
guanidinium chloride concentration of 4 M in the first dilution
step. Particular attention has been paid to the role of Ca2+,
which activates and stabilizes this archaeal pullulanase against
thermal inactivation. The enzyme binds two Ca2+ ions with a
Kd of 0.080 +/ 0.010 M and a Hill coefficient H of
1.00 +/ 0.10. This cation significantly enhances the stability of
the pullulanase against guanidinium-chloride-induced unfolding. The refolding of the pullulanase, on the other hand, was
not affected by Ca2+ [42]. Very recently, the genes encoding the
pullulanases from D. mucosus [43] and T. aggregans [44] have
been isolated and expressed in mesophilic hosts. The pullulanase from D. mucosus hydrolyzes -1,6-glycosidic linkages in
pullulan and also -1,4-glycosidic linkages in starch, amylose,
amylopectin and cyclodextrins producing maltotriose and maltose as main products. Because all thermoactive pullulanases
known so far from archaea are not active on cyclodextrins
and are in fact inhibited by these cyclic oligosaccharides, the
enzyme from D. mucosus should be considered as pullulanase
type II with a wider substrate specificity [43].
An unusual pullulanase has been recently found in
Thermococcus aggregans. This enzyme has different substrate
specificity as it attacks simultaneously -1,4- as well as
-1,6-glycosidic linkages in pullulan. On the basis of this
peculiar substrate specificity, it has been classified as pullulanhydrolase type III [44]. Interestingly, the gene sequence
showed only 26% homology to other bacterial family XIII
glycosyl hydrolases. Unlike pullulanase type II, to date
pullulanase type I has not been detected in archaea. The
first thermoactive debranching enzyme (pullulanase type I)
from an anaerobic thermophile was identified in the
bacterium Fervidobacterium pennivorans Ven5. This enzyme
was cloned and expressed in E. coli. In contrast to pullulanase type II, the enzyme from F. pennivorans Ven5 attacks
exclusively the -1,6-glycosidic linkages in polysaccharides
[45,46]. This thermostable debranching enzyme leads to
the formation of long-chain linear polysaccharides from
amylopectin. Other debranching enzymes have been
detected in T. maritima [47,48], in the less thermophilic
aerobic bacteria Bacillus flavocaldarius and Thermus caldophilus
GK-24 [49]. The pullulanase from T. caldophilus is optimally
active at 75C and pH 5.5, is thermostable up to 90C, and
does not require Ca2+ for either activity or stability.
Genes encoding branching enzyme have been isolated and the
enzymatic properties of this enzyme have been characterized,


Biocatalysis and biotransformation

Table 1
Starch-hydrolyzing enzymes from extremely thermophilic and hyperthermophilic archaea and bacteria.
Enzyme properties



Desulfurococcus mucosus (85)

Pyrococcus furiosus (100)

Pullulanase type I

Pullulanase type II

Optimal temp.

Pyrococcus sp. KOD1
Pyrococcus woesei (100)
Pyrodictium abyssi (98)
Staphylothermus marinus (90)
Sulfolobus solfataricus (88)

Thermococcus celer (85)

Thermococcus hydrothermalis (80)
Thermococcus profundus DT5432 (80)
Thermococcus profundus (80)
Thermococcus aggregans (85)
Dyctyoglomus thermophilum Rt46B.1(73) 90
Thermotoga maritima MSB8 (90)
Fervidobacterium pennavorans Ven5 (75) 80
Thermotoga maritima MSB8(90)
Thermus caldophilus GK24 (75)
Bacillus flavocaldarius (76)
Desulfurococcus mucosus(88)
Pyrococcus woesei (100)
Pyrococcus furiosus (100)
Pyrodictium abyssi (98)
Thermococcus celer (85)
Thermococcus litoralis (90)
Thermococcus hydrothermalis (80)
Thermoanaerobacter ethanolicus (85)
Thermotoga maritima (80)

Optimal pH

Mw (kDa)







190 (93)
93 (subunit)


Crude extract
Crude extract
Crude extract
Purified/cloned/Amy S
Purified/Amy L
Crude extract
Cloned/type I
Purified/cell associated
Purified/cell associated
Purified/cloned/cell associated
Purified/cloned/cell associated
Crude extract
Crude extract
Purified./cloned/requires cofactors

Pullulan hydrolase Thermococcus aggregans (85)

type III
Branching enzyme Rodothermus obamensis (80)
Thermoplasma acidophilum (60)
Picrophilus oshimae (60)
Picrophilus torridus (60)
Thermococcus sp. (75)
Anaerobranca gottschalkii (60)
Thermococcus strain AN1 (80)

Thermococcus hydrothermalis(80)
Pyrococcus furiosus (100)

Pyrococcus woesei (100)
Sulfolobus solfataricus (88)
Sulfolobus shibatae (88)

Thermoanaerobacter ethanolicus (85)
*Values in parentheses give the optimal growth temperature for each organism in C. , not determined.

mostly from moderate thermophilic microorganisms. Very

recently, a branching enzyme gene was isolated from an
extremely thermophilic bacterium, Rhodothermus obamensis.
The enzyme has successfully been expressed both in E. coli
and the fungus Aspergillus orizae. The enzyme, which converts
linear oligosaccharides to branched products, shows optimum
catalytic activity at pH 6.06.5 and 65C and is stable after
30 min of incubation at 80C. Branching activity of the enzyme
was higher toward amylose than amylopectin [50].
Thermostable CGTases are produced by the anaerobic
bacteria Thermoanaerobacter species, Thermoanaerobacterium
thermosulfurigenes and A. gottschalkii [6,51,52]. The latter
enzyme displays a very broad pH range for activity


(4.010.5) and the optimal temperature is 65C. When

incubated with starch, amylopectin or amylose, the
A. gottschalkii [53] CGTase produces -cyclodextrin as a
major product; only after prolonged incubation - and
-cyclodetrins are formed [3]. Recently, a CGTase, with
optimal temperature at 100C, was purified from a newly
isolated archaeon, Thermococcus sp. This is the first report
on the presence of a thermostable CGTase in a hyperthermophilic archaeon [54]. The enzyme from this strain has
been cloned and sequenced. The gene of 2217 nucleotides
encodes a protein with a molecular weight of 83 kDa. The
recombinant form of CGTase was obtained as an inclusion
body from E. coli and was purified to homogeneity, after
solubilization by 6 M urea and heat treatment at 80C for

Starch-hydrolyzing enzymes from thermophilic archaea and bacteria Bertoldo and Antranikian


Table 2
Regions conserved among thermophilic pullulanases.





Thermus sp.
























The underlined amino acid residues are those identified among all amylolytic enzymes. Q in region III of the Thermus pullulanase may be a G/C
sequencing error. The sequences are from the following sources: F. pennavorans Ven5 (GenBank n.AF096862), Thermotoga maritima (a/c
number AJ001087), Thermus sp. (a/c number R71616. WPI 95-100945/14), Caldicellulosiroptor saccharolyticus (a/c number L39876),
Desulfurococcus mucosus (GenBank a/c AF 247191), Thermococcus aggregans (a/c AJ251332).

20 min. Extreme thermophiles and hyperthermophiles

producing heat-stable glycosyl hydrolases are summarised
in Table 1.

Comparison of gene sequences of thermoactive

amylolytic enzymes
In general, starch-hydrolyzing enzymes contain four highly
conserved regions (IIV) that are implicated in catalytic
activity, substrate and metal ion binding [10]. Up to now, the
three-dimensional structures of four amylases (three from
mesophiles and one from a hyperthermophile) have been
determined, whereas no structure of pullanases is available.
However, it is likely that pullulanases are very similar to the
amylases, because most share the four conserved motifs and
have a similar arrangement of an (/)8 barrel structure. It
can be assumed that the catalytic mechanisms of the amylolytic enzymes are similar but the variations in substrate
specificity and products can be ascribed to the relationships
between their similar active centers and the different
residues surrounding the catalytic site [4,55].
The amino acid sequence alignment of -amylases has
been already presented [56,57]. Although some
sequences that are characteristic of certain specificities
have been identified in the pullulanases, it is, however, not
possible to offer a rational explanation for these sequencespecificity relationships. As shown in Table 2, a highly
conserved region of seven amino acids, YNWGYDP (using
single-letter amino acid code), is only found in type I
pullulanases (debranching enzymes) that are derived from
thermophilic and mesophilic bacteria and attack specifically
-1,6-glycosidic linkages in pullulan and branched polysaccharides. Because there is a strong selective pressure to
retain this motif in enzymes, which share little overall
homology, it can be speculated that this region is involved
in substrate binding or catalytic activity [46].
Among the Archaea, amino acid sequences of pullulanase
type II from T. hydrothermalis [41], P. abyssi and P. furiosus
[39] have been determined (Figure 2). Interestingly, the

YNWGYDP sequence and the four conserved regions

(IIV) are absent. The archaeal amylopullululanases,
which attack the -1,6 and -1,4 linkages in polysaccharides, however, are arranged into several well-defined
domains: a typical amino-terminal signal peptide, a catalytic
domain, and beyond the carboxyl terminus two almost
similar sequence repeats. This domain following the
carboxyl terminus has a complex structure probably
responsible of the enzyme anchor to the membrane [56].
Surprisingly, the enzyme from P. furiosus possesses only the
first two domains because the repeated motifs downstream
of the carboxyl terminus are absent (Figure 2). Because the
three strains are phylogenetically very related, we can
speculate that a mistake in determination of the sequence
of P. furiosus amylopullulanase occurred, and, presumably,
the multidomain structure is totally conserved. The other
archaeal enzymes from D. mucosus and T. aggregans, sharing
45.4% pairwise amino acid identity, do not contain this
multidomain structure and have low homology to the other
archaeal amylopullulanases. The four conserved regions
(IIV), however, are present (Table 2). This result is not
surprising, because both enzymes are characterized by an
unusual substrate specificity and product pattern. The
pullulanase from D. mucosus also hydrolyzes cyclodextrins
and the enzyme from T. aggregans (pullulan hydrolase
type III) attacks -1,4 and -1,6 linkages in pullulan [43,44].

A number of hyperthermophilic, thermoacidophilic and
thermoalkaliphilic microorganisms belonging to Archaea
and Bacteria produce unique amylolytic enzymes such as
-amylase, -glucosidase, pullulanase, glucoamylase and
CGTase. In addition to their industrial relevance [57],
these enzymes provide excellent model systems to study
structurefunction relationships of catalysts performing
reactions under extreme conditions. The molecular cloning
of the genes and their expression in heterologous hosts
allows circumventing of the problem of insufficient expression in the authentic extremophilic host. Thermoactive
pullulanases with different substrate specificity and


Biocatalysis and biotransformation

Figure 2





Y W K M A H Y L T E F P D I H V T I D L S G S L I A Q L A D Y M N G A K D I Y Q I I S E K I A N G E P L T Y D E K W F M L Q A P G G F 132
Y W K M A H Y L S Q Y P E V H A T I D L S G S L I A Q L A D Y M N G K K D T Y Q I I T E K I A N G E P L T V D E K W F M L Q A P G G F 133
Y W K M A H Y L S K Y P D V H V T I D L S G S L I A Q I A D Y M R G K K D I H Q I I T E K I A K G E P L T V E E K W F M L Q A P G G F 134



F D H T I P W N G E P V T D E N G N P I R D F W D R Y T E L K D K M L A A K Q K Y A N L P L E E Q K V A V T N E F T E Q D Y I D L A V 199
F D N T I P W N G E P I T D P N G N P I R D F W D R Y T E L K N K M L S A K A K Y A N F V T E S Q K V A V T N E F T E Q D Y I D L A V 200
F D H T I P W N G E P V A D K N G N P Y R R F W R R Y T Q L K D K M L D A K N K Y S N L P L E E Q K I A V T S E F T E Q D Y I D L A V 201



L F N L A W I D Y N Y I I S T P E L K A L Y D K V D E G G Y T R E D L K T V L Y H Q M W L L N N T F K E H E K I N L L L G N G N V E V 266
L F N L A W I D Y N Y I T S T P E F K A L Y D K V D E G G Y T R A D V K T V L D A Q I W L L N H T F E E H E K I N L L L G N G N V E V 267
L F N L A W I D Y D Y I M N T P E L K A L Y D K V D T G G Y T R K D V E T V L K H Q M W L L N H T F E E H E K I N L L L G N G N V E V 268



T V V P Y A H P I G P I L N D F G W S E D F D A H V K K A H E L Y K K Y L G G G V A T P R G G W A A E S A L N D K T L E I L A E N G W 333
T V V P Y A H P I G P I L N D F G W D S D F N D Q V K K A D E L Y K P Y L G G G T A V P K G G W A A E S A L N D K T L E I L A E N G W 334
T V V P Y T H P I G P I L N D F G W Y E D F D A Q V K K A N E L Y K E Y L G A G K V T P K G G W A A E S A L N D K T L E I L A E N G W 335



Q W V M T D Q M V L E R M G I P Y S I E N Y Y R P W V A E F N G K K I Y L F P R N H D L S D R V G F R Y S G M N Q Y E A V E D F I N E 400
E W V M T D Q M V L G K L G I E G T V E N Y H K P W V A E F N G K K I Y L F P R N H D L S D R V G F T Y S G M N Q Q Q A V E D F V N E 401
K W V M T D Q L V L E K L G V P K T I E S Y Y K P W V A Q F G D K K I Y L F P R N H D L S D R V G F R Y A G M N Q Y D A V K N F V E E 402



L L K I Q K Y N Y D G S L V Y V I T L D G E N P W E H Y P Y D G K L F L E T L Y K R L S E L Q E A G L I R T L T P T E Y I Q L Y G D K 467
L L K L Q K Q N Y D G S L V Y V V T L D G E N P V E N Y P Y D G E L F L T E L Y K K L T E L Q E Q G L I R T L T P S E Y I Q L Y G D K 468
L L K I Q K Q N Y D G S L V Y V I T L D G E N P W E H Y P F D G K L F L E E L Y R Q L E E L Q K K G L I R T V T P S E Y I E M F G D K 469



A N K L T P Q M M E R L D F T T E E R V E A L K V A N S L G E L Y D L A G V T E E M Q W P E S S W I D G T L S T W I G E P Q E N Y A W 534
A N K L T P R M M E R L D L T G D N - V N A L L K A Q S L G E L Y D M T G V K E E M Q W P E S S W I D G T L S T W I G E P Q E N Y G W 534
A N K L T P K M M K R L D F T T E D N V N A L L K A K T L G E L Y D M V G V T E E M Q W P E S S W I D G T L S T W I G E P Q E N I A W 536



Y W L Y L A R R T L M E N K D K M D S A S W E K A Y E Y L L R A E A S D W F W W Y G N D Q D S G Q D Y S F D R Y F K T Y L Y E I Y K L 601
Y W L Y M A R K A L M E N K D K M S Q A D W E K A Y E Y L L R A E A S D W F W W Y G S D Q D S G Q D Y T F D R Y L K T Y L Y E M Y K L 601
Y W L Y L A R K A L F E N K D - - N V K D W N K A Y E Y L F R A E G S D W F W W Y G R D Q N S M Q D Y V F D R Y F K L Y L Y E I Y K L 601



A G V E P P S Y L Y G N Y F P D G A P Y T V R A L E G L K E G D V K E Y S S L S P V A E G V K V F F D S Q G L H F I I K G R I D K F E 668
A G V E P P S Y L F G N Y F P D G E P Y T T R G L V G L K D G E M K N F S S M S P L A K G V S V Y F D G E G I H F I V K G N L D R F E 668
A G L E P P S Y L F G N Y Y P D G E P Y I V R A L V G L P E G V K K N W S S L S P L A K G I E V Y F D D E G L H F V V L T N R S - F E 667



I S I Y E K D K R I G N T F T L L Q K K P D K I R Y D V F P F V R D S V G L M I T K H I V Y K D G K A E I Y N A T D Y E G Y E K I G E 735
V S I W E K D E R V G N T F T R L Q E K P D E L S Y F M F P F S R D S V G L L I T K H V V Y E N G K A E I Y G A T D Y E K S E K L G E 735
I S I Y E P E K I I G N T F T V L Q K K P E E F R Y S E V P F S K D S V G L L I T T H I T V K G E R G E V F K A T S Y D N Y K K V G E 734



A Q V S V N G D E I E V I V P F E Y L E T P E D F Y F A V S T V D E L G M L E V I T T P V N L K L P V Q V K G V V L V D I A D P E G D 802
A T V K N T S E G I E V V L P F D Y I E N P S D F Y F A V S T V K D - G D L E V I S T P V E L K L P T E V K G V V I A D I T D P E G D 801
V K V N A I N G G Y E V V V P F D Y I E T P S D F Y F A V S T I N D N G S L E I I T T P I H L K L P K E I E G T L I T E I K D I E G D 801



D H G P G T Y T Y P T D K V F V E G A F D L L R F R M L E Q T D A Y V M E F Y F K E L G G N P W K W A - - - - - - - - - - - - - - - - 853
D H G P G N Y T Y P T D K V F K P G V F D L L R F R M L E Q T E S Y V M E F Y F K D L G G N P W N G P N G F S L Q I I E V Y L D F K D 868
D H G P G N Y T Y A T D K V F V E H H L D L L K V R L L E R P N S Y V F E F Y F K E L G D N P W N A P Y G F S L Q I M E V Y L D Y K E 868



- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 853
G G N S S A I K M F P D G P G A N V N L D P E H P W D V A F R I A G W D Y G N L I I L P N G T A I Q G E M Q I S A D P V K N A I I V K 935
G G N T S A I K M F P D G P G S N V D L D P E H P W D V A L R I A G W D Y G N I I V L A N G T T Y Q G E M K I S A D P V K N R I I V E 935



- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 853
V P K K Y I A I N E D Y G L W G D V L V G S Q D G Y G P D K W R T A A V D A E Q W K L G G A D P Q A V I N G V A P R V I D E L V P Q G 1002
V P K K Y L P K V P E F - - - M A V L V G S Q D G F G P D K W R P V S V K A E Q W V G G G A P A D A V I A G V A P R V Y D L L V P E G 999



- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 853
F E P T Q E E Q L S S Y D A N D M K L A T V K A L L L L K Q G - - - I V V T D P E G D D H G P G T Y T Y P T D K V F K P G V F D L L K 1066
F E P T Q E E Q L S S F D P K A G K R A V V K M I P V K A K T N V I V D M K D I E G D D H G P G T Y T Y A T D K V F V E H H L D L L R 1066



- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 853
F K V T E G S D D W T L E F H F K D L G G N P W N G P N G F S L Q I I E V Y F D F K E G G N V S A I K M F P D G P G S N V R L D P N H 1133
F R M L D T G D T Y T L E F Y F K E L G D N P W N A P Y G F S L Q I I E V Y L D F K E G G N T S A I K M F P D G P G S N V D L D P E H 1133



- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 853
P W D L A L R I A G W D Y G N L I I L P D G T A Y Q G E M Q I S A D P V K N A I I V K V P K K Y L N I S D - Y G L Y T A V I V G S Q D 1199
P W D V A L R I A G W D Y G N I I V P A N G T V Y T G E M K I S A D P I K N A I I V E V P K K F I S L D K N Y G L Y G A V L V G S Q D 1200



- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 853
G Y G P D K W R P V A A E A E Q W K L G G A D P Q A V I D N L V P R V V D E L V P E G F K P T Q E E Q L S S Y D L E K K T L A T V L M 1266
G F G P D K W R P V S V K A E Q W V G G G A S A E A V I A G V A P R V Y D L L V P Q G F R P T Q E E Q L S S F D P K A G K R A I V K M 1267



- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 853
V P L V N G T G G E E P - - - - - T P T E S P T E T T T T T P S E T T - - - T T T S T T T G P S S T T T S T P - - - - - - - - - - - - 1313
I P L W S V P K E E K P S Q T G T T T T R T P T K T S T P T P E K T T P K T T K T K T E T K E S P S P S Q T P P S A G A P P S G E E R 1334




M S - R K L S L L L V F L I F G S M L G A N - N I V K A E E P K P L N V I I V W H Q H Q P Y Y Y D P V Q G I Y T R P W V R L H A A N N 65
M R - R V V A L F I A I L M L G S I V G A N V K S V G A A E P K P L N V I I V W H Q H Q P Y Y Y D P V Q D V Y T R P W V R L H A A N N 66
M R G K F R A L F V L V I F V I S V L G P G I K S V N A A E P K P L N V I I V W H Q H Q P Y Y Y D P I L G V Y T R P W V R L H A A N N 67


Current Opinion in Chemical Biology

Starch-hydrolyzing enzymes from thermophilic archaea and bacteria Bertoldo and Antranikian


Figure 2
Amino acid sequence alignment of archaeal amylopullulanases
(pullulanase type II). Enzyme sources are abbreviated: PullP.furi
(a/c T08162) [39], PullT.hydro (a/c AF113969.1) [55] and
PullP.abys (GenBank a/c NC000868.1). Sequences are numbered
from the amino terminus, including the signal peptides. Identical

residues are framed. The sequence of the amylopullulanase from

P. furiosus ends at the amino acid 853 because the repeated
motifs downstream of the carboxyl terminus are absent. P. abys,
Pyrococcus abysii; P. furi, Pyrococcus furiosus; T. hydro,
Thermococcus hydrothermalis.

product pattern have been presented. The majority of

extremophiles investigated so far possess pullulanase
type II (amylopullulanase). The presence of pullulanase
type I (debranching enzyme) has been demonstrated in only
three thermophilic strains and pullulan hydrolase type III in
Thermococcus aggregans only. Novel archaeal glucoamylases,
which show reasonable activity at 70100C and pH 0.73.0,
have been found in three thermoacidophilic archaeal strains
belonging to the genera Picrophilus and Thermoplasma.
Thermo-stable and alkaline-stable CGTases are produced
by a hyperthermophilic archaeon and a thermoalkaliphilic
bacterium. Comparison of the primary sequence of the
pyrococcal pullulanase with other members of the glucosyl
hydrolase family revealed that significant differences are
responsible for the mode of action of these enzymes. In depth
information on the molecular properties of the enzymes and
their genes is necessary in order to understand the strategy
and mechanisms developed by this fascinating group of
microorganisms. Modern techniques, such as genomics,
proteomics, gene shuffling, structural genomics, gene
evolution and protein engineering will help to generate new
tailor-made enzymes with desired properties and unravel
the mechanisms involved in protein stabilization [55].


Sunna A, Moracci M, Rossi M, Antranikian G: Glycosyl hydrolases

from hyperthermophiles. Extremophiles 1996, 1:2-13.


Ladenstein R, Antranikian G: Proteins from hyperthermophiles:

stability and enzymatic catalysis close to the boiling point of
water. Adv Biochem Eng/Biotechnol 1998, 61:37-85.


Feller G, Narinx E, Arpigny JL, Aittaleb M, Baise E, Genicot S,

Gerday C: Enzyme from extremophilic microorganisms. FEMS
Microbiol Rev 1996, 18:189-202.


10. Vieille C, Zeikus GJ: Hyperthermophilic enzymes: sources,

uses and molecular mechanisms for thermostability. Microbiol

Mol Biol Rev 2001, 65:1-43.
An overview about the enzymes from hyperthermophilic microorganisms.
Special attention is given to the stability features, furthermore, the potential
applications of the enzymes are described.
11. Stetter KO: Hyperthermophiles: isolation, classification and
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Environments. Edited by Horikoshi K, Grant WD. New York:
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12. Huber H, Stetter KO: Hyperthermophiles and their possible
potential in biotechnology. J Biotechnol 1998, 64:39-52.
13. Stetter KO: Extremophiles and their adaptation to hot

environments. FEBS Lett 1999, 452:22-25.

KO Stetter is the most famous pioneer of thermophilic microorganisms. He
describes the properties of the thermophilic strains, their characteristics
(optimum temperature and pH growth, duplication time etc.) and their
adaptation at high temperature.
14. MacGregor EA, Janecek S, Svensson B: Relationship of sequence
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Thanks are due to the Deutsche Bundesstiftung Umwelt and to the Fonds
der Chemischen Industrie for financial support.

15. Horikoshi K: Alkaliphiles. In Extremophiles Microbial Life in

Extreme Environments. Edited by Horikoshi K, Grant WD. New York:
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References and recommended reading

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of special interest
of outstanding interest

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