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Introduction
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
152
Figure 1
CGTase
-Amylase
-Amylase
Debranching
enzymes
Glucoamylase
-glucosidase
-Limit dextrin
Branching
enzymes
Pullulanase II
Pullulanase I
Linear oligosacch.
Maltotriose
Isoamylase
Glucose
Maltose
Maltose and
-limit-dextrin
Glucose
Maltose
Glucose
Linear oligosaccharides
Pullulanase
type I and II
Pullulan
hydrolase type I
Panose
Pullulan
Pullulan
hydrolase type II
Isopanose
Pullulan
hydrolase type III
Maltose
Panose
Maltotriose
Current Opinion in Chemical Biology
Starch-hydrolyzing enzymes from thermophilic archaea and bacteria Bertoldo and Antranikian
153
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
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 3.2.1.1),
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 3.2.1.2) and glucose by glucoamylase
(3.2.1.3) [3] (Figure 1).
154
Starch-hydrolyzing enzymes from thermophilic archaea and bacteria Bertoldo and Antranikian
-Glucosidases
155
156
Table 1
Starch-hydrolyzing enzymes from extremely thermophilic and hyperthermophilic archaea and bacteria.
Enzyme properties
Enzymes
Organism*
-Amylase
Pullulanase type I
Pullulanase type II
Optimal temp.
(C)
100
100
100
Pyrococcus sp. KOD1
90
Pyrococcus woesei (100)
100
Pyrodictium abyssi (98)
100
Staphylothermus marinus (90)
100
Sulfolobus solfataricus (88)
Optimal pH
Mw (kDa)
Remarks
5.5
6.57.5
7.0
6.5
5.5
5.0
5.0
5.5
5.05.5
5.5
4.05.0
6.5
5.5
7.0
6
6.0
5.5
6.3
5.0
6.0
6.0
9.0
5.5
5.5
5.5
5.5
7.5
129
68
49.5
68
240
49
42
42
75
61
190 (93)
93 (subunit)
65
65
74
90
90
119
128
?
58
Purified/cloned
Purified/cloned/intracell.
Purified/cloned/extracell.
Purified/cloned/extracell.
Purified/Extracellular.
Crude extract
Crude extract
Extracellular
Crude extract
Cloned/purified
Purified/cloned/Amy S
Purified/Amy L
Crude extract
Purified/cloned/intracell.
Purified/cloned/lipoprotein
Purified/cloned
Cloned/type I
Purified/cell associated
Purified/cell associated
Purified/cloned
Purified/cloned/cell associated
Purified/cloned/cell associated
Crude extract
Crude extract
Purified/extracell./glycoprotein
Purified/cloned/glycoprotein
Purified/cloned
Purified./cloned/requires cofactors
Purified/cloned
63
Thermococcus hydrothermalis(80)
120
5.5
57
Pyrococcus furiosus (100)
5.06.0
125
Pyrococcus woesei (100)
100
5.05.5
90
Sulfolobus solfataricus (88)
>120
4.5
80
Sulfolobus shibatae (88)
5.5
313
Thermoanaerobacter ethanolicus (85)
75
5.05.5
?
*Values in parentheses give the optimal growth temperature for each organism in C. , not determined.
Purified/cloned
Purified
Purified
Purified
Purified
Purified
Purified/extracell./glycoprotein
Cloned
Purified/extracellular
Purified/cloned/intracellular
Extracellular
Extracellular
Purified/cloned
Starch-hydrolyzing enzymes from thermophilic archaea and bacteria Bertoldo and Antranikian
157
Table 2
Regions conserved among thermophilic pullulanases.
Source
YNWGYDP
II
III
IV
Fervidobacterium
pennivorans
Thermotoga
maritima
Thermus sp.
Caldicellul.
saccharolyticus
Desulfurococcus
mucosus
Thermococcus
aggregans
424 YNWGYDP
465 GIRVILDMVFPHT
538 DGFRFDQMGL
570 YGEPWGG
648 PQETINYVEVHDNHTLWD
430 YNWGYDP
473 FTGVIMDMVFPHT
548 DGFRFDQMGL
580 YGEPWGG
638 PEETINYAACHDNHTLWD
572 YNWGYNP
405 YNWGYDP
333 GLRVVMDAVYNHV
445 GIGVVMDVVFNHT
405 DGFRFDLMGV
520 DGFRFDLMGL
437 YGQGWDL
552 YGEGWVM
512 PRQSINYVECHDNHTFWD
629 PDECVNYVSCHDNLTLFD
272 GIKVIFDFVPDHV
458 DGLRIDTPLD
392 VGEIWDY
449 AGMGFNIIGSHDTSRVLT
375 GIRIIFDFVPNHS
461 DGIRIDAPQE
495 VGEIWEL
551 IAMGFNLVSSHDTSRVLT
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).
Conclusion
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
158
Figure 2
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67
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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
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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
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201
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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
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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
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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
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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
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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
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535
535
537
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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
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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
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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
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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
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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
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869
869
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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
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936
936
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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
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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
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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
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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
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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
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- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 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
PullP.furi
PullT.hydro.
PullP.abys
0
1314
1335
-------------------------------GGGICGPGIIAGLALIPLLLKRRN
TTQKTGGICGPAFVLVIVAIVAIARKRF
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
853
1337
1362
Starch-hydrolyzing enzymes from thermophilic archaea and bacteria Bertoldo and Antranikian
159
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
7.
8.
9.
Acknowledgements
Thanks are due to the Deutsche Bundesstiftung Umwelt and to the Fonds
der Chemischen Industrie for financial support.
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2.
6.
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160
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