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Information on EC 5.3.1.4 - L-arabinose isomerase and Organism(s) Geobacillus stearothermophilus and UniProt Accession Q9S467
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5.3.1.4 L-arabinose isomeraseIUBMB Comments
Requires a divalent metal ion (the enzyme from the bacterium Escherichia coli prefers Mn2+) . The enzyme binds beta-L-arabinopyranose and catalyses ring opening to generate a form of open-chain conformation that facilitates the isomerization reaction, which proceeds via an ene-diol mechanism . The enzyme can also convert alpha-D-galactose to D-tagatose with lower efficiency .
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Word Map
- 5.3.1.4
- d-tagatose
-
isomerization
- l-ribulose
- nutrition
- geobacillus
-
sweetener
- isomerases
- synthesis
- stearothermophilus
- food industry
- thermodenitrificans
-
low-calorie
- l-ribulokinase
-
packed-bed
-
arabad
- sakei
-
4-epimerase
- industry
The taxonomic range for the selected organisms is: Geobacillus stearothermophilus
The expected taxonomic range for this enzyme is: Bacteria, Archaea
The expected taxonomic range for this enzyme is: Bacteria, Archaea
Synonyms
l-arabinose isomerase, arabinose isomerase, l-ai us100, l-ai nc8, d-galactose isomerase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
GSAI
Geobacillus stearothermophilus L-arabinose isomease
L-arabinose aldose-ketose-isomerase
-
Isomerase, L-arabinose
-
-
-
-
L-arabinose isomerase
L-arabinose ketol-isomerase
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
isomerization
isomerization
intramolecular oxidoreduction
-
-
-
-
isomerization
-
-
-
-
PATHWAY SOURCE
PATHWAYS
BRENDA
-
-
SYSTEMATIC NAME
IUBMB Comments
beta-L-arabinopyranose aldose-ketose-isomerase
Requires a divalent metal ion (the enzyme from the bacterium Escherichia coli prefers Mn2+) [2]. The enzyme binds beta-L-arabinopyranose [4] and catalyses ring opening to generate a form of open-chain conformation that facilitates the isomerization reaction, which proceeds via an ene-diol mechanism [6]. The enzyme can also convert alpha-D-galactose to D-tagatose with lower efficiency [5].
CAS REGISTRY NUMBER
COMMENTARY
9023-80-7
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
L-arabinose
L-ribulose
other aldoses like D-fucose, D-ribose, D-allose, D-mannose and D-xylose are poor substrates for the wild type and the mutated enzymes
-
-
?
D-galactose
D-tagatose
-
at elevated temperatures in the presence of divalent metal ions
-
-
?
D-galactose
D-tagatose
-
the bioconversion yield of D-galactose to D-tagatose by the purified enzyme after 12 h at 65°C reaches 36%
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
D-galactose
D-tagatose
-
at elevated temperatures in the presence of divalent metal ions
-
-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Co2+
Mn2+
Mn2+
additional information
Co2+
activating, highest activity of the mutated enzyme at 1.0 mM
Co2+
increase in activity at 80°C only, no effect at 65°C
Mn2+
activating, highest activity of the wild type enzyme at 1.0 mM
Mn2+
increase in activity at 80°C only, no effect at 65°C
Mn2+
is closely bound to the protein even after treatment with EDTA
Mn2+
-
strong activator, approximately 0.06 eqiuvalents of Mn2+/subunit
Mn2+
-
404% relative activity at 1 mM, the enzyme has an absolute requirement for the divalent metal ion Mn2+ for both catalytic activity and thermostability
Mn2+
-
the enzyme is not metal-dependent for catalytic activity but Mn2+ significantly enhances the activity of its apo enzyme and increases thermostability
additional information
-
Ca2+, Ni2+, Zn2+, Mg2+, Fe2+, Cu2+ show no effect
additional information
Ca2+, Ni2+, Zn2+, Mg2+, Fe2+, Cu2+ show no effect
additional information
-
activity and thermostability are totally independent of metallic ions up to 65°C, above 65°C, the enzyme's activity is also independent of metallic ions of its activity, but its thermostability is improved in the presence of only 0.2 mM Co2+ and 1 mM Mn2+
additional information
activity and thermostability are totally independent of metallic ions up to 65°C, above 65°C, the enzyme's activity is also independent of metallic ions of its activity, but its thermostability is improved in the presence of only 0.2 mM Co2+ and 1 mM Mn2+
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
279
D-galactose
pH 8.0, 60°C, recombinant enzyme expressed in Bacillus subtilis
578
D-galactose
pH 8.0, 60°C, recombinant enzyme expressed in Escherichia coli
77
L-arabinose
pH 8.0, 60°C, recombinant enzyme expressed in Bacillus subtilis
100
L-arabinose
pH 8.0, 60°C, recombinant enzyme expressed in Escherichia coli
18.6
L-arabinose
-
in the presence of 0.8 mM Mn2+, at pH 7.5 and 60°C
22.6
L-arabinose
-
in the presence of 0.6 mM Mn2+, at pH 7.5 and 60°C
28
L-arabinose
-
in the presence of 0.4 mM Mn2+, at pH 7.5 and 60°C
39
L-arabinose
-
in the presence of 0.2 mM Mn2+, at pH 7.5 and 60°C
61
L-arabinose
-
in the presence of 0.1 mM Mn2+, at pH 7.5 and 60°C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
20.18
D-galactose
pH 8.0, 60°C, recombinant enzyme expressed in Escherichia coli
53.08
D-galactose
pH 8.0, 60°C, recombinant enzyme expressed in Bacillus subtilis
75.25
L-arabinose
pH 8.0, 60°C, recombinant enzyme expressed in Bacillus subtilis
107.9
L-arabinose
pH 8.0, 60°C, recombinant enzyme expressed in Escherichia coli
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.06
0.37
0.39
0.48
0.59
A408V/K475N mutant found in GSAI152 or GASI 153w D-galactose as substrate
2.3
of the recombinant wild type enzyme after purification
69
purified recombinant enzyme expressed in Bacillus subtilis, D-galactose isomerization
97
purified recombinant enzyme expressed in Bacillus subtilis, L-arabinose isomerization
0.9
-
purified enzyme, in the absence of Mn2+, at pH 7.5 and 60°C
6.9
-
purified enzyme, in the presence of 1 mM Mn2+, at pH 7.5 and 60°C
additional information
0.39
K475N mutant found in GSAI152, D-galactose as substrate
0.48
A475N mutant found in GSAI153, D-galactose as substrate
additional information
D-galactose bioconversion rates in% at different pH values at 65°C and 70°C with wild-type and mutant enzymes, overview
additional information
-
D-galactose bioconversion rates in% at different pH values at 65°C and 70°C with wild-type and mutant enzymes, overview
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
6 - 7
mutant Q268K/N175H, stable without addition of metal ions
7.5
7.5
7.5
Q408V mutant enzyme with higher isomerization activites than the wild type enzyme
7.5
R408V mutant enzyme with higher isomerization activites than the wild type enzyme
8
recombinant enzyme expressed in Escherichia coli or Bacillus subtilis, D-galactose isomerization and L-arabinose isomerization
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7 - 9
5 - 8.5
-
the enzyme exhibits 82% and 65% of its maximum activity at pH 6.0 and 5.0 respectively and maintains more than 80% activity at pH values ranging from 6.0 to 8.5
7 - 9
recombinant enzyme expressed in Bacillus subtilis
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
50 - 65
mutant Q268K/N175H, stable without addition of metal ions
60
65
70
80
60
recombinant enzyme expressed in Escherichia coli or Bacillus subtilis, D-galactose isomerization and L-arabinose isomerization
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
40 - 80
recombinant enzyme expressed in Bacillus subtilis
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
56000
112000
-
native gradient gel electrophoresis, weak band (dimer)
225000
56000
57000
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
tetramer
tetramer
alpha4, 4 * 56000, SDS-PAGE, native gel electrophoresis
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D228N/D384G/T393S/N428K/K475N
site directed mutagenesis, GSAI 152
M322V/S393T/V408A
error prone PCR mutagenesis using gali 152 as template, gali 153 with changes in 3 amino acids revealed a higher activity than gali 152
N175H
site-directed mutagenesis, the N175H mutant has a broad optimal temperature range from 50 to 65°C
Q268K
site-directed mutagenesis, the mutant enzyme shows increased acidotolerance and is more stable at acidic pH than the wild-type enzyme
Q268K/N175H
site-directed mutagenesis, the Q268K mutant is more acidotolerant, the N175H mutant has a broad optimal temperature range from 50 to 65°C
H175N
-
the mutant exhibits faster D-galactose bioconversion compared to the wild type enzyme
Q268K
-
the mutant shows a pH optimum of 6.0-6.5 and a higher stability at acidic pH compared to the wild type enzyme, the mutant exhibits faster D-galactose bioconversion compared to the wild type enzyme
Q268K/H175N
-
the mutant exhibits faster D-galactose bioconversion compared to the wild type enzyme
additional information
additional information
construction of three Bacillus stearothermophilus US100 L-arabinose isomerase mutants with increased activity for D-galactose and increased D-tagatose production
additional information
-
construction of three Bacillus stearothermophilus US100 L-arabinose isomerase mutants with increased activity for D-galactose and increased D-tagatose production
additional information
evaluation and optimization of Escherichia coli and Bacilus subtilis expression systems for toxic byproducts in L-ribulose and/or D-tagatose production by the recombinant engineered enzyme
additional information
-
five point mutations increase the activity 11fold in the first round of evolution, and three point mutations in the second round of evolution increase the activity 5fold beyond the activity from the first round
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
5
65°C, wild-type enzyme: half-life 13 h, mutant Q268K: half-life 43 h, mutant N175H: half-life 13 h, mutant Q268K/N175H: half-life 43 h
702482
7.5
65°C, wild-type enzyme: half-life 69 h, mutant Q268K: half-life 65 h, mutant N175H: half-life 69 h, mutant Q268K/N175H: half-life 64 h
702482
8.5
65°C, wild-type enzyme: half-life 51 h, mutant Q268K: half-life 38 h, mutant N175H: half-life 50 h, mutant Q268K/N175H: half-life 37 h
702482
6
-
half life for tagatose production in free cells: 2.7 h, in immobilized cells: 71.4 h
661387
7
-
half life for tagatose production in free cells: 3.0 h, in immobilized cells: 150 h
661387
8
-
half life for tagatose production in free cells: 2.8 h, in immobilized cells: 144 h
661387
9
-
half life for tagatose production in free cells: 1.3 h, in immobilized cells: 25.9 h
661387
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
50 - 65
100% activity in absence of metallic ions during 12 h
65
70
pH 7.5, wild-type enzyme: half-life 1.8 h, mutant Q268K: half-life 1.6 h, mutant N175H: half-life 0.7 h, mutant Q268K/N175H: half-life 0.6 h
70 - 80
decrease in activity in absence of metallic ions
75
pH 7.5, wild-type enzyme: half-life 1.0 h, mutant Q268K: half-life 0.9 h, mutant N175H: half-life 0.4 h, mutant Q268K/N175H: half-life 0.4 h
80
pH 7.5, wild-type enzyme: half-life 0.2 h, mutant Q268K: half-life 0.2 h, mutants N175H and Q268K/N175H are inactivated
50
-
half life for tagatose production in free cells: 8.7 h, in immobilized cells: 632 h
60
-
half life for tagatose production in free cells: 2.8 h, in immobilized cells: 144 h
65
-
half life for tagatose production in free cells: 1.4 h, in immobilized cells: 26.0 h
70
-
half life for tagatose production in free cells: 0.6 h, in immobilized cells: 12.5 h
80
additional information
65
until 65°C independent from metallic ions, above 65°C stability is improved in presence of 0.2 mM Co2+ and 1 mM Mn2+
65
pH 7.5, wild-type enzyme: half-life 70 h, mutant Q268K: half-life 69 h, mutant N175H: half-life 70 h, mutant Q268K/N175H: half-life 71 h
80
-
half life for tagatose production in free cells: 0.25 h, in immobilized cells: 7.0 h
additional information
-
EDTA-treated AI has a lower melting temperature (72°C) than the holoenzyme (78°C)
additional information
-
thermostability is totally independent of metallic ions up to 65°C, above 65°C, thermostability is improved in the presence of only 0.2 mM Co2+ and 1 mM Mn2+
additional information
thermostability is totally independent of metallic ions up to 65°C, above 65°C, thermostability is improved in the presence of only 0.2 mM Co2+ and 1 mM Mn2+
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
of the recombinant wild type and mutant enzymes
Hiprep 16/10 Q-XL column chromatography, phenyl-Sepharose column chromatography, Mono Q HR 5/5 column chromatography, and Superdex 200 gel filtration
-
His-bind resin column chromatography, and Superdex 200 gel filtration
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
expression in Escherichia coli of wild type and gali 153 mutant enzyme
expression of wild-type and mutant enzymes in Escherichia coli strain HB101
gene araA, subcloning in Escherichia coli strain DH5alpha, expression in Bacillus subtilis strain 168, ATCC 27370, and secretion at large quantities to the medium
expressed in Escherichia coli BL21(DE3) cells
-
expressed in Escherichia coli, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus
-
expression in Escherichia coli
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
food industry
synthesis
food industry
-
hyperthermophilic L-arabinose isomerase is useful in the commercial production of D-tagatose as a low-calorie bulk sweetener
nutrition
additional information
-
additionally, D-tagatose can potentially be used as a prescription drug additive to mask unpleasant tastes, and as a sweetener in toothpaste, mouthwash, and cosmetics such as flavoured lipstick
food industry
production of D-tagatose as a low-calorie sugar-substituting sweetener lower pH is preferable for industrial production
food industry
production of D-tagatose as a low-calorie sugar-substituting sweetener, the D-tagatose yield from the mutated enzyme is higher than from the wild type
synthesis
engineered enzyme mutants are useful for production of D-tagatose
synthesis
the enzyme is useful in production of low-calorie sweetener D-tagatose. Tagatose also may potentially be useful as a prescription drug additive, to mask unpleasant tastes, and as a sweetener in toothpaste, mouthwash, and cosmetics such as flavored lipstick
nutrition
-
the product D-tagatose is used as a low-calorie bulk sweetener
nutrition
-
there has been industrial interest in the end product D-tagatose as a low-calorie sugar-substituting sweetener
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Kim, P.
Current studies on biological tagatose production using L-arabinose isomerase: a review and future perspective
Appl. Microbiol. Biotechnol.
65
243-249
2004
Bacillus subtilis (P94523), Bacteroides thetaiotaomicron (Q8AAW1), Bifidobacterium longum (Q8G7J3), Clostridium acetobutylicum (Q97JE0), Clostridium acetobutylicum (Q97JE4), Escherichia coli (P08202), Escherichia coli (P58538), Escherichia coli (Q8FL89), Geobacillus stearothermophilus, Halalkalibacterium halodurans (Q9KBQ2), Klebsiella aerogenes, Lactiplantibacillus plantarum (Q88S84), Lactobacillus gayonii, Oceanobacillus iheyensis (Q8EMP4), Salmonella enterica subsp. enterica serovar Typhi (P58539), Salmonella enterica subsp. enterica serovar Typhimurium (P06189), Shigella flexneri (Q7UDT4), Thermoanaerobacter mathranii, Thermotoga maritima (Q9WYB3), Thermotoga neapolitana, Thermus sp., Vibrio parahaemolyticus, Yersinia pestis (P58540)
Lee, D.W.; Choe, E.A.; Kim, S.B.; Eom, S.H.; Hong, Y.H.; Lee, S.J.; Lee, H.S.; Lee, D.Y.; Pyun, Y.R.
Distinct metal dependence for catalytic and structural functions in the L-arabinose isomerases from the mesophilic Bacillus halodurans and the thermophilic Geobacillus stearothermophilus
Arch. Biochem. Biophys.
434
333-343
2005
Geobacillus stearothermophilus, Halalkalibacterium halodurans
Rhimi, M.; Bejar, S.
Cloning, purification and biochemical characterization of metallic-ions independent and thermoactive l-arabinose isomerase from the Bacillus stearothermophilus US100 strain
Biochim. Biophys. Acta
1760
191-199
2006
Geobacillus stearothermophilus, Geobacillus stearothermophilus (Q9S467), Geobacillus stearothermophilus US100
Jung, E.S.; Kim, H.J.; Oh, D.K.
Tagatose production by immobilized recombinant Escherichia coli cells containing Geobacillus stearothermophilus l-arabinose isomerase mutant in a packed-bed bioreactor
Biotechnol. Prog.
21
1335-1340
2005
Geobacillus stearothermophilus
Kim, H.J.; Kim, J.H.; Oh, H.J.; Oh, D.K.
Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase that increases the production rate of D-tagatose
J. Appl. Microbiol.
101
213-221
2006
Geobacillus stearothermophilus (Q9S467), Geobacillus stearothermophilus, Geobacillus stearothermophilus KCCM12265 (Q9S467)
Rhimi, M.; Juy, M.; Aghajari, N.; Haser, R.; Bejar, S.
Probing the essential catalytic residues and substrate affinity in the thermoactive Bacillus stearothermophilus US100 L-arabinose isomerase by site-directed mutagenesis
J. Bacteriol.
189
3556-3563
2007
Geobacillus stearothermophilus (Q9S467), Geobacillus stearothermophilus
Oh, D.K.; Oh, H.J.; Kim, H.J.; Cheon, J.; Kim, P.
Modification of optimal pH in L-arabinose isomerase from Geobacillus stearothermophilus for D-galactose isomerization
J. Mol. Catal. B
43
108-112
2006
Geobacillus stearothermophilus (Q9S467)
-
Rhimi, M.; Aghajari, N.; Juy, M.; Chouayekh, H.; Maguin, E.; Haser, R.; Bejar, S.
Rational design of Bacillus stearothermophilus US100 L-arabinose isomerase: potential applications for D-tagatose production
Biochimie
91
650-653
2009
Geobacillus stearothermophilus (Q9S467), Geobacillus stearothermophilus, Geobacillus stearothermophilus US100 (Q9S467)
Cheon, J.; Kim, S.; Park, S.; Han, J.; Kim, P.
Characterization of L-arabinose isomerase in Bacillus subtilis, a GRAS host, for the production of edible tagatose
Food Biotechnol.
23
8-16
2009
Geobacillus stearothermophilus (Q9S467)
-
Rhimi, M.; Chouayekh, H.; Gouillouard, I.; Maguin, E.; Bejar, S.
Production of D-tagatose, a low caloric sweetener during milk fermentation using L-arabinose isomerase
Biores. Technol.
102
3309-3315
2011
Geobacillus stearothermophilus, Geobacillus stearothermophilus US100
Cheng, L.; Mu, W.; Jiang, B.
Thermostable L-arabinose isomerase from Bacillus stearothermophilus IAM 11001 for D-tagatose production: gene cloning, purification and characterisation
J. Sci. Food Agric.
90
1327-1333
2010
Geobacillus stearothermophilus, Geobacillus stearothermophilus IAM 11001
Lee, Y.J.; Lee, S.J.; Kim, S.B.; Lee, S.J.; Lee, S.H.; Lee, D.W.
Structural insights into conserved L-arabinose metabolic enzymes reveal the substrate binding site of a thermophilic L-arabinose isomerase
FEBS Lett.
588
1064-1070
2014
Geobacillus stearothermophilus
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