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Information on EC 3.2.1.1 - alpha-amylase and Organism(s) Geobacillus stearothermophilus and UniProt Accession P06279

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IUBMB Comments
Acts on starch, glycogen and related polysaccharides and oligosaccharides in a random manner; reducing groups are liberated in the alpha-configuration. The term "alpha" relates to the initial anomeric configuration of the free sugar group released and not to the configuration of the linkage hydrolysed.
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Geobacillus stearothermophilus
UNIPROT: P06279
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The taxonomic range for the selected organisms is: Geobacillus stearothermophilus
The enzyme appears in selected viruses and cellular organisms
Synonyms
alpha-amylase, diastase, alpha amylase, pancreatic alpha-amylase, crustacean cardioactive peptide, maltogenic amylase, taka-amylase a, human salivary alpha-amylase, bacillus licheniformis alpha-amylase, alpha-amylase 2, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
AmyUS100
variant of the most thermoactive and thermostable maltohexaose forming alpha-amylase produced in Geobacillus stearothermophilus sp. US100
AmyUS100DELTAIG
recombinant protein
1,4-alpha-D-glucan glucanohydrolase
alpha-1,4-glucan-4-glucanohydrolase
-
-
Alpha-amylase carcinoid
-
-
-
-
Amy c6
-
-
-
-
AMY1
-
-
-
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Amylase THC 250
-
-
-
-
amylase, alpha-
-
-
-
-
Amylopsin
-
-
-
-
Bactosol TK
-
-
-
-
Buclamase
-
-
-
-
Clarase
-
-
-
-
Clone 103
-
-
-
-
Clone 168
-
-
-
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Clone PHV19
-
-
-
-
Clones GRAMY56 and 963
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-
-
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diastase
-
-
-
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endoamylase
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-
-
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Fortizyme
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-
-
-
G 995
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-
-
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glycogenase
-
-
-
-
High pI alpha-amylase
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-
-
-
Isozyme 1B
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-
-
-
Kleistase L 1
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-
-
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Low pI alpha-amylase
-
-
-
-
Maxamyl
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-
-
-
Maxilase
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-
-
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Meiotic expression upregulated protein 30
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-
-
-
Pancreatic alpha-amylase
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-
-
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Pivozin
-
-
-
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Ptyalin
-
-
-
-
Spitase CP 1
-
-
-
-
TAA
-
-
-
-
Taka-amylase A
-
-
-
-
Takatherm
-
-
-
-
Thermamyl
-
-
-
-
Thermolase
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
4-alpha-D-glucan glucanohydrolase
Acts on starch, glycogen and related polysaccharides and oligosaccharides in a random manner; reducing groups are liberated in the alpha-configuration. The term "alpha" relates to the initial anomeric configuration of the free sugar group released and not to the configuration of the linkage hydrolysed.
CAS REGISTRY NUMBER
COMMENTARY hide
9000-90-2
-
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
starch + H2O
maltohexaose + ?
show the reaction diagram
-
-
-
?
alpha-1,4-glucan + H2O
fragments of alpha-1,4-glucan
show the reaction diagram
-
-
-
-
?
amylose + H2O
?
show the reaction diagram
-
with potato amylose as substrate, the enzyme displays a high degree of multiple attack (the number of bonds broken during the lifetime of an enzyme-substrate complex minus one). The level of multiple attack decreases when temperature is raised
-
-
?
glycogen + H2O
?
show the reaction diagram
-
-
-
-
?
maltodextrin + H2O
?
show the reaction diagram
-
-
-
-
?
soluble starch + H2O
?
show the reaction diagram
-
-
-
-
?
starch + H2O
?
show the reaction diagram
starch + H2O
malto-oligosaccharides
show the reaction diagram
-
-
-
-
?
amylose + H2O
additional information
-
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
alpha-1,4-glucan + H2O
fragments of alpha-1,4-glucan
show the reaction diagram
-
-
-
-
?
glycogen + H2O
?
show the reaction diagram
-
-
-
-
?
starch + H2O
?
show the reaction diagram
alpha-amylases are classical calcium-binding enzymes, which randomly hydrolyze internal alpha-1,4-glucosidic linkages in starch to produce smaller molecular mass maltodextrins, maltooligosaccharides and glucose
-
-
?
starch + H2O
malto-oligosaccharides
show the reaction diagram
-
-
-
-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Cu2+
-
slightly improves enzymatic activity
Mg2+
-
slightly improves enzymatic activity
Mn2+
-
slightly improves enzymatic activity
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
EDTA
AmyUS100DELTAG retains 40 and 25% of its original activity at 40 and 60°C, respectively, when incubated with 200 mM of EDTA, compared to 12 and 0% for AmyUS100
linear alkylbenzene sulfonate
mutant M197A, concentration of 10% incubation at 60°C for 1 h, 71% residual relative activity, assay at 80°C and pH 5.6
SDS
mutant M197A, concentration of 10% incubation at 60°C for 1 h, 45% residual relative activity, assay at 80°C and pH 5.6
Triton X-100
mutant M197A, concentration of 10% incubation at 60°C for 1 h, 71% residual relative activity, assay at 80°C and pH 5.6
Tween-20
mutant M197A, concentration of 10% incubation at 60°C for 1 h, 94% residual relative activity, assay at 80°C and pH 5.6
Tween-80
mutant M197A, concentration of 10% incubation at 60°C for 1 h, 88% residual relative activity, assay at 80°C and pH 5.6
Na+
-
alpha-amylase in 1 M NaCl and 5 mM NaCl retains 73% and 43% of its original activity after 24 h at 4ºC, respectively
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Detergents
the mutant enzyme AmyUS100DELTAIG/M197A is found to be very stable in the presence of solid detergents (commercial detergents). The activity is increased in somes cases. Incubation of the enzyme at 40°C with Ariel, Skip, Dixan, Det, NewDet, Nadhif,m and OMO increases the activity between 10 and 50%. The activity of the engineered enzyme persists at 60°C of the majority of the commercial solid detergents. The incubation with the liquid detergents Lav+ and Nadhif increases the activity by 10-20%, respectively. The presence of the Dinol detergent, the activity decreases to 97 and 91% at 40 and 60°C, respectively
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
additional information
-
Km-values of wild-type and mutant enzymes
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
additional information
-
KM-values of wild-type and mutant enzymes
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 9
AmyUS100 and mutant M197A enzyme
6 - 13
-
pH 6.0: about 35% of maximal activity, pH 13: about 45% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45 - 55
-
cells grown at 35°C
60 - 70
-
cells grown at 55°C
70 - 100
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hydrolysis of maltodextrin
80
-
hydrolysis of starch
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 75
-
30°C: about 50% of maximal activity, 75°C: about 50% of maximal activity
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
sp. US100
Uniprot
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
alpha-amylases are classical calcium-binding enzymes, which randomly hydrolyze internal alpha-1,4-glucosidic linkages in starch to produce smaller molecular mass maltodextrins, maltooligosaccharides and glucose
additional information
homology modeling of native AmyS and mutant AmySxadDELTAR179-G180 structures are created using the wild-type BStA (PDB ID 1HVX) as a template. A small extra loop containing residues Arg179-Gly180 is located in domain B of AmyS
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION?
AMY_GEOSE
549
1
62671
Swiss-Prot
-
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
14000
-
the difference between the value determined by gel filtration and by nondenaturing gel electrophoresisis is due to hydrophobic interactions between the chromatography matrix and the enzyme molecule, gel filtration
41000
48000
-
equilibrium sedimentation
48000 - 52700
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equilibrium sedimentation
49000
52700
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meniscus depletion method
58000
-
x * 58000, SDS-PAGE
61500
-
the difference between the value determined by gel filtration and by nondenaturing gel electrophoresisis is due to hydrophobic interactions between the chromatography matrix and the enzyme molecule, nondenaturing gel electrophoresis
90000
-
x * 90000, SDS-PAGE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
tiny needle crystals are formed in 35 mM Na-acetate, pH 4.6, 35 mM CaCl2, 6.25% 2-propanol in the presence of 1.23% of the pseudo-oligosaccharide inhibitor acarbose at a protein concentration of 10 mg/ml, crystals diffract to 2.0 A resolution
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
M197A
site-directed mutagenesis. Studies of its catalytic properties show no effect on the thermostability, pH activity/stability, calcium demand and chelator resistance. Specific activity is decreased from 1000 to 845 U/mg. The profile of starch hydrolysis is affected. As a result hereof AmyUS100DELTAIG/M197A produces in majority maltose and maltotriose as major products compared to maltohexaose and maltopentaose produced by the wild-type and the AmyUS100DELTAIG variant. The mutant retains 85% of its original activity. 70% of the mutantM197A activity is retained after 60 min of treatment at 60°C in the presence of 1.8 M H2O2, whereas AmyUS100DELTAIG is totally inactivated. These results confirm the importance of Met197 in the oxidative sensibility, situated in the cavity of the active site
additional information
construction of a dexadletion mutant AmySDELTAR179-G180 by deleting Arg179 and Gly180 through site-directed mutagenesis. The thermostability of mutant AmySDELTAR179-G180 is enhanced and the half-life at 100°C significantly increased from 24 to 33 min. In addition, AmySDELTAR179-G180 exhibits greatxader acid resistance and lower calcium requirements to maintain alpha-amylase activity compared to the wild-type enzyme AmyS. The sexadcretory capacity of the recombinant strain is evaluated by fed-batch fermentation in a 7.5-litre fermenter in which high alpha-amylase activity is obtained. The highest activity reaches 3300 U/ml with a high productivity of 45.8 U/(ml*h). Structural comparison of the AmyS model with that of the AmySDELTAR179-G180 mutant model shows that the deletion of R179-G180 causes a slight structural rearrangement and a decrease in AmySDELTAR179-G180 calcium requirements
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 11
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
100
purified recombinant wild-type enzyme AmyS, half-life is 24 min, for the purified recombinant mutant AmySxadDELTAR179-G180 half-life is 33 min
25
-
24 h, completely stable
4
-
24 h, 50% loss of activity
additional information
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
activity of AmyUS100DELTAIG and Termamyl300 (commercial amylase used in detergent) are instantaneously decreased in the presence of 1.8 M H2O2, whereas AmyUSDELTAIG/M197A retains 85% of its original activity. 70% of the mutantM197A activity is retained after 60 min of treatment at 60°C. Termamyl300 retains 20% of its original activity and AmyUS100DELTAIG is totally inactivated. These results confirm the importance of Met197 in the oxidative sensibility, situated in the cavity of the active site
691421
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
spry-dried enzyme, after 6 months in liquid detergent, 90% of its original activity retained. After 1 year the activity decreases to 61%. The activity of the enzyme in solid detergent is almost invariant. In presence of commercial proteases salvinase and alcalase the enzyme activity decreases by 22 and 18%, respectively, after 6 months
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant alpha-amylase
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli ER2566
expression in Bacillus subtilis
-
expression in Escherichia coli strain MK79, which also contains the hemoglobin gene from Vitreoscilla sp.. The hemoglobin gene enhances production of alpha-amylase
-
gene ami, recombinant expression of wild-type enzyme and deletion mutant in Bacillus subtilis strain 1A751
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
detergent
AmyUS100DELTAIG is designed to improve the thermostability of the thermoactive and thermostable maltohexaose forming alpha-amylase produced in Geobacillus stearothermophilus sp. US100, AmyUS100
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Takagi, T.; Toda, H.; Isemura, T.
Bacterial and mold amylases
The Enzymes, 3rd Ed. (Boyer, P. D. , ed. )
5
235-271
1971
Aspergillus niger, Aspergillus oryzae, Geobacillus stearothermophilus, Bacillus subtilis
-
Manually annotated by BRENDA team
Brumm, P.J.; Hebeda, R.E.; Teague, W.M.
Purification and properties of a new, commercial, thermostable Bacillus stearothermophilus alpha-amylase
Food Biotechnol.
2
67-80
1988
Geobacillus stearothermophilus
-
Manually annotated by BRENDA team
Kindle, K.L.
Characterization and production of thermostable alpha-amylase
Appl. Biochem. Biotechnol.
8
153-170
1983
Acinetobacter sp., Alicyclobacillus acidocaldarius, Aspergillus niger, Geobacillus stearothermophilus, Bacillus amyloliquefaciens, Weizmannia coagulans, Priestia megaterium, Bacillus subtilis, Bacillus licheniformis, Bacillus sp. (in: Bacteria), Thermochaetoides thermophila, Humicola brevis, Trichocladium griseum, Humicola insolens, Thermomyces lanuginosus, Humicola stellata, Malbranchea pulchella, Micromonospora vulgaris, Rhizomucor miehei, Rhizomucor pusillus, Thermothelomyces myriococcoides, Rhizopus microsporus, Mycothermus thermophilus, Thermomyces dupontii, Thermoactinomyces vulgaris, Thermoascus aurantiacus, Thermoascus crustaceus, Thermomonospora curvata, Thermomonospora viridis, Thermomonospora vulgaris, Bacillus licheniformis CUMC305, Weizmannia coagulans CUMC512, Bacillus sp. (in: Bacteria) 11-15
-
Manually annotated by BRENDA team
Ingle, M.B.; Erikson, R.J.
Bacterial alpha-amylases
Adv. Appl. Microbiol.
24
257-278
1978
Alicyclobacillus acidocaldarius, Geobacillus stearothermophilus, Weizmannia coagulans, Bacillus licheniformis, Bacillus sp. (in: Bacteria), Clostridium acetobutylicum, Thermomonospora curvata, Alicyclobacillus acidocaldarius 104-1A, Bacillus sp. (in: Bacteria) A-40-2, Alicyclobacillus acidocaldarius Agnano 101
Manually annotated by BRENDA team
Svensson, B.
Protein engineering in the alpha-amylase family: catalytic mechanism, substrate specificity and stability
Plant Mol. Biol.
25
141-157
1994
Aspergillus oryzae, Geobacillus stearothermophilus, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus licheniformis, Drosophila melanogaster, Escherichia coli, Saccharomycopsis fibuligera, Homo sapiens, Hordeum vulgare, Mus musculus, Oryza sp., Vigna radiata, Streptomyces hygroscopicus, Sus scrofa, Triticum aestivum, Xanthomonas campestris
Manually annotated by BRENDA team
Pfueller, S.L.; Elliott, W.H.
The extracellular alpha-amylase of Bacillus stearothermophilus
J. Biol. Chem.
244
48-54
1969
Geobacillus stearothermophilus
Manually annotated by BRENDA team
Ogasahara, K.; Imanishi, A.; Isemura, T.
Studies on thermophilic alpha-amylase from Bacillus stearothermophilus. I. Some general and physico-chemical properties of thermophilic alpha-amylase
J. Biochem.
67
65-75
1970
Geobacillus stearothermophilus
Manually annotated by BRENDA team
Dercova, K.; Augustin, J.; Krajcova, D.
Cell growth and alpha-amylase production characteristics of Bacillus subtilis
Folia Microbiol. (Praha)
37
17-23
1991
Geobacillus stearothermophilus, Bacillus subtilis, Bacillus licheniformis, Bacillus licheniformis CCM 2145, Geobacillus stearothermophilus CCM 2183, Bacillus subtilis DP 1
Manually annotated by BRENDA team
Liu, S.C.; Ogretmen, B.; Chuang, Y.Y.; Stark, B.C.
Selection and characterization of alpha-amylase-overproducing recombinant Escherichia coli containing the bacterial hemoglobin gene
Appl. Microbiol. Biotechnol.
38
239-242
1992
Geobacillus stearothermophilus
Manually annotated by BRENDA team
Suvd, D.; Fujimoto, Z.; Takase, K.; Matsumura, M.; Mizuno, H.
Crystal structure of Bacillus stearothermophilus alpha-amylase: possible factors determining the thermostability
J. Biochem.
129
461-468
2001
Geobacillus stearothermophilus
Manually annotated by BRENDA team
Bijttebier, A.; Goesaert, H.; Delcour, J.A.
Temperature impacts the multiple attack action of amylases
Biomacromolecules
8
765-772
2007
Aspergillus oryzae, Geobacillus stearothermophilus, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus licheniformis, Sus scrofa, Thermoactinomyces vulgaris
Manually annotated by BRENDA team
Al-Qodah, Z.
Production and characterization of thermostable alpha-amylase by thermophilic Geobacillus stearothermophilus
Biotechnol. J.
1
850-857
2006
Geobacillus stearothermophilus, Geobacillus stearothermophilus JT2
Manually annotated by BRENDA team
Khemakhem, B.; Ali, M.B.; Aghajari, N.; Juy, M.; Haser, R.; Bejar, S.
Engineering of the alpha-amylase from Geobacillus stearothermophilus US100 for detergent incorporation
Biotechnol. Bioeng.
102
380-389
2009
Geobacillus stearothermophilus (P06279), Geobacillus stearothermophilus
Manually annotated by BRENDA team
Prakash, O.; Jaiswal, N.
alpha-Amylase: an ideal representative of thermostable enzymes
Appl. Biochem. Biotechnol.
160
2401-2414
2010
Alicyclobacillus acidocaldarius, Geobacillus stearothermophilus, Bacillus amyloliquefaciens, Anoxybacillus flavithermus, Bacillus subtilis, Lederbergia lentus, Bacillus licheniformis, Chloroflexus aurantiacus, Thermothelomyces heterothallicus, Desulfurococcus mucosus, Dictyoglomus thermophilum, Thermomyces lanuginosus, Lactiplantibacillus plantarum, Lipomyces kononenkoae, Pyrococcus furiosus, Pyrococcus woesei, Pyrodictium abyssi, Rhizopus sp., Rhodothermus marinus, Mycothermus thermophilus, Staphylothermus marinus, Thermoactinomyces vulgaris, Thermococcus fumicolans, Thermococcus hydrothermalis, Thermococcus litoralis, Thermococcus profundus, Thermotoga maritima, Thermus filiformis, Lactobacillus amylovorus, Halothermothrix orenii, Thermococcus aggregans, Thermococcus celer, Thermococcus guaymasensis
Manually annotated by BRENDA team
Gai, Y.; Chen, J.; Zhang, S.; Zhu, B.; Zhang, D.
Property improvement of alpha-amylase from Bacillus stearothermophilus by deletion of amino acid residues arginine 179 and glycine 180
Food Technol. Biotechnol.
56
58-64
2018
Geobacillus stearothermophilus (O31193)
Manually annotated by BRENDA team