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Information on EC 5.4.99.11 - isomaltulose synthase and Organism(s) Serratia plymuthica and UniProt Accession D0VX20
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5.4.99.11 isomaltulose synthaseIUBMB Comments
The enzyme simultaneously produces isomaltulose (6-O-alpha-D-glucopyranosyl-D-fructose) and smaller amounts of trehalulose (1-O-alpha-D-glucopyranosyl-beta-D-fructose) from sucrose.
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Word Map
- 5.4.99.11
- synthesis
- erwinia
- rhapontici
- palatinose
-
protaminobacter
-
2.4.1.1
- pantoea
-
1,4-alpha-d-glucan:orthophosphate
- molasses
- plymuthica
- food industry
-
vacuole-targeted
- dispersa
- whiteflies
- bemisia
- honeydew
The taxonomic range for the selected organisms is: Serratia plymuthica
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
sucrose isomerase, siase, alpha-glucosyltransferase, isomaltulose synthase, trehalulose synthase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
alpha-Glucosyltransferase
-
-
-
-
Isomaltulose synthetase
-
-
-
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Sucrose 6-glucosylmutase
-
-
-
-
Sucrose alpha-glucosyltransferase
Sucrose mutase
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-
-
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Synthase, isomaltulose
-
-
-
-
Trehalulose synthase
Sucrose alpha-glucosyltransferase
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-
-
-
Trehalulose synthase
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-
-
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Sucrose = 6-O-alpha-D-glucopyranosyl-D-fructofuranose
Glu295 plays a role as the general acid catalyst to protonate the oxygen of the glycosidic linkage for substrate hydrolysis, the Odelta2 of Asp241 acts as the nucleophile to attack the C1 of the D-glucosyl and forms a bond with C1 to obtain beta-glucosyl-enzyme intermediate, and the isomaltulose is produced when the O-6 of the hydrolysis product D-fructose nucleophilically binds to the C1 of the D-glucosyl group of the intermediate
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
isomerization
isomerization
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-
-
-
SYSTEMATIC NAME
IUBMB Comments
Sucrose glucosylmutase
The enzyme simultaneously produces isomaltulose (6-O-alpha-D-glucopyranosyl-D-fructose) and smaller amounts of trehalulose (1-O-alpha-D-glucopyranosyl-beta-D-fructose) from sucrose.
CAS REGISTRY NUMBER
COMMENTARY
159940-49-5
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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
sucrose
1-O-alpha-D-glucopyranosyl-D-fructofuranose + alpha-D-glucosylpyranosyl-1,6-D-fructofuranose
sucrose
alpha-D-glucopyranosyl-1,1-D-fructofuranose
-
-
plus small amounts of glucose, fructose and eventually isomaltose as by-products
-
?
sucrose
1-O-alpha-D-glucopyranosyl-D-fructofuranose + alpha-D-glucosylpyranosyl-1,6-D-fructofuranose
-
-
-
?
sucrose
1-O-alpha-D-glucopyranosyl-D-fructofuranose + alpha-D-glucosylpyranosyl-1,6-D-fructofuranose
-
-
-
-
?
sucrose
1-O-alpha-D-glucopyranosyl-D-fructofuranose + alpha-D-glucosylpyranosyl-1,6-D-fructofuranose
activity involves residues D241, E295, H368, and D369
-
-
?
sucrose
6-O-alpha-D-Glucopyranosyl-D-fructofuranose
isomerization of sucrose to isomaltulose is a highly efficient reaction, the formation of the beta-glucopyranosyl-enzyme complex followed by the nucleophilic attack of the 6'-OH group of fructose is concerted
-
-
?
sucrose
6-O-alpha-D-Glucopyranosyl-D-fructofuranose
-
i.e. beta-D-glucopyranosyl-D-fructofuranose
isomaltulose + glucose,fructose 1,1'-disaccharide
?
additional information
?
-
the enzyme catalyzes the isomerization of sucrose (alpha-D-glucosylpyranosyl-1,2-beta-D-fructofuranose) to trehalulose (alpha-D-glucosylpyranosyl-1,1-D-fructofuranose) and isomaltulose (alpha-D-glucosylpyranosyl-1,6-D-fructofuranose)
-
-
?
additional information
?
-
-
the enzyme catalyzes the isomerization of sucrose (alpha-D-glucosylpyranosyl-1,2-beta-D-fructofuranose) to trehalulose (alpha-D-glucosylpyranosyl-1,1-D-fructofuranose) and isomaltulose (alpha-D-glucosylpyranosyl-1,6-D-fructofuranose)
-
-
?
additional information
?
-
-
the enzyme catalyzes the enzymatic rearrangement of the alpha-1,2 linkage between glucose and fructose to an alpha-1,6 linkage (producing isomaltulose) or alpha-1,4 linkage (producing trehalulose). In addition, the enzyme hydrolyzes sucrose to produce small amounts of glucose and fructose monosaccharides
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-
?
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
sucrose
1-O-alpha-D-glucopyranosyl-D-fructofuranose + alpha-D-glucosylpyranosyl-1,6-D-fructofuranose
sucrose
1-O-alpha-D-glucopyranosyl-D-fructofuranose + alpha-D-glucosylpyranosyl-1,6-D-fructofuranose
-
-
-
?
sucrose
1-O-alpha-D-glucopyranosyl-D-fructofuranose + alpha-D-glucosylpyranosyl-1,6-D-fructofuranose
-
-
-
-
?
additional information
?
-
the enzyme catalyzes the isomerization of sucrose (alpha-D-glucosylpyranosyl-1,2-beta-D-fructofuranose) to trehalulose (alpha-D-glucosylpyranosyl-1,1-D-fructofuranose) and isomaltulose (alpha-D-glucosylpyranosyl-1,6-D-fructofuranose)
-
-
?
additional information
?
-
-
the enzyme catalyzes the isomerization of sucrose (alpha-D-glucosylpyranosyl-1,2-beta-D-fructofuranose) to trehalulose (alpha-D-glucosylpyranosyl-1,1-D-fructofuranose) and isomaltulose (alpha-D-glucosylpyranosyl-1,6-D-fructofuranose)
-
-
?
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Glutaraldehyde
-
the application of 1 or 2% glutaraldehyde after immobilization of Protaminobacter rubrum in alginate results in the complete destruction of the isomaltulose synthase activity
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
sucrose
-
the production of isomaltulose with alginate-immobilized Protaminobacter rubrum at 30 °C is influenced by sucrose concentration. Isomaltulose yields above 90% in 8 h are reached in 40-65% sucrose solutions, and yields up to 82% in 24 h were obtained with 70% sucrose. At higher concentrations (75%) a significant decrease in the isomaltulose yield (42.7-55%) is observed
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0324
sucrose
-
pH and temperature not specified in the publication, recombinant enzyme
additional information
additional information
Michaelis-Menten kinetics of wild-type and mutant enzymes, overview
-
additional information
additional information
-
Michaelis-Menten kinetics of wild-type and mutant enzymes, overview
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1301
sucrose
-
pH and temperature not specified in the publication, recombinant enzyme
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
1017.6
purified recombinant mutant E175N pH 6.0, 30°C, substrate sucrose
1045.7
purified recombinant mutant K576D, pH 6.0, 30°C, substrate sucrose
1218.9
purified recombinant mutant E175N/K576D, pH 6.0, 30°C, substrate sucrose
957.5
purified recombinant wild-type enzyme, pH 6.0, 30°C, substrate sucrose
additional information
additional information
-
the specific activity of the Protaminobacter rubrum immobilized pellets in calcium alginate at 30°C range from 1.6 to 4.0 g isomaltulose/g pellet/h, respectively with 70% and 65% sucrose solution, while in lower sucrose concentration have higher specific activities presumably due to substrate inhibition of the isomaltulose synthase in higher sucrose concentrations
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
30
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
20 - 50
temperature profiles of wild-type and mutant enzymes, overview
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
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physiological functions of isomaltulose and biochemical properties of sucrose isomerases, as well as biological isomaltulose production from sucrose using sucrose isomerase, overview
additional information
additional information
enzyme-substrate complex, docking study with sucrose and fructose
additional information
-
enzyme-substrate complex, docking study with sucrose and fructose
additional information
proteins AS9 PalI and SmuA differ in only one amino acid, structure of the wild-type sucrose isomerases is obtained by single point mutation through pymol using the crystal structure of SmuA from Protaminobacter rubrum strain CBS 547.77 (PDB ID 3GBD). Homology models of structures of the mutant sucrose isomerases are constructed based on the crystal structure of SmuA, using the EMBL-EBI server
additional information
-
proteins AS9 PalI and SmuA differ in only one amino acid, structure of the wild-type sucrose isomerases is obtained by single point mutation through pymol using the crystal structure of SmuA from Protaminobacter rubrum strain CBS 547.77 (PDB ID 3GBD). Homology models of structures of the mutant sucrose isomerases are constructed based on the crystal structure of SmuA, using the EMBL-EBI server
additional information
-
the unique RLDRD motif is located in a loop adjacent to the active site cleft and is determined to play an important role in the isomerization process and in the control of product specificity
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). PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
?
x 67560, sequence calculation, x * 65000, recombinant enzyme, SDS-PAGE
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
in complex with deoxynojirimycin, sitting drop vapor diffusion method, using
crystal structure determination, PDB IDs GBD and GBE, with ethylene glycol, citrate, 1,2-ethanediol, and 1-deoxynojirimycin as interacting compounds
-
native enzyme, crystals belong to space group P212121 and diffract to 1.95 A resolution
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
E175N
site-directed mutagenesis, the mutant shows increased activity and exhibits an identical pH optimum and a slightly increased optimal temperature (35°C) compared to wild-type enzyme (30°C)
E175N/K576
site-directed mutagenesis, the mutant shows increased activity and exhibits an identical pH optimum and a slightly increased optimal temperature (35°C) compared to wild-type enzyme (30°C)
E428D
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
F297A
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
F297P
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
F297P/R333K
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
F321A
site-directed mutagenesis, the mutant shows only hydrolytic activity
F321A/F319A
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
G176D
site-directed mutagenesis, the mutant shows reduced activity compared to wild-type
K174D
site-directed mutagenesis, the mutant shows reduced activity compared to wild-type
K576D
site-directed mutagenesis, the mutant shows increased activity and exhibits an identical pH optimum and a slightly increased optimal temperature (35°C) compared to wild-type enzyme (30°C)
N577K
site-directed mutagenesis, the mutant's activity is similar to the wild-type enzyme
R333K
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
S575D
site-directed mutagenesis, the mutant's activity is similar to the wild-type enzyme
additional information
additional information
engineering of the enzyme to exhibit isomelezitose synthase activity for production of the probiotically nutrial component, overview
additional information
-
engineering of the enzyme to exhibit isomelezitose synthase activity for production of the probiotically nutrial component, overview
additional information
the half-lives of the enzyme mutants E175N, K576D and E175N/K576D are 2.30, 1.78 and 7.65 times greater than that of the wild-type enzyme at 45°C, respectively. The Km values for the E175N, K576D and E175N/K576D mutants decrease by 6.6%, 2.0% and 11.0%, respectively, and their kcat/Km values increase by 38.2%, 4.2% and 19.4%, respectively, compared with those of the wild-type enzyme. After optimizing the conditions for isomaltulose production at 45°C, the E175N, K576D and E175N/K576D mutants display slightly improved isomaltulose yields, compared with the wild-type enzyme. The catalytic efficiencies (kcat/Km values) of E175N, K576D and E175N/K576D are increased by 38.2%, 4.2% and 19.4%, respectively
additional information
-
the half-lives of the enzyme mutants E175N, K576D and E175N/K576D are 2.30, 1.78 and 7.65 times greater than that of the wild-type enzyme at 45°C, respectively. The Km values for the E175N, K576D and E175N/K576D mutants decrease by 6.6%, 2.0% and 11.0%, respectively, and their kcat/Km values increase by 38.2%, 4.2% and 19.4%, respectively, compared with those of the wild-type enzyme. After optimizing the conditions for isomaltulose production at 45°C, the E175N, K576D and E175N/K576D mutants display slightly improved isomaltulose yields, compared with the wild-type enzyme. The catalytic efficiencies (kcat/Km values) of E175N, K576D and E175N/K576D are increased by 38.2%, 4.2% and 19.4%, respectively
additional information
-
immobilization of cells expressing the recombinant wild-type enzyme on alginate cell pellets for isomaltulose production, overview. The operational stability and enzyme half-life are significantly improved
additional information
-
immobilization of cells expressing the wild-type enzyme on alginate cell pellets for isomaltulose production, overview. The operational stability and enzyme half-life are significantly improved
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
45
at 45°C and pH 6.0, the half-life of wild-type enzyme is 39.2 minutes. Whereas, the half-lives of mutants E175N, K576D, and E175N/K576D are 90.2 minutes, 69.8 minutes, and 300 minutes, respectively, which are 2.30, 1.78 and 7.65 times greater than that of the wild-type enzyme
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
the isomaltulose synthase immobilized only with alginate remains stable for eight 24 h batch cycles or 192 h total time
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant wild-type enzyme and mutants E175N, K576D, and E175N/K576D from Escherichia coli strain BL21(DE3) by ammonium sulfate fractionation, dialysis, and cation exchange chromatography, to homogeneity
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene smuA, DNA and amino acid sequence determination and analysis, phylogenetic analysis, cloning of full-length gene including the periplasmic signal sequence and expression of wild-type and insertional disruption mutant in Escherichia coli strain BL21 (DE3)
gene smuA, sequence comparison, expression of wild-type and mutant enzymes
gene smuA, synthetic gene, sequence comparisons, recombinant expression of codon-optimized wild-type and mutant enzymes in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain JM109
recombinant enzyme expression, Protaminobacter sucrose isomerase is totally susceptible to inactivation and cannot be solubly expressed with a tac vector either but may fold correctly and is effectively expressed from the L-rhamnose-inducible vector
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
food industry
sucrose isomerase activity is used industrially for the conversion of sucrose into isomers, particularly isomaltulose or trehalulose, which have properties advantageous over sucrose for some food uses.The industrial potential may be further enhanced by selection for variants that do not catabolize the sucrose substrate
synthesis
immobilized cells of Protaminobacter rubrum strain CBS 574.77 are commonly used for the synthesis of isomaltulose from sucrose on an industrial scale
synthesis
additional information
-
in contrast to sucrose, isomaltulose is scarcely fermented by oral microbes and effectively inhibits the formation of water-insoluble glucans, showing that it is particularly suitable as a noncariogenic sucrose replacement
synthesis
-
production of palatinose, which is used as a sweetener and as a substitute for sucrose
synthesis
-
isomaltulose (alpha-D-glucopyranosyl-1,6-D fructofuranose) is a carbohydrate used as ideal sucrose substitute. Sucrose isomerase is widely used in industries for the production of isomaltulose. The enzyme catalyzes the isomerization of sucrose into isomaltulose and trehalulose and may hydrolyze sucrose to produce small amounts of glucose and fructose
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
McAllister, M.; Kelly, C.T.; Doyle, E.; Fogarty, W.M.
The isomaltoluse synthesising enzyme of Serratia plymuthica
Biotechnol. Lett.
12
667-672
1990
Serratia plymuthica
-
Egerer, P.
Downstream processing of sucrose 6-glucosylmutase and production of isomaltulose, a non-cariogenic, low-caloric sweetener
Hindustan Antibiot. Bull.
36
65-77
1994
Serratia plymuthica, Serratia plymuthica Z12
Park, Y.K.; Uekane, R.T.; Pupin, A.M.
Conversion of sucrose to isomaltose by microbial glucosyltransferase
Biotechnol. Lett.
14
547-551
1992
Serratia plymuthica
-
Ravaud, S.; Watzlawick, H.; Haser, R.; Mattes, R.; Aghajari, N.
Overexpression, purification, crystallization and preliminary diffraction studies of the Protaminobacter rubrum sucrose isomerase SmuA
Acta Crystallogr. Sect. F
F62
74-76
2006
Serratia plymuthica
de Oliva-Neto, P.; Menao, P.T.
Isomaltulose production from sucrose by Protaminobacter rubrum immobilized in calcium alginate
Biores. Technol.
100
4252-4256
2009
Serratia plymuthica, Serratia plymuthica CBS 574.77
Ravaud, S.; Robert, X.; Watzlawick, H.; Haser, R.; Mattes, R.; Aghajari, N.
Structural determinants of product specificity of sucrose isomerases
FEBS Lett.
583
1964-1968
2009
Serratia plymuthica (D0VX20), Serratia plymuthica
Goerl, J.; Timm, M.; Seibel, J.
Mechanism-oriented redesign of an isomaltulose synthase to an isomelezitose synthase by site-directed mutagenesis
ChemBioChem
13
149-156
2012
Serratia plymuthica (D0VX20), Serratia plymuthica, Serratia plymuthica CBS 574.77 (D0VX20)
Goulter, K.C.; Hashimi, S.M.; Birch, R.G.
Microbial sucrose isomerases: producing organisms, genes and enzymes
Enzyme Microb. Technol.
50
57-64
2012
Serratia plymuthica (D0VX20), Serratia plymuthica, Rhizobium sp. (M1E1F3), Rhizobium sp. (M1E1F6), Rhizobium sp. (M1E1F7), Rhizobium sp., Serratia plymuthica L0W308 (D0VX20)
Mu, W.; Li, W.; Wang, X.; Zhang, T.; Jiang, B.
Current studies on sucrose isomerase and biological isomaltulose production using sucrose isomerase
Appl. Microbiol. Biotechnol.
98
6569-6582
2014
Erwinia rhapontici, Erwinia rhapontici (D9MPF2), Erwinia rhapontici (Q6XNK5), Erwinia rhapontici (Q9AI64), Serratia plymuthica, no activity in Bemisia argentifolii, Erwinia sp. D12, Enterobacter sp. FMB-1 (B5ABD8), Rhizobium sp. MX-45 (M1E1F6), Paraburkholderia acidicola (Q2PS28), Klebsiella pneumoniae (Q4L2Q1), Raoultella planticola (Q6XKX6), Pantoea dispersa (Q6XNK6), Klebsiella sp. LX3 (Q8KR84), Erwinia rhapontici DSM 4484 (Q9AI64), Klebsiella pneumoniae NK33-98-8 (Q4L2Q1), Raoultella planticola UQ14S (Q6XKX6), Erwinia rhapontici NCPPB 1578, Pantoea dispersa UQ68J (Q6XNK6), Serratia plymuthica CBS 574.77, Erwinia rhapontici wac2928 (Q6XNK5), Paraburkholderia acidicola MX-45 (Q2PS28), Serratia plymuthica ATCC 15928, Erwinia rhapontici NX-5 (D9MPF2)
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