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Information on EC 3.2.1.135 - neopullulanase and Organism(s) Geobacillus stearothermophilus and UniProt Accession P38940
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3.2.1.135 neopullulanaseIUBMB Comments
cf. EC 3.2.1.41 (pullulanase ) and EC 3.2.1.57 (isopullulanase).
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Word Map
- 3.2.1.135
- starch
- stearothermophilus
-
amylolytic
- alpha-amylases
- cyclomaltodextrinase
-
maltogenic
- amylopectin
-
transglycosylation
- amylose
-
alpha-1,4
- amylopullulanase
-
debranching
- oligo-1,6-glucosidase
-
glucanotransferase
-
thermoactinomyces
-
alpha-1,6-glucosidic
- isopanose
-
cdase
-
saccharifying
- pullulanases
- cyclodextrinase
- maltooligosaccharides
- isoamylase
- isopullulanase
- gamma-cyclodextrins
- biotechnology
- industry
- synthesis
The taxonomic range for the selected organisms is: Geobacillus stearothermophilus
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Synonyms
neopullulanase, tetapum955, env npu193a, tetapur855, apuadelta, type iii pullulan hydrolase, neopullulanase-like enzyme, pullulan hydrolase type i, bsnpl, pullulanase ii, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
pullulanase II
-
-
-
-
pullulanase, neo-
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hydrolysis of pullulan to panose (6-alpha-D-glucosylmaltose)
structure-function relationship
-
hydrolysis of pullulan to panose (6-alpha-D-glucosylmaltose)
residues E357, D424, and D328 are involved in the catalytic reaction at the active site, residues H423, E332, and N331 are involved in substrate recognition
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hydrolysis of O-glycosyl bond
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
pullulan 4-D-glucanohydrolase (panose-forming)
cf. EC 3.2.1.41 (pullulanase ) and EC 3.2.1.57 (isopullulanase).
CAS REGISTRY NUMBER
COMMENTARY
119632-58-5
-
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
acarbose + H2O
pseudotrisaccharide + D-glucose
-
and transglycosylation of D-glucose to isoacarbose
?
amylose + H2O
maltose + D-glucose
-
-
small amount D-glucose
?
pullulan + H2O
6-alpha-D-glucosylmaltose + ?
-
-
i.e. panose
-
?
pullulan + H2O
D-glucose + maltose + maltotriose
-
the enzyme attacks both alpha-D-(1->4) and alpha-D-(1->6) glycosidic linkages
-
-
?
beta-cyclomaltodextrin + H2O
maltose + D-glucose
-
-
-
?
pullulan + H2O
panose + ?
-
-
in the first step, the enzyme hydrolyzes only alpha-1,4-glucosidic linkages on the nonreducing side of alpha-1,6-linkages of pullulan and produces panose and several intermediate products composed of some panose units. In the second step, taking 62-O-alpha-(63-O-alpha-glucosyl-maltotriosyl)-maltose as an example of one of the intermediate products, the enzyme hydrolyzes either alpha-1,4-, or alpha-1,6-linkages and produces panose or 63-O-alpha-glucosyl-maltotriose plus maltose, respectively. In the third step, the alpha-1,4-linkage of the 63-O-alpha-glucosyl-maltotriose is hydrolyzed by the enzyme, and D-glucose and another panose are produced, + maltose + D-glucose, in a molar ratio of 3:1:1, small amount
?
pullulan + H2O
panose + ?
-
-
+ maltose + D-glucose, in a molar ratio of 3:1:1, small amount
?
additional information
?
-
4 reactions are catalyzed by the enzyme: 1. hydrolysis of alpha-1,4-glucosidic linkage, 2. hydrolysis of alpha-1,6-glucosidic linkage, 3. transglycosylation to form alpha-1,4-glucosidic linkage, 4. transglycosylation to form alpha-1,6-glucosidic linkage
-
-
?
additional information
?
-
-
4 reactions are catalyzed by the enzyme: 1. hydrolysis of alpha-1,4-glucosidic linkage, 2. hydrolysis of alpha-1,6-glucosidic linkage, 3. transglycosylation to form alpha-1,4-glucosidic linkage, 4. transglycosylation to form alpha-1,6-glucosidic linkage
-
-
?
additional information
?
-
the dual specificity of the enzyme toward alpha-1,4-, and alpha-1,6-glucosidic linkages based on structural analyses of the complexes with the enzyme and substrates is demonstrated
-
-
?
additional information
?
-
-
the dual specificity of the enzyme toward alpha-1,4-, and alpha-1,6-glucosidic linkages based on structural analyses of the complexes with the enzyme and substrates is demonstrated
-
-
?
additional information
?
-
the enzyme catalyzes the hydrolysis of alpha-1,4- and alpha-1,6-glucosidic linkages (of pullulan) of transglycosylations to form both alpha-1,4- and alpha-1,6-glucosidic bonds
-
-
?
additional information
?
-
the enzyme catalyses condensation between two maltose molecules and subsequent hydrolysis of the resulting 6-O-alpha-maltosylmaltose to D-glucose and panose, when maltose concentration is inceased to 20%
-
-
?
additional information
?
-
-
the enzyme catalyses condensation between two maltose molecules and subsequent hydrolysis of the resulting 6-O-alpha-maltosylmaltose to D-glucose and panose, when maltose concentration is inceased to 20%
-
-
?
additional information
?
-
-
analysis of the active centre, one active centre participates in the dual activity toward alpha-1,4-, and alpha-1,6-glucosidic linkages
-
-
?
additional information
?
-
-
4 reactions are catalyzed by the enzyme: 1. hydrolysis of alpha-1,4-glucosidic linkage, 2. hydrolysis of alpha-1,6-glucosidic linkage, 3. transglycosylation to form alpha-1,4-glucosidic linkage, 4. transglycosylation to form alpha-1,6-glucosidic linkage
-
-
?
additional information
?
-
-
4 reactions are catalyzed by the enzyme: 1. hydrolysis of alpha-1,4-glucosidic linkage, 2. hydrolysis of alpha-1,6-glucosidic linkage, 3. transglycosylation to form alpha-1,4-glucosidic linkage, 4. transglycosylation to form alpha-1,6-glucosidic linkage
-
-
?
additional information
?
-
enzyme hydrolyzes starch
-
-
?
additional information
?
-
-
enzyme hydrolyzes not only alpha-1,4-glucosidic linkages but also specific alpha-1,6-glucosidic linkages of several branched oligosaccharides
-
-
?
additional information
?
-
-
the enzyme is unable to degrade alpha-cyclodextrin and panose
-
-
?
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
pullulan + H2O
6-alpha-D-glucosylmaltose + ?
-
-
i.e. panose
-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
16.6
-
maltotriose as substrate, wild-type enzyme, comparison with mutant enzymes
28.6
-
pullulan as substrate, wild-type enzyme, comparison with mutant enzymes
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
5 - 7
5 - 7
-
pH 5: about 25% of activity maximum, pH 7: about 40% of activity maximum
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
40 - 80
-
40°C: about 50% of activity maximum, 80°C: about 25% of activity maximum
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
analysis and verification of neopullulanases belonging to the alpha-amylase family GH13, subfamily GH13_20, but also subfamily GH13_36, an intermediary group, which is defined using the sequence of the fifth conserved sequence region (CSR) as a selection marker. Evolutionary relationships and tree, and conserved sequence regions of GH13 subfamilies, detailed overview. The GH13_20 neopullulanase from Bacillus stearothermophilus in complex with maltotetraose is a representative of the neopullulanase subfamily. One of the most clear sequence signatures of true members of the neopullulanase subfamily subfamily GH13_20 is the stretch VANE succeeding the catalytic nucleophile in the CSR II, although not every GH13_20 member has to possess this segment in its entirety. GH13_36 enzymes contain a histidine at the end of the CSR II (strand beta4) and a tryptophan (or at least an aromatic residue) two residues succeeding the catalytic proton donor in the CSR III (strand beta5)
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
62000
65000
enzyme likely to be present in monomer-dimer equilibrium with a molecular ratio of 1:9 in 50 mM sodium acetate buffer, pH 6.0, x * 65000, SDS-PAGE, x * 70156, MALDI-TOF MS analyzis
70156
enzyme likely to be present in monomer-dimer equilibrium with a molecular ratio of 1:9 in 50 mM sodium acetate buffer, pH 6.0, x * 65000, SDS-PAGE, x * 70156, MALDI-TOF MS analyzis
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
the active enzyme forms a dimer in the crystalline states and in solution
monomer
additional information
additional information
enzyme likely to be present in monomer-dimer equilibrium with a molecular ratio of 1:9 in 50 mM sodium acetate buffer, pH 6.0, x * 65000, SDS-PAGE, x * 70156, MALDI-TOF MS analyzis
additional information
-
enzyme likely to be present in monomer-dimer equilibrium with a molecular ratio of 1:9 in 50 mM sodium acetate buffer, pH 6.0, x * 65000, SDS-PAGE, x * 70156, MALDI-TOF MS analyzis
additional information
-
analysis of conserved regions in the enzyme primary sequence, overview
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystal structures of enzyme an dits complexes with panose, maltoteraose and isopanose are detemined
structure analysis and modeling of GH13_20 neopullulanase in complex with maltotetraose, PDB ID 1J0J
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
A416I
t1/2 of mutant enzyme at 70°C is 17 min, compared to 15 min for wild-type enzyme. Mutation does not compromise the catalytic activity
A566L
t1/2 of mutant enzyme at 70°C is 27 min, compared to 15 min for wild-type enzyme. Mutation does not compromise the catalytic activity
D46E
t1/2 of mutant enzyme at 70°C is 100 min, compared to 15 min for wild-type enzyme. Mutation does not compromise the catalytic activity
N413Q
t1/2 of mutant enzyme at 70°C is 32 min, compared to 15 min for wild-type enzyme. Mutation does not compromise the catalytic activity
S407T
t1/2 of mutant enzyme at 70°C is 66 min, compared to 15 min for wild-type enzyme. Mutation does not compromise the catalytic activity
V239L
t1/2 of mutant enzyme at 70°C is 103 min, compared to 15 min for wild-type enzyme. Mutation does not compromise the catalytic activity
V374I
t1/2 of mutant enzyme at 70°C is 14 min, compared to 15 min for wild-type enzyme. Mutation does not compromise the catalytic activity
V404L
t1/2 of mutant enzyme at 70°C is 191 min, compared to 15 min for wild-type enzyme. Mutation does not compromise the catalytic activity
V533L
t1/2 of mutant enzyme at 70°C is 8 min, compared to 15 min for wild-type enzyme. Mutation does not compromise the catalytic activity
I358V
-
mutation decreases the preference for alpha(1-6)-branched oligosaccharides and pullulan as substrates
I358W
-
mutation reduces the acceptability of alpha(1-6)-branched oligo- and polysaccharides
M375L
-
mutation increases transglycosylation activity in comparison to wild-type enzyme
S422V
-
mutation increases transglycosylation activity in comparison to wild-type enzyme
Y377D
-
mutation decreases transglycosylation activity in comparison to wild-type enzyme
Y377F
-
mutation increases transglycosylation activity in comparison to wild-type enzyme
Y377S
-
mutation decreases transglycosylation activity in comparison to wild-type enzyme
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
70
t1/2: 15 min (wild-type enzyme), 100 min (mutant enzyme D46E), 103 min (mutant enzyme V239L), 14 min (mutant enzyme V374I), 191 min (mutant enzyme V404L), 66 min (mutant enzyme S407T), 32 min (mutant enzyme N413Q), 17 min (mutant enzyme A416I), 8 min (mutant enzyme V533L), 27 min (mutant enzyme A566L)
60 - 70
-
the irreversible thermoinactivation of the enzyme is recorded in 50 mM sodium acetate, pH 5.5 at 60, 70 and 80°C. Pullulanase retains 85% of its activity after 50 min of incubation at 60°C, its residual activities at 70 and 80°C after 50 min are 70 and 15%, respectively
additional information
-
EDTA stabilizes against thermal inactivation
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
2 mM EDTA and 2 mM phytic acid decreases the stability of the enzyme
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitation, DEAE-Sepharose column chromatography, and Sephadex G-100 gel filtration
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
industry
potentially valuable enzyme for starch and detergent industries
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Lee, H.S.; Kim, M.S.; Cho, H.S.; Kim, J.I.; Kim, T.J.; Choi, J.H.; Park, C.; Oh, B.H.; Park, K.H.
Cyclomaltodextrinase, neopullulanase, and maltogenic amylase are nearly indistinguishable from each other
J. Biol. Chem.
277
21891-21897
2002
Geobacillus stearothermophilus
Imanaka, T.; Kuriki, T.
Pattern of action of Bacillus stearothermophilus neopullulanase on pullulan
J. Bacteriol.
171
369-374
1989
Geobacillus stearothermophilus
Kuriki, T.; Okada, S.; Imanaka, T.
New type of pullulanase from Bacillus stearothermophilus and molecular cloning and expression of the gene in Bacillus subtilis
J. Bacteriol.
170
1554-1559
1988
Geobacillus stearothermophilus
Kuriki, T.; Takata, H.; Okada, S.; Imanaka, T.
Analysis of the active center of Bacillus stearothermophilus neopullulanase
J. Bacteriol.
173
6147-6152
1991
Geobacillus stearothermophilus
Kuriki, T.; Kaneko, H.; Yanase, M.; Takata, H.; Shimada, J.; Handa, S.; Takada, T.; Umeyama, H.; Okada, S.
Controlling substrate preference and transglycosylation activity of neopullulanase by manipulating steric constraint and hydrophobicity in active center
J. Biol. Chem.
271
17321-17329
1996
Geobacillus stearothermophilus, Geobacillus stearothermophilus TRS40
Kamasaka, H.; Sugimoto, K.; Takata, H.; Nishimura, T.; Kuriki, T.
Bacillus stearothermophilus neopullulanase selective hydrolysis of amylose to maltose in the presence of amylopectin
Appl. Environ. Microbiol.
68
1658-1664
2002
Geobacillus stearothermophilus, Geobacillus stearothermophilus TRS40
Hondoh, H.; Kuriki, T.; Matsuura, Y.
Three-dimensional structure and substrate binding of Bacillus stearothermophilus neopullulanase
J. Mol. Biol.
326
177-188
2003
Geobacillus stearothermophilus (P38940), Geobacillus stearothermophilus, Geobacillus stearothermophilus TRS40 (P38940)
Park, K.H.; Kim, T.J.; Cheong, T.K.; Kim, J.W.; Oh, B.H.; Svensson, B.
Structure, specificity and function of cyclomaltodextrinase, a multispecific enzyme of the alpha-amylase family
Biochim. Biophys. Acta
1478
165-185
2000
Bacillus sp. KSM-1876, Bacteroides thetaiotaomicron, Bacteroides thetaiotaomicron 95-1, Geobacillus stearothermophilus, Klebsiella pneumoniae, Paenibacillus polymyxa
Cheong, K.A.; Kim, T.J.; Yoon, J.W.; Park, C.S.; Lee, T.S.; Kim, Y.B.; Park, K.H.; Kim, J.W.
Catalytic activities of intracellular dimeric neopullulanase on cyclodextrin, acarbose and maltose
Biotechnol. Appl. Biochem.
35
27-34
2002
Geobacillus stearothermophilus (Q9AIV2), Geobacillus stearothermophilus, Geobacillus stearothermophilus IMA6503 (Q9AIV2)
Lamminmaki, U.; Vihinen, M.
Structural consequences of neopullulanase mutations
Biochim. Biophys. Acta
1295
195-200
1996
Geobacillus stearothermophilus
Doman-Pytka, M.; Bardowski, J.
Pullulan degrading enzymes of bacterial origin
Crit. Rev. Microbiol.
30
107-121
2004
Alicyclobacillus acidocaldarius, Bacteroides thetaiotaomicron, Bacteroides thetaiotaomicron 95-1, Geobacillus stearothermophilus, Paenibacillus polymyxa, Thermoactinomyces vulgaris, Thermoactinomyces vulgaris R-47
Zareian, S.; Khajeh, K.; Ranjbar, B.; Dabirmanesh, B.; Ghollasi, M.; Mollania, N.
Purification and characterization of a novel amylopullulanase that converts pullulan to glucose, maltose, and maltotriose and starch to glucose and maltose
Enzyme Microb. Technol.
46
57-63
2010
Geobacillus stearothermophilus, Geobacillus stearothermophilus L14
-
Majzlova, K.; Pukajova, Z.; Jane?ek, S.
Tracing the evolution of the alpha-amylase subfamily GH13_36 covering the amylolytic enzymes intermediate between oligo-1,6-glucosidases and neopullulanases
Carbohydr. Res.
367
48-57
2013
Geobacillus stearothermophilus (P38940)
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