Information on EC 3.2.1.135 - neopullulanase

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The expected taxonomic range for this enzyme is: Bacteria, Archaea

EC NUMBER
COMMENTARY hide
3.2.1.135
-
RECOMMENDED NAME
GeneOntology No.
neopullulanase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
hydrolysis of pullulan to panose (6-alpha-D-glucosylmaltose)
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis
hydrolysis of O-glycosyl bond
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-
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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 hide
119632-58-5
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain ATCC 27009, gene Cda
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Manually annotated by BRENDA team
NA-1
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-
Manually annotated by BRENDA team
strain UCC2003
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Manually annotated by BRENDA team
strain NCFB2243
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Manually annotated by BRENDA team
strain NCFB2243
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-
Manually annotated by BRENDA team
strains CCUG45868, CCUG36569, NCDO2205
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-
Manually annotated by BRENDA team
strains NCIMB2244, DSM20095 and DSM20092
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-
Manually annotated by BRENDA team
strain JCM7027
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Manually annotated by BRENDA team
strain JCM7027
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-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
OR-1
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-
Manually annotated by BRENDA team
strain R-47, genes TVAI and TVAII
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Manually annotated by BRENDA team
strain R-47, genes TVAI and TVAII
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Manually annotated by BRENDA team
strain 39E, TetApuM955 gene of 2.9 kb
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Manually annotated by BRENDA team
strain 39E, TetApuM955 gene of 2.9 kb
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-
Manually annotated by BRENDA team
AL662
SwissProt
Manually annotated by BRENDA team
gene Env Npu193A of environmental soil DNA sample from a hot spring in Thailand and expressed in yeast Pichia pastoris KM71 with plasmid pPICZalphaA
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
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)
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
acarbose + H2O
pseudotrisaccharide + D-glucose
show the reaction diagram
alpha-cyclomaltodextrin + H2O
maltose + D-glucose
show the reaction diagram
-
-
-
-
?
amylopectin + H2O
?
show the reaction diagram
amylose + H2O
maltose + D-glucose
show the reaction diagram
amylose + H2O
maltotriose + maltotetraose + maltopentaose
show the reaction diagram
beta-cyclomaltodextrin + H2O
maltose + D-glucose
show the reaction diagram
gamma-cyclodextrin + H2O
maltose + ?
show the reaction diagram
not alpha or beta-cyclodextrins
no glucose
-
?
gamma-cyclomaltodextrin + H2O
maltose + D-glucose
show the reaction diagram
-
-
-
-
?
glycogen + H2O
?
show the reaction diagram
glycogen + H2O
maltooligosaccharides
show the reaction diagram
linear maltooligosaccharides + H2O
maltose + ?
show the reaction diagram
-
no glucose
-
?
maltodextrin 10 + H2O
?
show the reaction diagram
-
-
-
?
maltoheptaose + H2O
?
show the reaction diagram
maltoheptaose + H2O
D-glucose + maltose
show the reaction diagram
maltohexaose + H2O
?
show the reaction diagram
maltohexaose + H2O
D-glucose + maltose
show the reaction diagram
maltopentaose + H2O
?
show the reaction diagram
maltopentaose + H2O
D-glucose + maltose
show the reaction diagram
maltotetraose + H2O
?
show the reaction diagram
maltotetraose + H2O
D-glucose + maltose
show the reaction diagram
maltotriose + H2O
?
show the reaction diagram
-
-
-
-
?
maltotriose + H2O
D-glucose + maltose
show the reaction diagram
pullulan + H2O
6-alpha-D-glucosylmaltose
show the reaction diagram
pullulan + H2O
D-glucose
show the reaction diagram
-
-
-
-
?
pullulan + H2O
D-glucose + maltose + maltotriose
show the reaction diagram
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the enzyme attacks both alpha-D-(1->4) and alpha-D-(1->6) glycosidic linkages
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-
?
pullulan + H2O
isomaltose
show the reaction diagram
-
-
-
-
?
pullulan + H2O
maltohexaose
show the reaction diagram
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strains CCUG43878 and CCUG34405
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-
?
pullulan + H2O
maltooligosaccharides
show the reaction diagram
pullulan + H2O
maltotriose
show the reaction diagram
pullulan + H2O
maltotriose + ?
show the reaction diagram
-
no panose
-
?
pullulan + H2O
maltotriose + maltopentaose + maltohexaose + maltoheptaose
show the reaction diagram
pullulan + H2O
panose
show the reaction diagram
soluble and raw starch + H2O
maltose + maltotriose + maltotetraose + maltopentaose + maltohexaose + maltoheptaose + ?
show the reaction diagram
soluble starch + H2O
D-glucose + maltose
show the reaction diagram
-
-
-
-
?
soluble starch + H2O
maltotriose + maltotetraose
show the reaction diagram
starch + H2O
maltose + ?
show the reaction diagram
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
pullulan + H2O
6-alpha-D-glucosylmaltose
show the reaction diagram
pullulan + H2O
panose
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
CaCl2
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1 mM C-terminal truncated enzyme ApuADELTA: amylase activity: 105%, pullulanase activity: 94%; 1 mM full length enzyme: amylase activity: 111%, pullulanase activity: 101%
CoCl2
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1 mM C-terminal truncated enzyme ApuADELTA: amylase activity: 128%, pullulanase activity: 130%; 1 mM full length enzyme: amylase activity: 150%, pullulanase activity: 140%
KCl
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50 mM C-terminal truncated enzyme ApuADELTA: amylase activity: 106%, pullulanase activity: 93%; 50 mM full length enzyme: amylase activity: 112%, pullulanase activity: 99%
MnCl2
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1 mM C-terminal truncated enzyme ApuADELTA: amylase activity: 109%, pullulanase activity: 121%; 1 mM full length enzyme: amylase activity: 133%, pullulanase activity: 133%
NaCl
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50 mM C-terminal truncated enzyme ApuADELTA: amylase activity: 103%, pullulanase activity: 94%; 50 mM full length enzyme: amylase activity: 107%, pullulanase activity: 95%
NiCl2
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1 mM C-terminal truncated enzyme ApuADELTA: amylase activity: 87%, pullulanase activity: 100%; 1 mM full length enzyme: amylase activity: 116%, pullulanase activity: 112%
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
6-Azido-6-deoxypullulan
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6-Chloro-6-deoxypullulan
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alpha-cyclodextrin
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moderate inhibition by 10 mM, 30C, 1 h
formaldehyde
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gamma-cyclodextrin
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moderate inhibition by 10 mM, 30C, 1 h
Guanidine-HCl
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inhibition of both enzyme variants at 1 M, 30C, 1 h
N-bromosuccinimide
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strong inhibition by 0.1 mM, 30C, 1 h
Urea
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inhibition of both enzyme variants at 5 M, 30C, 1 h
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.22
Pullulan
optimal pH 7.0, optimal temperature 75°C
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
828.3 - 62000
Pullulan
111000
soluble starch
Geobacillus stearothermophilus
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298.3 - 313.3
starch
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00000431
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full length enzyme
0.00000596
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C-terminal truncated enzyme ApuADELTA
0.15
Vmax, substrate starch, 50 mM MOPS buffer, pH 7.0, 2 mM CaCl2, 75C, 45 min
16.6
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maltotriose as substrate, wild-type enzyme, comparison with mutant enzymes
23.24
Vmax, substrate pullulan, 50 mM MOPS buffer, pH 7.0, 2 mM CaCl2, 75C, 45 min
28.6
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pullulan as substrate, wild-type enzyme, comparison with mutant enzymes
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4
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full length enzyme and C-terminal truncated enzyme ApuADELTA with optimum at pH 4-4.5, 0.1 M buffers pH 2.5-4: glycine-HCl, pH 3.5-6: acetate, pH 6-8: phosphate, substrate: soluble starch
7
substrate pullulan
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2 - 7
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40C, 0.1 M buffers pH 2.5-4: glycine-HCl, pH 3.5-6: acetate, pH 6-8: phosphate, substrate: soluble starch
4 - 11
substrate pullulan: at least 50% activity retained between pH 5 and 9, about 20% activity at pH 4 and beyond pH 10. buffers of 100 mM pH 4: sodium citrate, pH 5-6: acetic acid, pH 6.5-7: MOPS, pH 7-9: Tris-HCl, pH 9-11: glycine
5 - 8
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pH 5 and 8: more than 50% activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
optimal growth temperature is 30-70C
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 60
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C-terminal truncated enzyme ApuADELTA with only slightly higher activities than full length enzyme, especially at higher than optimal temperatures, about 100% activity at 40-45C, 60-90% activity at 20-35C and 50C, 20-40% activity at 55-60C, 0.1 M sodium acetate butter, pH 4.5, starch as substrate
30 - 70
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30C: about 70% of activity maximum, 70C: about 50% of activity maximum
30 - 90
substrate pullulan: more than 80% activity from 65-80C, 70-80% activity from 55-60°C, about 40% activity between 40 and 50°C, about 20-30% activity at 30C and beyond 80°C
40 - 80
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40C: about 50% of activity maximum, 80C: about 25% of activity maximum
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
additional information
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pullulanase secreton (type II secretion system) components PulM and PulL do not localize specifically to the cell poles, Pul secreton is distributed over the cell surface. GFP-PulM is evenly distributed over the cell envelope, whereas GFP-PulL is barely detectable in the envelope. When produced together with all other secreton components, GFP-PulL is circumferentially distributed, with numerous brighter patches. Envelope-association of GFP-PulL is almost completely abolished when native PulL is also produced. GFP-PulM and GFP-PulL both appear in spherical polar foci when overexpressed
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Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
53000
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SDS-PAGE
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
85000
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theoretically calculated mass of truncated TetApuM855, corroborated by SDS-PAGE
100000
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1 * 100000, SDS-PAGE
107800
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calculated mass of matured full length enzmye TetAbuM955, corroborated by SDS-PAGE, 13 N-terminal amino acids (signal peptide) cleaved off proteolytically, DNA fragment 2882 bp
107818
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x * 107818, SDS-PAGE
175000
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matured enzyme linked to peptidoglycan at carboxy terminus, premature 182360 Da product of 5127 bp apuB gene with 1708 amino acids, signal sequence of 34 amino acids with cleavage site located between Ala34 and Asp35, 4 highly conserved regions, calculated from sequence
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
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
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2 - 8
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C-terminal truncated enzyme ApuADELTA, pre-incubation for 1 h, 40C, 0.1 M buffers pH 2.5-8, 90-100% activity from pH 2.5-6.6, about 80% activity at pH 7, about 60% at pH 7.5, about 30% activity at pH 8; full length enzyme slightly less stable than C-terminal truncated enzyme ApuADELTA, pre-incubation for 1 h, 40C, 0.1 M buffers pH 2.5-8, 90-100% activity from pH 2.5-6, about 80% activity at pH 6.5, about 50% at pH 7, between 20-30% activity at pH 7.5 and 8
699051
3 - 9
substrate pullulan, 25C, 4 h pre-incubation at pH 3: 50 mM sodium citrate, pH 5: 50 mM sodium acetate, pH 7: 50 mM Tris-HCl, pH 9: 50 mM glycin, more than 50% activity retained at pH 3, more than 80% at pH 5, about 100% at pH 7 and 9
696810
6
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30 min, 50C, stable
646822
additional information
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-
646821
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 60
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C-terminal truncated enzyme ApuADELTA, 100% activity at 20-30C, 50% at 45°C, pre-incubation at given temperatures for 1 h, pH 4.5, residual activity measured at 40C, starch as substrate; full length enzyme, 100% activity at 20-30C, 50% activity at 41C, pre-incubation at given temperatures for 1 h, pH 4.5, residual activity measured at 40C, starch as substrate
45 - 75
substrate pullulan, pre-incubation at 45, 50, 60, 65, 70, 75°C for 30, 60, and 120 min. about 80% activity after 120 min between 45 and 60°C, more than 60% activity after 120 min at 65C, about 50% after 120 min at 70C, around 20% activity after 120 min at 75°C
60 - 70
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the irreversible thermoinactivation of the enzyme is recorded in 50 mM sodium acetate, pH 5.5 at 60, 70 and 80C. Pullulanase retains 85% of its activity after 50 min of incubation at 60C, its residual activities at 70 and 80C after 50 min are 70 and 15%, respectively
70
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60 min, about 80% loss of activity
80
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no temperature sensitivity difference between truncated and full length enzyme below and at 80°C
88
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onset of denaturation of full length TetApuM955
90
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onset of denaturation of truncated TetApuR885, it is more thermostable than full length TetApuM955 above 90°C
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
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 20% glycerol, stable
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitation, DEAE-Sepharose column chromatography, and Sephadex G-100 gel filtration
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BD TALON spin column for 6x His-tag protein affinity purification, simultaneous SDS-PAGE zymogram with 1% soluble starch
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gene purification by agarose gel electrophoresis, enzyme purification by size exclusion chromatography, elution via Superdex 200 10/300 GL column with 50 mM MOPS, pH7, 50 mM NaCl
Lactococcus lactis cytoplasmic fraction containing the alpha-amylase and pullulanase domain proteins, respectively, is purified by using Ni-nitrilotriacetic acid matrices, elution fractions analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
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supernatant ultrafiltrated, DEAE Sepharose CLl-4B, Superose 6
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
all pul genes expressed in Escherichia coli K-12 strains, all pull genes except pulL expressed from plasmid pCHAP1217, pulL expressed from plasmid pCHAP710. Overexpression of gfp-pulL or gfp-pulM in Escherichia coli
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Bifidobacterium breve cultured in reinforced clostridial medium, anaeorbic, 37°C. Escherichia coli cultured in Luria Bertani broth, agitated, 37°C. Lactococcus lactis cultured in M17 broth containing 0.5% glucose, 30°C for amplification of vectro pNZ8048 containing only alpha-amylase or pullulanase encoding domains, respectively. Selection of recombinant Escherichia coli with pORI19-apuB on agar containing erythromycin and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside and isopropyl-beta-D-galactopyranoside
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Escherichia coli DH5alphaF' grown in Luria-Bertani broth, 37°C with plasmid pTZ57R/T, expression in yeast Pichia pastoris with recombinant plasmid pPICZ-BK193 in Yeast Extract Peptone Dextrose
Escherichia coli NovaBlue and Rosetta (DE3) pLysS, cloning and expression host, respectively, with plasmid pET-20b(+), in LB broth, 37°C
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expression in Bacillus subtilis
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expression in bacteria
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expression in Escherichia coli
expression in Saccharomyces cerevisiae
NCL21 with plasmid pRN-PulSa, pRN-Puldelta3
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D328H
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site-directed mutagenesis, inactive mutant
D328N
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site-directed mutagenesis, inactive mutant
D424H
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site-directed mutagenesis, inactive mutant
D424N
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site-directed mutagenesis, inactive mutant
E357H
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site-directed mutagenesis, inactive mutant
E357Q
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site-directed mutagenesis, inactive mutant
I358V
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mutation decreases the preference for alpha(1-6)-branched oligosaccharides and pullulan as substrates
I358W
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mutation reduces the acceptability of alpha(1-6)-branched oligo- and polysaccharides
M375L
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mutation increases transglycosylation activity in comparison to wild-type enzyme
S422V
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mutation increases transglycosylation activity in comparison to wild-type enzyme
Y377D
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mutation decreases transglycosylation activity in comparison to wild-type enzyme
Y377F
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mutation increases transglycosylation activity in comparison to wild-type enzyme
Y377S
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mutation decreases transglycosylation activity in comparison to wild-type enzyme
I358V
-
mutation decreases the preference for alpha(1-6)-branched oligosaccharides and pullulan as substrates
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I358W
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mutation reduces the acceptability of alpha(1-6)-branched oligo- and polysaccharides
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M375L
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mutation increases transglycosylation activity in comparison to wild-type enzyme
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S422V
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mutation increases transglycosylation activity in comparison to wild-type enzyme
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Y377F
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mutation increases transglycosylation activity in comparison to wild-type enzyme
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I358V
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site-directed mutagenesis, the mutant shows increased activity hydrolyzing alpha-1,6-glucosidic bonds, producing maltotriose, compared to the wild-type enzyme
I358W
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site-directed mutagenesis, the mutant shows decreased activity hydrolyzing alpha-1,6-glucosidic bonds, producing maltotriose, compared to the wild-type enzyme
I358V
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site-directed mutagenesis, the mutant shows increased activity hydrolyzing alpha-1,6-glucosidic bonds, producing maltotriose, compared to the wild-type enzyme
-
I358W
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site-directed mutagenesis, the mutant shows decreased activity hydrolyzing alpha-1,6-glucosidic bonds, producing maltotriose, compared to the wild-type enzyme
-
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis
Show AA Sequence (881 entries)
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