Information on EC 3.2.1.133 - glucan 1,4-alpha-maltohydrolase

Word Map on EC 3.2.1.133
Please wait a moment until all data is loaded. This message will disappear when all data is loaded.
Specify your search results
Select one or more organisms in this record:
Show additional data
Do not include text mining results
Include (text mining) results (more...)
Include results (AMENDA + additional results, but less precise; more...)


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

EC NUMBER
COMMENTARY hide
3.2.1.133
-
RECOMMENDED NAME
GeneOntology No.
glucan 1,4-alpha-maltohydrolase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
hydrolysis of (1->4)-alpha-D-glucosidic linkages in polysaccharides so as to remove successive alpha-maltose residues from the non-reducing ends of the chains
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis
hydrolysis of O-glycosyl bond
-
-
-
-
transglycosylation
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Metabolic pathways
-
-
Starch and sucrose metabolism
-
-
SYSTEMATIC NAME
IUBMB Comments
4-alpha-D-glucan alpha-maltohydrolase
Acts on starch and related polysaccharides and oligosaccharides. The product is alpha-maltose; cf. EC 3.2.1.2 beta-amylase.
CAS REGISTRY NUMBER
COMMENTARY hide
160611-47-2
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
-
UniProt
Manually annotated by BRENDA team
strain TS-25; TS-25
-
-
Manually annotated by BRENDA team
SUH4-2
-
-
Manually annotated by BRENDA team
SUH4-2
-
-
Manually annotated by BRENDA team
gene GcaTK4MA; strain TK4, gene GcaTK4MA
UniProt
Manually annotated by BRENDA team
gene GcaTK4MA; strain TK4, gene GcaTK4MA
UniProt
Manually annotated by BRENDA team
-
UniProt
Manually annotated by BRENDA team
-
UniProt
Manually annotated by BRENDA team
-
UniProt
Manually annotated by BRENDA team
-
UniProt
Manually annotated by BRENDA team
GSS1
-
-
Manually annotated by BRENDA team
strain IM6501
-
-
Manually annotated by BRENDA team
fenugreek
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
4-nitrophenyl alpha-D-maltohexaoside + H2O
4-nitrophenol + maltohexaose
show the reaction diagram
-
-
-
?
4-nitrophenyl beta-D-glucoside + H2O
4-nitrophenol + D-glucose
show the reaction diagram
poor substrate
-
-
?
4-nitrophenyl maltopyranoside + H2O
4-nitrophenol + maltose
show the reaction diagram
excellent substrate
-
-
?
acarbose + alpha-D-glucose
isoacarbose
show the reaction diagram
acarbose + H2O
acarviosine-glucose
show the reaction diagram
-
-
-
-
?
acarbose + H2O
acarviosine-glucose + alpha-D-glucose
show the reaction diagram
acarbose + H2O
alpha-maltose + ?
show the reaction diagram
acarbose + H2O
D-glucose + acarviosine-glucose
show the reaction diagram
acarbose + H2O
glucose + acarviosine-glucose
show the reaction diagram
-
-
-
-
?
alpha-(1,4)-glycosidic linked cyclodextrins + H2O
maltooligosaccharide
show the reaction diagram
-
main depolymerization of outer amylopectin branches
-
-
?
alpha-cyclodextrin + H2O
?
show the reaction diagram
alpha-cyclodextrin + H2O
alpha-maltose + alpha-D-glucose
show the reaction diagram
-
-
molar ratio 10:1
?
alpha-cyclodextrin + H2O
maltose + D-glucose
show the reaction diagram
-
-
-
?
alpha-Schardinger dextrin + H2O
alpha-maltose + alpha-D-glucose
show the reaction diagram
-
-
-
-
?
amylopectin + H2O
?
show the reaction diagram
amylopectin + H2O
alpha-maltose + ?
show the reaction diagram
-
hydrolytic release of maltose residues, wild-type, double and triple mutant enzymes studied to determine substrate size and geometric shape of catalytic site
-
-
?
amylopectin + H2O
fragments of amylopectin
show the reaction diagram
-
main depolymerization of outer amylopectin branches
mainly short amylopectin chains from degradation of outer branches, inhibiting amylopectin retrogradation, and therefore, amorphous starch network and week amylose network of freshly baked bread are retained
-
?
amylopectin + H2O
fragments of amylopectin + dextrin
show the reaction diagram
-
main depolymerization of outer amylopectin branches
mainly short amylopectin chains from degradation of outer branches, inhibiting amylopectin retrogradation, and therefore, amorphous starch network and week amylose network of freshly baked bread are retained
-
?
amylopectin + H2O
maltose + ?
show the reaction diagram
amylopectin + H2O
maltose + alpha-D-glucose
show the reaction diagram
-
-
in the initial stages of hydrolysis enzyme produces maltotetraose, maltotriose and maltose, as the reaction progresses, the maltotriose and maltotetraose disappears, glucose being formed by the splitting of maltotriose into equimolar amounts of maltose and glucose
?
amylopectin + H2O
maltose + D-glucose
show the reaction diagram
amylopectin + H2O
maltose + maltotriose
show the reaction diagram
amylose + H2O
?
show the reaction diagram
amylose + H2O
alpha-maltose + ?
show the reaction diagram
-
substrate size and geometric shape of catalytic site analyzed, wild-type, double and triple mutant enzymes tested, wild-type enzyme hydrolyzed amylose more favourably than amylopectin
-
-
?
amylose + H2O
maltose + ?
show the reaction diagram
amylose + H2O
maltose + D-glucose
show the reaction diagram
azurine cross-linked amylose + H2O
maltose + ?
show the reaction diagram
-
-
-
-
?
beta-cyclodextrin + H2O
?
show the reaction diagram
beta-cyclodextrin + H2O
alpha-maltose + ?
show the reaction diagram
beta-cyclodextrin + H2O
alpha-maltose + alpha-D-glucose
show the reaction diagram
beta-cyclodextrin + H2O
alpha-maltose + glucose
show the reaction diagram
beta-cyclodextrin + H2O
D-glucose + maltose + maltotriose + maltooligosaccharides
show the reaction diagram
100% activity
-
-
?
beta-cyclodextrin + H2O
maltooligosaccharide
show the reaction diagram
-
-
-
-
?
beta-cyclodextrin + H2O
maltose
show the reaction diagram
beta-cyclodextrin + H2O
maltose + ?
show the reaction diagram
beta-cyclodextrin + H2O
maltose + D-glucose
show the reaction diagram
-
-
-
?
cyclomaltodextrin + H2O
alpha-maltose + alpha-D-glucose
show the reaction diagram
-
-
-
-
?
D-tagatose + maltotriose
maltosyl-tagatose
show the reaction diagram
-
transglycosylation
-
-
?
gamma-cyclodextrin + H2O
?
show the reaction diagram
gamma-cyclodextrin + H2O
alpha-maltose + alpha-D-glucose
show the reaction diagram
-
maximal activity (100%)
-
-
?
gelatinised starch + H2O
maltose + ?
show the reaction diagram
-
-
-
-
?
gelatinised waxy maize starch + H2O
alpha-maltose + ?
show the reaction diagram
-
-
main product
-
?
gelatinized corn starch + H2O
?
show the reaction diagram
-
-
-
-
?
gelatinized rice starch + H2O
maltose + ?
show the reaction diagram
-
-
-
-
?
glycogen + H2O
?
show the reaction diagram
glycogen + H2O
maltose + ?
show the reaction diagram
maize starch + H2O
maltose + ?
show the reaction diagram
-
-
-
-
?
maltoheptaose + H2O
maltose + ?
show the reaction diagram
-
-
-
-
?
maltoheptaose + H2O
maltose + D-glucose
show the reaction diagram
-
-
mutant enzyme A290I produces mostly maltose, while wild-type enzyme produces glucose (32.8%) as well as maltose
-
?
maltohexaose + H2O
?
show the reaction diagram
-
-
-
-
?
maltohexaose + H2O
maltose + ?
show the reaction diagram
-
-
-
-
?
maltopentaose + H2O
2 maltose + D-glucose
show the reaction diagram
the enzyme displays dual hydrolysis activity toward alpha-1,4- and alpha-1,6-glycosidic linkages, the catalytic efficiency of 6-O-maltosyl-beta-cyclodextrin is 16fold higher than that of maltotriose. Compared to the kcat/Km value toward maltotriose, the values for longer substrates such as maltotetraose and maltopentaose are negligible
-
-
?
maltopentaose + H2O
?
show the reaction diagram
-
-
-
-
?
maltopentaose + H2O
alpha-maltose + ?
show the reaction diagram
-
-
-
-
?
maltopentaose + H2O
maltose + D-glucose
show the reaction diagram
maltose + H2O
D-glucose
show the reaction diagram
-
-
-
-
?
maltotetraose + H2O
2 maltose
show the reaction diagram
the enzyme displays dual hydrolysis activity toward alpha-1,4- and alpha-1,6-glycosidic linkages, the catalytic efficiency of 6-O-maltosyl-beta-cyclodextrin is 16fold higher than that of maltotriose. Compared to the kcat/Km value toward maltotriose, the values for longer substrates such as maltotetraose and maltopentaose are negligible
-
-
?
maltotetraose + H2O
alpha-maltose + ?
show the reaction diagram
-
-
-
-
?
maltotetraose + H2O
alpha-maltose + alpha-D-glucose
show the reaction diagram
-
-
-
-
?
maltotetraose + H2O
maltose
show the reaction diagram
-
-
-
?
maltotetraose + H2O
maltose + D-glucose
show the reaction diagram
-
-
mutant enzyme A290I produces mostly maltose, while wild-type enzyme produces glucose (24.8%) as well as maltose
-
?
maltotriose + H2O
?
show the reaction diagram
maltotriose + H2O
alpha-maltose + alpha-D-glucose
show the reaction diagram
maltotriose + H2O
isomaltose + isopanose + panose + branched glucooligosaccharides
show the reaction diagram
-
-
transfer products of transglycosylation
-
?
maltotriose + H2O
maltose + D-glucose
show the reaction diagram
polished rice grain + H2O
maltose + ?
show the reaction diagram
-
-
-
-
?
potato starch + H2O
?
show the reaction diagram
puerarin + beta-cyclodextrin
daidzein 8-C-glucosyl-(alpha-glucosyl)n-1
show the reaction diagram
-
transglycosylation activity
-
-
?
pullulan + H2O
?
show the reaction diagram
pullulan + H2O
alpha-maltose + ?
show the reaction diagram
pullulan + H2O
maltose + D-glucose + panose
show the reaction diagram
-
relative hydrolytic activity towards beta-cyclodextrin, soluble starch and pullulan are 8:1:1.9
mainly maltose and glucose with relatively minor quantity of panose and other maltooligosaccharides
-
?
pullulan + H2O
panose
show the reaction diagram
pullulan + H2O
panose + ?
show the reaction diagram
rice meal + H2O
maltose + ?
show the reaction diagram
-
-
-
-
?
simmondsin + acarviosine-glucose
acarviosine-simmondsin + alpha-D-glucose
show the reaction diagram
-
transglycosylation
novel compound in which acarviosine is attached to the glucose-moiety of simmondsin by an alpha-(1,6)-glycosidic linkage, with both antiobesity and hypoglycemic activity
?
soluble starch + H2O
?
show the reaction diagram
soluble starch + H2O
maltose + ?
show the reaction diagram
starch + H2O
?
show the reaction diagram
starch + H2O
alpha-maltose
show the reaction diagram
starch + H2O
alpha-maltose + ?
show the reaction diagram
starch + H2O
maltooligosaccharide
show the reaction diagram
-
-
-
-
?
starch + H2O
maltose
show the reaction diagram
starch + H2O
maltose + ?
show the reaction diagram
wheat 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
acarbose + H2O
glucose + acarviosine-glucose
show the reaction diagram
-
-
-
-
?
alpha-(1,4)-glycosidic linked cyclodextrins + H2O
maltooligosaccharide
show the reaction diagram
-
main depolymerization of outer amylopectin branches
-
-
?
amylopectin + H2O
fragments of amylopectin + dextrin
show the reaction diagram
-
main depolymerization of outer amylopectin branches
mainly short amylopectin chains from degradation of outer branches, inhibiting amylopectin retrogradation, and therefore, amorphous starch network and week amylose network of freshly baked bread are retained
-
?
amylopectin + H2O
maltose + ?
show the reaction diagram
-
-
-
-
?
amylopectin + H2O
maltose + maltotriose
show the reaction diagram
amylose + H2O
maltose + ?
show the reaction diagram
-
-
-
-
?
beta-cyclodextrin + H2O
maltooligosaccharide
show the reaction diagram
-
-
-
-
?
maltotriose + H2O
isomaltose + isopanose + panose + branched glucooligosaccharides
show the reaction diagram
-
-
transfer products of transglycosylation
-
?
pullulan + H2O
panose
show the reaction diagram
-
-
-
-
?
starch + H2O
?
show the reaction diagram
starch + H2O
alpha-maltose
show the reaction diagram
starch + H2O
alpha-maltose + ?
show the reaction diagram
starch + H2O
maltooligosaccharide
show the reaction diagram
-
-
-
-
?
starch + H2O
maltose
show the reaction diagram
-
the enzyme shows higher affinity to the starch at negative pressure (-200 mbar) compared to the atmospheric pressure
-
-
?
starch + H2O
maltose + ?
show the reaction diagram
-
-
-
-
?
additional information
?
-
-
BSMA preferentially hydrolyzes longer branch chains, releasing maltose and glucose from the non-reducing end of the branch chains, and transfers the resulting maltooligosaccharides to the non-reducing ends of the shorter branch chains by forming alpha-1,6-glucosidic linkages
-
-
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Fe2+
-
increases the activity at low concentrations (1 and 2 mM)
K+
about 186.6% activity at 1 mM
Li+
about 166.5% activity at 1 mM
Na+
123% activity at 1 mM
Sn4+
about 121% activity at 1 mM
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
69.9% residual activity at 10 mM
acarbose
Al3+
about 3.0% residual activity at 10 mM
Cr3+
about 22.2% residual activity at 10 mM
diethyl dicarbonate
24.5% residual activity at 10 mM
maltose
N-bromosuccinamide
complete inhibition at 1 mM
Na+
about 1.7% residual activity at 10 mM
Pb+
about 2% residual activity at 5 mM
-
phenylmethylsulfony fluoride
62.1% residual activity at 10 mM
-
phenylmethylsulfonyl fluoride
about 2% residual activity at 1% (v/v)
SDS
about 6% residual activity at 1% (v/v)
Sn4+
about 89.3% residual activity at 10 mM
sodium stearoyl lactylate
-
-
Woodward's reagent K
72.4% residual activity at 10 mM
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
about 120% activity at 1% (v/v)
Ca2+
the enzyme is activated by 15% at 5 mM
DMSO
-
enzyme activity increases to 136% in the presence of 10% (v/v) DMSO
EDTA
-
increases activity of wild-type enzyme and of mutant enzyme R26Q/S169N/I333V/A398V/Q411L/P453L
ethanol
-
enzyme activity increases to 113% in the presence of 10% (v/v) ethanol
methanol
-
enzyme activity increases to 104.4% in the presence of 10% (v/v) methanol
Mn2+
the enzyme is activated by 51% at 5 mM
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.426 - 1.08
acarbose
0.43
alpha-cyclodextrin
pH and temperature not specified in the publication
0.05 - 2.03
beta-cyclodextrin
2.68
gamma-cyclodextrin
pH and temperature not specified in the publication
1.5 - 9.21
maltoheptaose
3.8 - 7.53
maltohexaose
3.5 - 21.2
maltopentaose
63.43 - 427
maltose
1.6 - 18.8
maltotetraose
1.13 - 8.4
maltotriose
4.1 - 13
soluble starch
-
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
18.7 - 67.8
acarbose
0.1 - 500
alpha-cyclodextrin
0.005 - 15.38
amylopectin
0.022 - 62.82
amylose
0.00000343 - 280.8
beta-cyclodextrin
29.1 - 225.6
gamma-cyclodextrin
2 - 384.1
maltoheptaose
9.2 - 160.3
maltohexaose
0.0017 - 197
maltopentaose
0.04 - 0.29
maltose
0.0033 - 574.1
maltotetraose
0.037 - 652.5
maltotriose
0.021 - 9.9
soluble starch
-
0.000245 - 457
starch
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3.37
alpha-cyclodextrin
Staphylothermus marinus
A3DM60
pH and temperature not specified in the publication
455
3.62
beta-cyclodextrin
Staphylothermus marinus
A3DM60
pH and temperature not specified in the publication
302
10.86
gamma-cyclodextrin
Staphylothermus marinus
A3DM60
pH and temperature not specified in the publication
640
0.000078 - 0.24
maltopentaose
272
0.000193 - 0.22
maltotetraose
269
0.76 - 2.7
maltotriose
188
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.7
maltose
-
pH 5.0, 90C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.2
-
pH 6.0, 60C, beta-cyclodextrin as substrate, variant DM
0.3
-
pH 6.0, 60C, beta-cyclodextrin as substrate, variant 4B74
1
-
pH 6.0, 60C, beta-cyclodextrin as substrate, variant 4B78
1.25
-
pH 5.0, 90C, substrate: maltotriose
1.5
-
pH 6.0, 60C, beta-cyclodextrin as substrate, wild-type
1.7
-
pH 6.0, 60C, beta-cyclodextrin as substrate, variants 2A39, 4A48 and 3C71
2
-
pH 6.0, 60C, beta-cyclodextrin as substrate, variant 1B100
2.04
-
-
2.1
-
pH 6.0, 60C, beta-cyclodextrin as substrate, variant 1B76
5.4
-
beta-cyclodextrin as substrate, dimer
14.2
-
starch as substrate
21.1
-
starch as substrate, 0.2 M KCl
24
-
starch as substrate, 0.4 M KCl
26
-
starch as substrate, 0.6 M KCl
26.3
-
starch as substrate, 0.8 M KCl
27.6
-
starch as substrate, 1.0 M KCl
30.5
-
soluble starch as substrate, monomer
58.7
-
hydrolysis of beta-cyclodextrin
91
-
after 3fold purification, using gamma-cyclodextrin as substrate, at 90C in 50 mM sodium acetate buffer (pH 5.5), with 5 mM CuCl2
93.6
-
after 3fold purification, using gamma-cyclodextrin as substrate, at 90C in 50 mM sodium acetate buffer (pH 5.5), with 5 mM HgCl2
95.2
-
after 3fold purification, using gamma-cyclodextrin as substrate, at 90C in 50 mM sodium acetate buffer (pH 5.5), with 5 mM ZnCl2
98.5
-
after 3fold purification, using gamma-cyclodextrin as substrate, at 90C in 50 mM sodium acetate buffer (pH 5.5), with 5 mM FeSO4
177.4
-
cell extract, using gamma-cyclodextrin as substrate, at 90C in 50 mM sodium acetate buffer (pH 5.5)
194.1
-
beta-cyclodextrin as substrate, monomer
206.8
-
beta-cyclodextrin as substrate, 1.0 M KCl
210
-
beta-cyclodextrin as substrate, 0.8 M KCl
227.2
-
beta-cyclodextrin as substrate, 0.6 M KCl
242.9
-
beta-cyclodextrin as substrate, 0.4 M KCl
257.8
-
beta-cyclodextrin as substrate, 0.2 M KCl
268.6
-
after 3fold purification, using gamma-cyclodextrin as substrate, at 90C in 50 mM sodium acetate buffer (pH 5.5), with 5 mM MnCl2
278
-
beta-cyclodextrin as substrate
342.7
-
beta-cyclodextrin as substrate, dimer
413.6
-
after 3fold purification, using gamma-cyclodextrin as substrate, at 90C in 50 mM sodium acetate buffer (pH 5.5), with 5 mM BaCl2
535.1
-
after 3fold purification, using gamma-cyclodextrin as substrate, at 90C in 50 mM sodium acetate buffer (pH 5.5)
558.6
-
after 3fold purification, using gamma-cyclodextrin as substrate, at 90C in 50 mM sodium acetate buffer (pH 5.5), with 10% (v/v) methanol
568.8
-
after 3fold purification, using gamma-cyclodextrin as substrate, at 90C in 50 mM sodium acetate buffer (pH 5.5), with 5 mM CoCl2
592.9
-
after 3fold purification, using gamma-cyclodextrin as substrate, at 90C in 50 mM sodium acetate buffer (pH 5.5), with 10% (v/v) DMSO
604.7
-
after 3fold purification, using gamma-cyclodextrin as substrate, at 90C in 50 mM sodium acetate buffer (pH 5.5), with 10% (v/v) ethanol
695.6
-
after 3fold purification, using gamma-cyclodextrin as substrate, at 90C in 50 mM sodium acetate buffer (pH 5.5), with 5 mM MgCl2
728.8
-
after 3fold purification, using gamma-cyclodextrin as substrate, at 90C in 50 mM sodium acetate buffer (pH 5.5), with 5 mM CaCl2
785.5
-
after 3fold purification, using gamma-cyclodextrin as substrate, at 90C in 50 mM sodium acetate buffer (pH 5.5), with 5 mM AlCl3
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4
-
or pH 5.0, assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3.5 - 7
-
about 65% of activity maximum at pH 3.5, about 35% of activity maximum at pH 7.3
5 - 9.5
more than 80% of enzyme activity retained at pH ranges from 5 to 7, recombinant maltogenic amylase stable at pH ranging from 5 to 9.5 and mainly between 6 and 8
5 - 6.5
-
more than 50% of the maximum activity is retained in the range between pH 5.0 and pH 6.5
6 - 9
activity range, profile, overview
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45
-
truncated mutant ThMA-DELTA124
85
alpha-1,4-glycosidic linkage hydrolysis
90 - 95
-
-
98
alpha-1,6-glycosidic linkage hydrolysis
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10 - 60
kinetics shown, more than 50% of activity retained at the temperature range of 1050C, rapid decrease in activity above 60C
20 - 55
-
more than 50% activity between 20 and 50C, at 55C the enzyme retains 38% of activity
20 - 80
-
about 20% of activity maximum at 30C, about 60% of activity maximum at 80C
30 - 80
profile, overview
30 - 60
-
30C: about 20% of maximal activity with starch, about 25% of maximal activity with beta-cyclodextrin and about 35% of maximal activity with pullulan, 60C: about 40% of maximal activity with pullulan, about 50% of maximal activity with starch anf about 80% of maximal activity with beta-cyclodextrin
40 - 70
about 40% activity at 40C, about 80% activity at 50C, 100% activity at 60C, about 30% activity at 70C
40 - 100
about 35% activity at 40C, about 60% activity at 50C, about 80% activity at 90C, about 35% activity at 70C, 100% activity at 80C, about 97% activity at 90C, and about 75% activity at 100C
40 - 65
-
more than 50% activity between 40 and 65C
60 - 95
60C; about 50% of maximal activity, 60C: about 60% of maximal activity, alpha-1,4-glycosidic linkage hydrolysis of maltotriose
75 - 80
-
high thermostability, dependent on oligomer structure of the enzyme, optimal activity at 75C and 80C for beta-cyclodextrin and soluble starch, half-lifes of 61 min at 75C and of 24 min at 80C
80 - 98
activity at 80C is about 50% compared to the activity at 98C, alpha-1,6-glycosidic linkage hydrolysis of 6-O-maltotetraosyl-beta-cyclodextrin
80 - 105
-
at 105C, almost 50% of the maximum activity is detected
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8.5
-
determined by thin layer gel-electrofocusing
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
additional information
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50000
x * 50000, SDS-PAGE
53000
-
x * 53000, SDS-PAGE
64000
-
SDS-PAGE
66000
-
SDS-PAGE, recombinant and wild-type protein
66630
-
deduced from sequence
68830
-
predicted from nucleotide sequence data
68900
-
MALDI-TOF-MS spectra
70030
x * 70030, sequence calculation
70191
-
4 * 70191, calculated from sequence
72060
-
MALDI-TOF
72090
-
deduced from sequence
72500
2 * 72500, SDS-PAGE
74000
-
x * 74000, SDS-PAGE
74252
-
x * 74252, calculated from amino acid sequence
94000 - 112000
-
gel filtration
104700
calculated from gel filtration
109000
-
native BbmA, gel filtration
135000
SDS-PAGE, gel filtration
145000
dimer, gel filtration
211000
-
gel filtration
260000
-
gel filtration
725000
1 * 725000, SDS_PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
homotetramer
monomer
oligomer
tetramer
-
4 * 65000, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
sitting drop vapor diffusion method, using 1.0 M ammonium citrate dibasic, 0.8 M sodium acetate trihydrate pH 4.6
-
sitting drop vapor diffusion method, using 12% (w/v) polyethylene glycol 4000, 2% (v/v) isopropyl alcohol, 0.1 M ADA, pH 6.5, and 0.1 M Li2SO4
sitting drop vapor diffusion method
crystals obtained by vapor diffusion, space group P6(1), cell dimensions of a = b = 118.04 A, c = 266.88 A
-
mutant E357L, space group P6(1), a = b = 119.09, c = 270.20
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5 - 5.5
-
stable
646794
5 - 8
there is no loss in the activity of the enzyme till 14 days of incubation at pH 7.0 and 8.0, but it decreases drastically after 8 days at pH 5.0 and 6.0
732746
5 - 10
-
the enzyme retains 100% of its activity when incubated for 24 h in a pH range between 5.0 and 10.0
731907
7 - 9
the enzyme is highly active and stable in a pH range from 7.0 to 9.0
732767
additional information
determined in different buffers ranging from pH 5 to pH 10 for 12 h at 4C followed by activity assay
681861
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
-20 - 40
the enzyme retains more than 50% of its activity at 40C and 30C for 10 min. The half-lives of the enzyme are 239.02 h, 126.03 h and 108.30 h at -20C, 4C and 20C, respectively. However, the half-lives are dramatically reduced to 0.37 h and 0.04 h at 30C and 40C, respectively
20 - 70
-
the enzyme shows 80% of the initial activity after an incubation time of 30 min without substrate in the temperature range of 20-70C
40 - 70
-
the enzyme is stable up to 40C at atmospheric pressure, but the activity of the maltogenic alpha-amylase is drastically reduced above 40C. Hhigher temperatures (60 and 70C) are destructive to the enzyme even in the presence of substrate starch
50 - 55
55
-
half-life at 55C is about 15 min
55 - 75
the enzyme remains stable for up to 60 min at 55C, after 60 min at 60 and 65C the enzyme shows 75% activity, while after 20 min at 70 and 75C the enzyme is inactive
60 - 70
-
stable at pH 5.5 at 60C, 25% loss of activity at 70C
64
-
Tm of Novamyl wild-type at pH 4.0
67
melting temperature at pH 4.0
77
-
Tm-value at pH 8 is 76.7C
78
half-life of mutant III-1 about 20 times greater than half-life of the wild-type at 78C
82
-
Tm-value of mutant R26Q/S169N/I333V/A398V/Q411L/P453L is 81.5C
83
-
Tm of Novamyl wild-type at pH 5.0
85 - 100
-
purified enzyme is extremely thermostable with a half-life of 60 min at an optimal temperature of 95C. The enzyme retains about 80% relative activity after 60 min of incubation at 85C and 90C, about 50% relative activity after 60 min of incubation at 95C, and about 25% relative activity after 60 min of incubation at 100C
85
-
Tm-value of mutant enzyme R26Q/I152N/S153N/S169N/I333V/A398V/Q411L/P453L, Tm-value decreases to 79.9C in presence of EDTA. Tm-value increases to 87.4C in presence of CaCl2
88
-
TD of variant NM447 at pH 4.0, an increase of 10C relative to the wild-type Novamyl
90
the half-life values of the monomeric enzyme at 80, 90 and 100C are 35.7, 8.2 and 3.12 h, respectively. The half-life values of the dimeric enzyme at 80, 90 and 100C are 55.7, 12.63 and 3.58 h, respectively
91
melting temperature at pH 5.0
95
-
Tm of variant NM447 at pH 5.0
105
melting temperature at pH 6.0
109
109C melting temperature with enzymatic activity under acidic conditions (pH 3.5-5.0)
210
-
baking at 210C for 40 min leaves the enzyme active
additional information
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
at atmospheric pressure the enzyme is unstable at 60 and 70C, while under negative pressure (_-200 mbar) the enzyme shows its activity on the starch at up to 70C
-
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ethanol
Methanol
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitation and fast-performance liquid chromatography
-
ammonium sulfate precipitation and MonoQ Sepharose column chromatography
-
ammonium sulfate precipitation and UNO-Q12 anion exchange column chromatography
-
His-tagged protein purified via Ni-NTA colummns
His-tagged recombinant protein purified via Ni-NTA colummns
-
HiTrap SP column chromatography and Superdex 200 gel filtration
mutagenic PCR fragment purification with Quiaquick Gel Extract kit, and agarose electrophoresis, then cloned into plasmid pHP13amp, fermentation in bioreactors, enzyme variants purified on alpha-cyclodextrin coupled to agarose column, corroborated by SDS-PAGE; recombinant protein, the variants NM319, NM326, NM398, NM404, and NM447 are grown in fermenters and the expressed enzymes are purified
-
mutant E357L
-
Ni-NTA agarose column chromatography
Ni-NTA column chromatography
Ni-NTA column chromatography and Superdex 200 gel filtration
Ni2+-NTA agarose resin column chromatography, and Sephacryl S-200 gel filtration
purification of ThMA variants with N-terminal six-histidines by nickel-nitrilotriacetic acid column chromatography
-
recombinant enzyme
-
recombinant protein, from cell crude extracts with a yield of 23%
recombinant protein, gel filtration
-
truncated mutant ThMA-DELTA124
-
wild-type and mutant enzyme A290I
-
wild-type and mutant enzymes
wild-type and recombinant protein, gel filtration
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
amplified in Escherichia coli DH5alpha, heterogenous expression in Lactococcus lactis MG1363, P170 expression system for production of recombinant LGMA
-
cloned and expressed in Escherichia coli
-
cloned and expressed in Escherichia coli BL21 (DE3)
-
cloned and overexpressed in Escherichia coli
-
cloned and overproduced in Escherichia coli MC1061
-
cloning and expression in Escherichia coli MC 1061 with p6xHTMX, transformants grown in Luria-Bertani medium with ampicillin at 37°C
-
expressed in Escherichia coli
-
expressed in Escherichia coli BL21(DE3) cells
expressed in Escherichia coli BL21-CodonPlus(DE3)-RP cells
-
expressed in Escherichia coli DH5alpha cells
-
expressed in Escherichia coli DH5alpha using expression vectors pBMS1-4, open reading frame of 1749 bp encoding a protein of 582 residues, low sequence similarity of 60% to maltogenic amylase of Thermus sp., four known conserved regions at position 241 to 246, 324 to 331, 355 to 360, and 418 to 423 identified
expressed in Escherichia coli MC1061
-
expressed in Escherichia coli MC1061 cells
expressed in Escherichia coli MC1061, His-tagged, recombinant protein
-
expressed in Escherichia coli MC1061, His-tagged, wild-type and mutant protein
expressed in the periplasm of Escherichia coli BL21(DE3) cells
-
expressed the gene in Escherichia coli
expression in Escherichia coli
-
gene amyM cloned in Escherichia coli, transferred with plasmid pDN400 carrier to a Bacillus subtilis 168 host and expressed heterogenously
-
gene GcaTK4MA, DNA and amino acid sequence determination and analysis, sequence comparison and phylogenetic tree, expression of His6-tagged TK4MA in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain JM101
heterogenous expression in Bacillus subtilis host strain ISW1214
Novamyl libraries are iniatially made in Escherichia coli, then shuttled and expressed in the screen host Bacillus subtilis. The variants NM319, NM326, NM398, NM404, and NM447 are grown in fermenters, and the expressed enzymes are purified and tested for anti-staling properties in standard bread pH 5.5-5.9 and/or bread at sourdough (pH 4.0-4.3); shuffled libraries of Novamyl are produced in in Escherichia coli, shuttled to screening host B. subtilis strain A164 delta 5, plasmid pHP13amp
-
wild-type and mutant ThMAs expressed in Escherichia coli
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D30A/K40R/D261G
-
Novamyl variant NM398, similar to wild-type at pH 4.0; variant NM398, medium thermotolerance due to D261G mutation
D46G
-
the mutant behaves similar to the wild type enzyme regarding thermoactivity, thermostability and pH profile. The Km values of the mutant for all substrates are strongly increased compared to the wild type enzyme
D46N
-
the mutant behaves similar to the wild type enzyme regarding kinetic parameters, thermoactivity, thermostability and pH profile
D46V
-
the mutant behaves similar to the wild type enzyme regarding thermoactivity, thermostability and pH profile. The affinity and catalytic efficiency of the mutant toward beta-cyclodextrin are increased 5fold as compared with the wild type enzyme
D46V/P78L/V145A/K548E
-
the half-life times for the mutant at 50C and 55C are 70 min and 25 min, compared to 30 min and 13 min for the wild type, respectively
F188L/D261G/T288P
-
Novamyl variant NM447, outperform wild-type Novamyl in bread at pH 4.3. Wild-type Novamyl requires a nearly 30fold protein dosage increase over NM447 to obtain anti-staling bread properties. NM447 appears to have a broad pH functionality profile and performs better than wild-type Novamyl in standard bread to pH 5.9. It seems to be be generally tehrmally stabilized, both at pH 4.0 and 5.0.; variant NM447, most thermotolerant, probably due to F188L mutation, but reduced activity, medium thermotolerance due to D261G mutation, improved anti-staling performance in application test, about 70% activity retained after incubation at 80°C at pH 4.3 for 25 min
G312A
-
the mutant has an optimal temperature of 45C instead of the 40C for the wild type enzyme
G312A/K436R
-
the half-life time at 55C increase from 15 to 25 min for the double mutant
K436R
-
the mutant has an optimal temperature of 45C instead of the 40C for the wild type enzyme
N115D/F188L
-
Novamyl variant NM319, similar to wild-type at pH 4.0, at pH 5.5 the variant outperform wild-type Novamyl, significantly more thermally stable that wild-type; variant NM319, most thermotolerant, probably due to F188L mutation, but reduced activity, improved anti-staling performance in application test, about 40% activity retained after incubation at 80°C at pH 4.3 for 25 min
T142A
-
Novamyl variant NM326, similar to wild-type at pH 4.0; variant NM326, inducing 20% activity increase and 50% thermal stability increase
T142A/D261G/N327S/K425E/K520R/N5951
-
variant NM404, medium thermotolerance due to D261G mutation
T142A/D261G/N327S/K425E/K520R/N595I
-
Novamyl variant NM404, similar to wild-type at pH 4.0
F188L/D261G/T288P
-
Novamyl variant NM447, outperform wild-type Novamyl in bread at pH 4.3. Wild-type Novamyl requires a nearly 30fold protein dosage increase over NM447 to obtain anti-staling bread properties. NM447 appears to have a broad pH functionality profile and performs better than wild-type Novamyl in standard bread to pH 5.9. It seems to be be generally tehrmally stabilized, both at pH 4.0 and 5.0.; variant NM447, most thermotolerant, probably due to F188L mutation, but reduced activity, medium thermotolerance due to D261G mutation, improved anti-staling performance in application test, about 70% activity retained after incubation at 80°C at pH 4.3 for 25 min
-
T142A
-
Novamyl variant NM326, similar to wild-type at pH 4.0; variant NM326, inducing 20% activity increase and 50% thermal stability increase
-
D46G
-
the mutant behaves similar to the wild type enzyme regarding thermoactivity, thermostability and pH profile. The Km values of the mutant for all substrates are strongly increased compared to the wild type enzyme
-
D46N
-
the mutant behaves similar to the wild type enzyme regarding kinetic parameters, thermoactivity, thermostability and pH profile
-
D46V
-
the mutant behaves similar to the wild type enzyme regarding thermoactivity, thermostability and pH profile. The affinity and catalytic efficiency of the mutant toward beta-cyclodextrin are increased 5fold as compared with the wild type enzyme
-
D46V/P78L/V145A/K548E
-
the half-life times for the mutant at 50C and 55C are 70 min and 25 min, compared to 30 min and 13 min for the wild type, respectively
-
G312A
-
the mutant has an optimal temperature of 45C instead of the 40C for the wild type enzyme
-
G312A/K436R
-
the half-life time at 55C increase from 15 to 25 min for the double mutant
-
K436R
-
the mutant has an optimal temperature of 45C instead of the 40C for the wild type enzyme
-
N147D/F195L/N263S/D311G/A344V/F397S/N508D
mutant III-1, seven mutations, generated by random mutagenesis after three rounds of DNA shuffling and recombination, lineage of shuffling mutants indicated
N147D/F195L/N263S/D311G/A344V/F397S/N508D/M375T
additional exchange M375T of mutant III-2 responsible for decreased specific activity, lineage of shuffling mutants shown
N147D/F195L/N263S/D311G/A344V/F397S/N508D
-
mutant III-1, seven mutations, generated by random mutagenesis after three rounds of DNA shuffling and recombination, lineage of shuffling mutants indicated
-
N147D/F195L/N263S/D311G/A344V/F397S/N508D/M375T
-
additional exchange M375T of mutant III-2 responsible for decreased specific activity, lineage of shuffling mutants shown
-
D109A
the mutant shows increased affinity towards amylose, amylopectin and starch, and a decreased affinity towards alpha- and beta-cyclodextrin
D109E
the mutant does not show any effect on the binding affinity and substrate hydrolytic efficiency towards alpha- and beta-cyclodextrin but a strong decline in the affinity and substrate hydrolytic efficiency of the mutant enzyme towards amylopectin
E580Q
-
strongly reduced activity compared to the wild type enzyme
F218A
-
the mutant shows wild type activity with alpha-1,6-glycosidic bond hydrolysis and about 4fold increased activity with alpha-1,4-glycosidic bond hydrolysis compared to the wild type enzyme
F452A
-
inactive
W453A
-
strongly reduced activity compared to the wild type enzyme
E580Q
-
strongly reduced activity compared to the wild type enzyme
-
F218A
-
the mutant shows wild type activity with alpha-1,6-glycosidic bond hydrolysis and about 4fold increased activity with alpha-1,4-glycosidic bond hydrolysis compared to the wild type enzyme
-
F452A
-
inactive
-
W453A
-
strongly reduced activity compared to the wild type enzyme
-
A290I
-
mutant enzyme A290I produces mostly maltose from maltotetraose, while wild-type enzyme produces glucose as well as maltose. kcat/KM of mutant enzyme 290I for maltose is 48times less than that of wild-type enzyme. kcat/Km for maltotriose is 18.5fold lower than wild-type enzyme. kcat/Km for maltotetraose is 2.9fold lower than wild-type enzyme. kcat/Km for maltopentaose is 2.5fold lower than wild-type enzyme. kcat/Km for maltohexaose is 1.9fold lower than wild-type enzyme. kcat/Km for maltoheptaose is 4.7fold lower than wild-type enzyme
A330G/N331C/E332C
-
F-18, strong reduction of all substrate hydrolyzing activities, lower relative specificity to beta-cyclodextrin and pullulan than to starch and to maltotriose than to acarbose compared to wild-type
A330G/N331G/E332C
-
C-20, strong reduction of all substrate hydrolyzing activities, lower relative specificity to beta-cyclodextrin and pullulan than to starch and to maltotriose than to acarbose compared to wild-type
A330G/N331G/E332G
-
G-91, strong reduction of all substrate hydrolyzing activities, higher relative specificity to beta-cyclodextrin than to starch compared to wild-type, lower relative specificity to maltotriose than to acarbose compared to wild-type, transglycosylation: high amount of branched tetraose and pentaose
A330G/N331G/E332S
-
G-22, strong reduction of all substrate hydrolyzing activities, lower relative specificity to beta-cyclodextrin and pullulan than to starch and to maltotriose than to acarbose compared to wild-type
A330G/N331P/E332G
-
C-43, strong reduction of all substrate hydrolyzing activities, lower relative specificity to beta-cyclodextrin and pullulan than to starch and to maltotriose than to acarbose compared to wild-type
A330G/N331V/E332G
-
G-90, strong reduction of all substrate hydrolyzing activities, lower relative specificity to beta-cyclodextrin and pullulan than to starch and to maltotriose than to acarbose compared to wild-type
A330M/N331G/E332C
-
B-96, strong reduction of all substrate hydrolyzing activities, lower relative specificity to beta-cyclodextrin and pullulan than to starch and to maltotriose than to acarbose compared to wild-type
A330S/N331A
-
F-80, strong reduction of all substrate hydrolyzing activities, higher relative specificity to beta-cyclodextrin and pullulan than to starch compared to wild-type, lower relative specificity to maltotriose than to acarbose compared to wild-type, transglycosylation: high amount of branched tetraose and pentaose
A330S/N331G/E332T
-
K-37, strong reduction of all substrate hydrolyzing activities, lower relative specificity to beta-cyclodexxtrin and pullulan than to starch and to maltotriose than to acarbose compared to wild-type
A398V
-
random mutagenesis, using DNA shuffling
E332D
-
site-directed mutagenesis, significantly decreased transglycosylation activity
E332H
-
site-directed mutagenesis, replacing Glu 332 with histidine reduces transglycosylation activity significantly, but enhances hydrolysis activity on alpha-(1,3)-, alpha-(1,4)- and alpha-(1,6) glycosidic bonds relative to the wild-type
E332Q
-
site-directed mutagenesis
E357L
-
site-directed mutagenesis
G50I/D109E
-
double mutation, two main residues of the catalytic binding pocket, site-directed mutagenesis
G50I/D109E/V431I
-
triple mutation of three main residues of the catalytic binding pocket, site-directed mutagenesis
I333V