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Enzyme Nomenclature
Information on EC 3.2.1.133 - glucan 1,4-alpha-maltohydrolase
for references in articles please use BRENDA:EC3.2.1.133
Word Map on EC 3.2.1.133
- 3.2.1.133
- starch
- stearothermophilus
-
transglycosylation
-
amylases
- acarbose
- amylopectin
- maltotriose
- pullulan
-
amylolytic
- alpha-amylases
- maltooligosaccharides
- acarviosine-glucose
- panose
- neopullulanase
- synthesis
- beta-cds
- cyclomaltodextrins
- cgtases
-
alpha-1,6-glycosidic
-
staling
- food industry
-
glucanotransferase
-
antistaling
- maltotetraose
-
stale
-
alpha-1,4
- acarviosine
- biotechnology
- nutrition
- medicine
- pharmacology
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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria, Archaea
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EC NUMBER 
COMMENTARY 
3.2.1.133
-
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RECOMMENDED NAME
GeneOntology No.
glucan 1,4-alpha-maltohydrolase
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REACTION
REACTION DIAGRAM
COMMENTARY
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
-
-
-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hydrolysis
hydrolysis of O-glycosyl bond
-
-
-
-
transglycosylation
transglycosylation
-
-
-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
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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.
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CAS REGISTRY NUMBER
COMMENTARY
160611-47-2
-
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
646794, 646797, 646798, 646802, 664522, 666955, 698428, 710128, 714654, 731579, 731845, 731847, 732280
-
-
maltogenic alpha-amylase precursor, amyM gene
SwissProt
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
physiological function
-
the enzyme may have a role in regulating the concentration of maltose during maltodextrin metabolism
physiological function
-
the enzyme may have a role in regulating the concentration of maltose during maltodextrin metabolism
-
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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
4-nitrophenyl alpha-D-maltohexaoside + H2O
4-nitrophenol + maltohexaose
-
-
-
?
4-nitrophenyl beta-D-glucoside + H2O
4-nitrophenol + D-glucose
poor substrate
-
-
?
4-nitrophenyl maltopyranoside + H2O
4-nitrophenol + maltose
excellent substrate
-
-
?
alpha-(1,4)-glycosidic linked cyclodextrins + H2O
maltooligosaccharide
-
main depolymerization of outer amylopectin branches
-
-
?
alpha-cyclodextrin + H2O
alpha-maltose + alpha-D-glucose
-
-
molar ratio 10:1
?
alpha-Schardinger dextrin + H2O
alpha-maltose + alpha-D-glucose
-
-
-
-
?
amylopectin + H2O
alpha-maltose + ?
-
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
-
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
-
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 + alpha-D-glucose
-
-
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
?
amylose + H2O
alpha-maltose + ?
-
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
-
-
?
beta-cyclodextrin + H2O
D-glucose + maltose + maltotriose + maltooligosaccharides
100% activity
-
-
?
D-tagatose + maltotriose
maltosyl-tagatose
-
transglycosylation
-
-
?
gamma-cyclodextrin + H2O
alpha-maltose + alpha-D-glucose
-
maximal activity (100%)
-
-
?
gelatinised waxy maize starch + H2O
alpha-maltose + ?
-
-
main product
-
?
maltoheptaose + H2O
maltose + D-glucose
-
-
mutant enzyme A290I produces mostly maltose, while wild-type enzyme produces glucose (32.8%) as well as maltose
-
?
maltopentaose + H2O
2 maltose + D-glucose
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
2 maltose
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
maltose + D-glucose
-
-
mutant enzyme A290I produces mostly maltose, while wild-type enzyme produces glucose (24.8%) as well as maltose
-
?
maltotriose + H2O
isomaltose + isopanose + panose + branched glucooligosaccharides
-
-
transfer products of transglycosylation
-
?
puerarin + beta-cyclodextrin
daidzein 8-C-glucosyl-(alpha-glucosyl)n-1
-
transglycosylation activity
-
-
?
pullulan + H2O
maltose + D-glucose + panose
-
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
-
?
simmondsin + acarviosine-glucose
acarviosine-simmondsin + alpha-D-glucose
-
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
?
acarbose + alpha-D-glucose
isoacarbose
-
transglycosylation
-
-
?
acarbose + H2O
acarviosine-glucose + alpha-D-glucose
-
-
-
-
?
acarbose + H2O
acarviosine-glucose + alpha-D-glucose
-
hydrolysis
-
-
?
alpha-cyclodextrin + H2O
?
80% activity compared to beta-cyclodextrin
-
-
?
alpha-cyclodextrin + H2O
?
high specificity for alpha-cyclodextrin
-
-
?
alpha-cyclodextrin + H2O
?
-
64.7% activity compared to gamma-cyclodextrin
-
-
?
amylopectin + H2O
?
less than 2% activity compared to beta-cyclodextrin
-
-
?
amylopectin + H2O
?
less than 2% activity compared to beta-cyclodextrin
-
-
?
amylopectin + H2O
?
-
the maltogenic Bacillus stearothermophilus alpha-amylase preferentially hydrolyses the exterior chains of amylopectin. However, during the later phases, the enzyme also hydrolyses inner chains, presumably with a high multiple attack action
-
-
?
amylopectin + H2O
maltose + ?
the enzyme recognized maltose units with alpha-1,4 and alpha-1,6 linkages in polysaccharides (e.g., starch, amylopectin, and glycogen) and hydrolyzes pullulan very poorly. Branched cyclodextrin is only hydrolyzed along its branched maltooligosaccharides. 6-O-D-glucosyl-beta-cyclodextrin and beta-cyclodextrin are resistant. Exo-type glucan hydrolase with alpha-1,4- and alpha-1,6-glucan hydrolytic activities
maltose is the primary end product of hydrolysis
-
?
amylopectin + H2O
maltose + ?
the enzyme recognized maltose units with alpha-1,4 and alpha-1,6 linkages in polysaccharides (e.g., starch, amylopectin, and glycogen) and hydrolyzes pullulan very poorly. Branched cyclodextrin is only hydrolyzed along its branched maltooligosaccharides. 6-O-D-glucosyl-beta-cyclodextrin and beta-cyclodextrin are resistant. Exo-type glucan hydrolase with alpha-1,4- and alpha-1,6-glucan hydrolytic activities
maltose is the primary end product of hydrolysis
-
?
amylopectin + H2O
maltose + ?
the enzyme only releases maltose from polymers such as soluble starch, amylopectin, and glycogen, while maltose is rarely detected from reaction with amylose and pullulan
-
-
?
amylopectin + H2O
maltose + maltotriose
-
-
wild-type enzyme produces 93% maltose (2 homomers) and 5% maltotriose compared to 66% maltose and 20% maltotriose of mutant F188L/D261G/T288P, wild-type enzyme produces 93% maltose (2 homomers) and 5% maltotriose compared to 66% maltose and 20% matotriose of mutant F188L/D261G/T288P
-
?
amylopectin + H2O
maltose + maltotriose
-
-
wild-type enzyme produces 93% maltose (2 homomers) and 5% maltotriose compared to 66% maltose and 20% maltotriose of mutant F188L/D261G/T288P, wild-type enzyme produces 93% maltose (2 homomers) and 5% maltotriose compared to 66% maltose and 20% matotriose of mutant F188L/D261G/T288P
-
?
amylose + H2O
maltose + ?
-
high preference toward amylose compared to amylopectin
-
-
?
beta-cyclodextrin + H2O
?
-
78.1% activity compared to gamma-cyclodextrin
-
-
?
beta-cyclodextrin + H2O
alpha-maltose + ?
main product alpha-maltose
-
-
?
beta-cyclodextrin + H2O
alpha-maltose + ?
main product alpha-maltose
-
-
?
beta-cyclodextrin + H2O
alpha-maltose + ?
-
recombinant rLGMA, expressed in Escherichia coli and in Lactococcus lactis MG1363
-
-
?
beta-cyclodextrin + H2O
alpha-maltose + ?
-
recombinant rLGMA, expressed in Escherichia coli and in Lactococcus lactis MG1363
-
-
?
beta-cyclodextrin + H2O
alpha-maltose + alpha-D-glucose
highest catalytic efficiency
-
-
?
beta-cyclodextrin + H2O
alpha-maltose + alpha-D-glucose
highest catalytic efficiency
-
-
?
beta-cyclodextrin + H2O
alpha-maltose + alpha-D-glucose
-
-
-
-
?
beta-cyclodextrin + H2O
alpha-maltose + alpha-D-glucose
-
-
molar ratio 3:1
?
beta-cyclodextrin + H2O
alpha-maltose + alpha-D-glucose
-
hydrolytic activity
-
-
?
beta-cyclodextrin + H2O
alpha-maltose + alpha-D-glucose
-
high thermostability, substrate preference dependent on oligomeric state
-
-
?
beta-cyclodextrin + H2O
alpha-maltose + alpha-D-glucose
-
high thermostability, substrate preference dependent on oligomeric state
-
-
?
beta-cyclodextrin + H2O
alpha-maltose + alpha-D-glucose
-
prefers cyclodextrins to starch or pullulan as substrate
-
-
?
beta-cyclodextrin + H2O
alpha-maltose + glucose
substrate determination for recombinant enzyme MAUS149
-
-
?
beta-cyclodextrin + H2O
alpha-maltose + glucose
substrate determination for recombinant enzyme MAUS149
-
-
?
beta-cyclodextrin + H2O
maltose
-
-
mainly hydrolyzed to maltose
-
?
beta-cyclodextrin + H2O
maltose
-
-
mainly hydrolyzed to maltose
-
?
beta-cyclodextrin + H2O
maltose + ?
-
relative hydrolytic activity towards beta-cyclodextrin, soluble starch and pullulan are 8:1:1.9
-
-
?
gamma-cyclodextrin + H2O
?
10% activity compared to beta-cyclodextrin
-
-
?
gamma-cyclodextrin + H2O
?
10% activity compared to beta-cyclodextrin
-
-
?
glycogen + H2O
maltose + ?
the enzyme recognized maltose units with alpha-1,4 and alpha-1,6 linkages in polysaccharides (e.g., starch, amylopectin, and glycogen) and hydrolyzes pullulan very poorly. Branched cyclodextrin is only hydrolyzed along its branched maltooligosaccharides. 6-O-D-glucosyl-beta-cyclodextrin and beta-cyclodextrin are resistant.Exo-type glucan hydrolase with alpha-1,4- and alpha-1,6-glucan hydrolytic activities
maltose is the primary end product of hydrolysis
-
?
glycogen + H2O
maltose + ?
the enzyme only releases maltose from polymers such as soluble starch, amylopectin, and glycogen, while maltose is rarely detected from reaction with amylose and pullulan
-
-
?
maltotriose + H2O
?
-
recombinant rLGMA, expressed in Escherichia coli and in Lactococcus lactis MG1363
-
-
?
maltotriose + H2O
?
-
recombinant rLGMA, expressed in Escherichia coli and in Lactococcus lactis MG1363
-
-
?
maltotriose + H2O
maltose + D-glucose
highest hydrolysis activities are on the alpha-1,4-glycosidic linkage of maltotriose (1.25 U/mg) and the alpha-1,6-glycosidic bond of 6-O-maltosyl-beta-cyclodextrin
-
-
?
maltotriose + H2O
maltose + D-glucose
highest hydrolysis activities are on the alpha-1,4-glycosidic linkage of maltotriose (1.25 U/mg) and the alpha-1,6-glycosidic bond of 6-O-maltosyl-beta-cyclodextrin
-
-
?
maltotriose + H2O
maltose + D-glucose
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
-
-
?
pullulan + H2O
alpha-maltose + ?
substrate determination for recombinant enzyme MAUS149
-
-
?
pullulan + H2O
alpha-maltose + ?
substrate determination for recombinant enzyme MAUS149
-
-
?
pullulan + H2O
alpha-maltose + ?
-
recombinant rLGMA, expressed in Escherichia coli and in Lactococcus lactis MG1363
-
-
?
pullulan + H2O
alpha-maltose + ?
-
recombinant rLGMA, expressed in Escherichia coli and in Lactococcus lactis MG1363
-
-
?
soluble starch + H2O
?
45% activity compared to beta-cyclodextrin
-
-
?
soluble starch + H2O
?
45% activity compared to beta-cyclodextrin
-
-
?
soluble starch + H2O
maltose + ?
the enzyme displays less hydrolytic action on raw starches than on soluble starch
-
-
?
soluble starch + H2O
maltose + ?
-
relative hydrolytic activity towards beta-cyclodextrin, soluble starch and pullulan are 8:1:1.9
-
-
?
soluble starch + H2O
maltose + ?
the enzyme only releases maltose from polymers such as soluble starch, amylopectin, and glycogen, while maltose is rarely detected from reaction with amylose and pullulan
-
-
?
starch + H2O
?
-
the enzyme shows a substrate hydrolysis preference for cyclodextrins over starch
-
-
?
starch + H2O
alpha-maltose
substrate determination for recombinant enzyme MAUS149
-
-
?
starch + H2O
alpha-maltose
substrate determination for recombinant enzyme MAUS149
-
-
?
starch + H2O
alpha-maltose
-
carbohydrate metabolism in the cytoplasm
-
?
starch + H2O
alpha-maltose
-
carbohydrate metabolism in the cytoplasm
-
?
starch + H2O
alpha-maltose
-
exo-acting maltogenic alpha-amylase, removes maltose units from the non-reducing chain ends
-
?
starch + H2O
alpha-maltose
-
catalyzes the hydrolysis of starch material, central role in carbohydrate metabolism
-
?
starch + H2O
alpha-maltose + ?
maltogenic amylase from Bacillus sp.
-
-
?
starch + H2O
alpha-maltose + ?
maltogenic amylase from Bacillus sp.
-
-
?
starch + H2O
alpha-maltose + ?
utilization of BSMA for production of highly branched amylopectin and amylose from enzymatically modified rice starch, branching by transglycosylation mediated by BSMA, increased number of branched side chains in modified amylopectin clusters determined
-
-
?
starch + H2O
alpha-maltose + ?
-
wild-type LGMA and recombinant rLGMA, reaction products determined by thin-layer chromatography and gel filtration
-
-
?
starch + H2O
alpha-maltose + ?
-
wild-type LGMA and recombinant rLGMA, reaction products determined by thin-layer chromatography and gel filtration
-
-
?
starch + H2O
alpha-maltose + ?
-
transglycosylation pattern opposite to that of bacterial maltogenic amylases, predominant formation of alpha-1,4-glycosidic linked transfer products than of alpha-1,6-linked products
-
-
?
starch + H2O
alpha-maltose + ?
-
transglycosylation pattern opposite to that of bacterial maltogenic amylases, predominant formation of alpha-1,4-glycosidic linked transfer products than of alpha-1,6-linked products
-
-
?
starch + H2O
maltose
-
the enzyme shows higher affinity to the starch at negative pressure (-200 mbar) compared to the atmospheric pressure
-
-
?
starch + H2O
maltose + ?
the enzyme recognized maltose units with alpha-1,4 and alpha-1,6 linkages in polysaccharides (e.g., starch, amylopectin, and glycogen) and hydrolyzes pullulan very poorly. Branched cyclodextrin is only hydrolyzed along its branched maltooligosaccharides. 6-O-D-glucosyl-beta-cyclodextrin and beta-cyclodextrin are resistant.Exo-type glucan hydrolase with alpha-1,4- and alpha-1,6-glucan hydrolytic activities
maltose is the primary end product of hydrolysis
-
?
starch + H2O
maltose + ?
the enzyme recognized maltose units with alpha-1,4 and alpha-1,6 linkages in polysaccharides (e.g., starch, amylopectin, and glycogen) and hydrolyzes pullulan very poorly. Branched cyclodextrin is only hydrolyzed along its branched maltooligosaccharides. 6-O-D-glucosyl-beta-cyclodextrin and beta-cyclodextrin are resistant.Exo-type glucan hydrolase with alpha-1,4- and alpha-1,6-glucan hydrolytic activities
maltose is the primary end product of hydrolysis
-
?
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
-
-
-
additional information
?
-
-
the enzyme forms highly branched products from branched glucan and branching enzyme-treated tapioca starch
-
-
-
additional information
?
-
the dimeric enzyme transglycosylates hydrolytic products of G4/G5 and acarbose, while the monomeric form does not because of the lack of extra sugar-binding space formed due to dimerization
-
-
-
additional information
?
-
-
enzyme shows hydrolytic activity towards alpha-1,6-glycosidic linkage
-
-
-
additional information
?
-
an exo-type maltose-forming alpha-amylase acting on the non-reducing end of the substrates and requires at least a G2 unit at its working sites of substrates. When the length of the branch is longer than G2 in the substrate, the enzyme primarily attacks alpha-1,4-glycosidic linkages in the long branch and cleaves off G2 unit until it reaches the final G2, and then it performs a debranching reaction by acting on alpha-1,6-glycosidic bonds at branching points
-
-
-
additional information
?
-
-
the enzyme hydrolyzes both alpha-1,4-glucosidic and alpha-1,6-glucosidic linkages of substrates, recognizing only maltose units, in an exo-type manner
-
-
-
additional information
?
-
an exo-type maltose-forming alpha-amylase acting on the non-reducing end of the substrates and requires at least a G2 unit at its working sites of substrates. When the length of the branch is longer than G2 in the substrate, the enzyme primarily attacks alpha-1,4-glycosidic linkages in the long branch and cleaves off G2 unit until it reaches the final G2, and then it performs a debranching reaction by acting on alpha-1,6-glycosidic bonds at branching points
-
-
-
additional information
?
-
-
the enzyme hydrolyzes both alpha-1,4-glucosidic and alpha-1,6-glucosidic linkages of substrates, recognizing only maltose units, in an exo-type manner
-
-
-
additional information
?
-
-
maltooligosaccharides G3-G7 show 5.4-24.1% relative activity compared to gamma-cyclodextrin
-
-
-
additional information
?
-
does not hydrolyze cyclodextrin, pullulan and acarbose
-
-
-
additional information
?
-
-
exhibits dual activity of alpha-D-(1,4)- and alpha-D-(1,6)- glycosidic bond cleavages, shows activity of alpha-D(1,4)- to alpha-D-(1,3), alpha-D-(1,4), or alpha-D-(1,6)-transglycosylation and cleaves acarbose, a pseudotetrasaccharide competitive inhibitor of alpha-amylases
-
-
-
additional information
?
-
-
nearly indistinguishable from cyclomaltodextrinase from Bacillus sp. and Bacillus stearothermophilus neopullulanase, distinguished from typicsl alpha-amylases by containing a novel N-terminal domain and exhibiting preferential substrate specificities for cyclomaltodextrins over starch
-
-
-
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NATURAL SUBSTRATES
NATURAL PRODUCTS
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
alpha-(1,4)-glycosidic linked cyclodextrins + H2O
maltooligosaccharide
-
main depolymerization of outer amylopectin branches
-
-
?
amylopectin + H2O
fragments of amylopectin + dextrin
-
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
-
?
maltotriose + H2O
isomaltose + isopanose + panose + branched glucooligosaccharides
-
-
transfer products of transglycosylation
-
?
starch + H2O
maltose
-
the enzyme shows higher affinity to the starch at negative pressure (-200 mbar) compared to the atmospheric pressure
-
-
?
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
-
-
-
amylopectin + H2O
maltose + maltotriose
-
-
wild-type enzyme produces 93% maltose (2 homomers) and 5% maltotriose compared to 66% maltose and 20% maltotriose of mutant F188L/D261G/T288P
-
?
amylopectin + H2O
maltose + maltotriose
-
-
wild-type enzyme produces 93% maltose (2 homomers) and 5% maltotriose compared to 66% maltose and 20% maltotriose of mutant F188L/D261G/T288P
-
?
starch + H2O
alpha-maltose
-
carbohydrate metabolism in the cytoplasm
-
?
starch + H2O
alpha-maltose
-
carbohydrate metabolism in the cytoplasm
-
?
starch + H2O
alpha-maltose
-
exo-acting maltogenic alpha-amylase, removes maltose units from the non-reducing chain ends
-
?
starch + H2O
alpha-maltose
-
catalyzes the hydrolysis of starch material, central role in carbohydrate metabolism
-
?
starch + H2O
alpha-maltose + ?
A7DWA8
maltogenic amylase from Bacillus sp.
-
-
?
starch + H2O
alpha-maltose + ?
A7DWA8
maltogenic amylase from Bacillus sp.
-
-
?
starch + H2O
alpha-maltose + ?
-
transglycosylation pattern opposite to that of bacterial maltogenic amylases, predominant formation of alpha-1,4-glycosidic linked transfer products than of alpha-1,6-linked products
-
-
?
starch + H2O
alpha-maltose + ?
-
transglycosylation pattern opposite to that of bacterial maltogenic amylases, predominant formation of alpha-1,4-glycosidic linked transfer products than of alpha-1,6-linked products
-
-
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Al3+
Ca2+
Co2+
Mg2+
Mn2+
additional information
Ca2+
-
calcium can stabilize the enzyme activity up to 50°C at atmospheric pressure (% of residual activities at 50, 60, and 70°C are 100, 4.93, 0, respectively)
Ca2+
-
mutant R26Q/I152N/S153N/S169N/I333V/A398V/Q411L/P453L shows highly improved thermostability and catalytic activity in presence of Ca2+.
additional information
K+, Ni2+, Ca2+, and Ba2+ do not exert any observable effect on enzyme activity
additional information
5 mM Zn2+ has no effect on the enzyme activity
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
phenylmethylsulfony fluoride
62.1% residual activity at 10 mM
-
phenylmethylsulfonyl fluoride
about 2% residual activity at 1% (v/v)
additional information
-
no inhibition by sulfhydryl reagents, no inhibition with p-chloromercuribenzoate or Schardinger dextrins
-
additional information
EGTA and EDTA have no observable effect on the activity
-
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.43
alpha-cyclodextrin
pH and temperature not specified in the publication
2.68
gamma-cyclodextrin
pH and temperature not specified in the publication
0.16
beta-cyclodextrin
-
pH 6.0, 60°C, hydrolysis, wild-type and variants 3C71, 4B78 and 4A48
0.36
beta-cyclodextrin
-
pH 6.0, 60°C, hydrolysis, truncated mutant ThMA DELTA124, 1M KCl
0.461
beta-cyclodextrin
-
pH 6.0, 60°C, hydrolysis, truncated mutant ThMA- DELTA124
1.48
beta-cyclodextrin
pH and temperature not specified in the publication
1.5
maltoheptaose
-
kinetic parameters for C14-labelled maltooligosaccharides
3.8
maltohexaose
-
kinetic parameters for C14-labelled maltooligosaccharides
4.4
maltopentaose
-
kinetic parameters for C14-labelled maltooligosaccharides
1.6
maltotetraose
-
kinetic parameters for C14-labelled maltooligosaccharides
3.6
maltotriose
-
kinetic parameters for C14-labelled maltooligosaccharides
4.1
soluble starch
-
estimated for monomeric conformation, in presence of 1 M NaCl
-
additional information
additional information
-
Km 49.7 mg/ml, starch as substrate, pH 6.0, 60°C, wild-type, 1 M KCl; Km 5.34 mg mL-1, starch as substrate, pH 6.0, 60°C, hydrolysis, truncated mutant ThMA-DELTA124; Km 61.0 mg/ml, starch as substrate, pH 6.0, 60°C, hydrolysis, wild-type; Km 7.72 mg/ml, starch as substrate, pH 6.0, 60°C, truncated mutant ThMA-DELTA124, 1M KCl
-
additional information
additional information
-
Km 131 mg/ml, starch as substrate, mutant W47A; Km 73.5 mg/ml, starch as substrate, wild-type
-
additional information
additional information
-
lower affinity for binding of amylopectin but higher affinity for amylose in mutant enzymes demonstrated, sterospecific substrate binding properties of triple mutant enzyme determined by molecular modelling
-
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.1
alpha-cyclodextrin
monomeric enzyme form, at pH 7.0 and 80°C
0.75
alpha-cyclodextrin
mutant enzyme D109A, at pH 7.0 and 80°C
1.39
alpha-cyclodextrin
mutant enzyme D109E, at pH 7.0 and 80°C
1.45
alpha-cyclodextrin
pH and temperature not specified in the publication
2.78
alpha-cyclodextrin
dimeric enzyme form, at pH 7.0 and 80°C
500
alpha-cyclodextrin
in sodium-acetate buffer (50 mM, pH 6.0), at 60°C
0.17
amylopectin
monomeric enzyme form, at pH 7.0 and 80°C
0.8
amylopectin
pH and temperature not specified in the publication
0.59
amylose
pH and temperature not specified in the publication
0.00000343
beta-cyclodextrin
-
pH 6.0, 60°C, hydrolysis, truncated mutant ThMA-DELTA124
0.00000367
beta-cyclodextrin
-
pH 6.0, 60°C, hydrolysis, truncated mutant ThMA- DELTA124, 1 M KCl
0.12
beta-cyclodextrin
monomeric enzyme form, at pH 7.0 and 80°C
0.49
beta-cyclodextrin
mutant enzyme D109A at pH 7.0 and 80°C
0.66
beta-cyclodextrin
mutant enzyme D109E at pH 7.0 and 80°C
1.33
beta-cyclodextrin
dimeric enzyme form, at pH 7.0 and 80°C
5.36
beta-cyclodextrin
pH and temperature not specified in the publication
280.8
beta-cyclodextrin
in sodium-acetate buffer (50 mM, pH 6.0), at 60°C
29.1
gamma-cyclodextrin
pH and temperature not specified in the publication
225.6
gamma-cyclodextrin
in sodium-acetate buffer (50 mM, pH 6.0), at 60°C
0.021
soluble starch
mutant enzyme D109E, at pH 7.0 and 80°C
-
0.04
soluble starch
dimeric enzyme form, at pH 7.0 and 80°C
-
0.12
soluble starch
mutant enzyme D109A, at pH 7.0 and 80°C
-
0.2
soluble starch
monomeric enzyme form, at pH 7.0 and 80°C
-
0.000332
starch
-
pH 6.0, 60°C, hydrolysis, truncated mutant ThMA- DELTA124, 1 M KCl
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
3.37
alpha-cyclodextrin
pH and temperature not specified in the publication
3.62
beta-cyclodextrin
pH and temperature not specified in the publication
10.86
gamma-cyclodextrin
pH and temperature not specified in the publication
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
91
-
after 3fold purification, using gamma-cyclodextrin as substrate, at 90°C in 50 mM sodium acetate buffer (pH 5.5), with 5 mM CuCl2
93.6
-
after 3fold purification, using gamma-cyclodextrin as substrate, at 90°C in 50 mM sodium acetate buffer (pH 5.5), with 5 mM HgCl2
95.2
-
after 3fold purification, using gamma-cyclodextrin as substrate, at 90°C in 50 mM sodium acetate buffer (pH 5.5), with 5 mM ZnCl2
98.5
-
after 3fold purification, using gamma-cyclodextrin as substrate, at 90°C in 50 mM sodium acetate buffer (pH 5.5), with 5 mM FeSO4
177.4
-
cell extract, using gamma-cyclodextrin as substrate, at 90°C in 50 mM sodium acetate buffer (pH 5.5)
268.6
-
after 3fold purification, using gamma-cyclodextrin as substrate, at 90°C in 50 mM sodium acetate buffer (pH 5.5), with 5 mM MnCl2
413.6
-
after 3fold purification, using gamma-cyclodextrin as substrate, at 90°C in 50 mM sodium acetate buffer (pH 5.5), with 5 mM BaCl2
535.1
-
after 3fold purification, using gamma-cyclodextrin as substrate, at 90°C in 50 mM sodium acetate buffer (pH 5.5)
558.6
-
after 3fold purification, using gamma-cyclodextrin as substrate, at 90°C 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 90°C in 50 mM sodium acetate buffer (pH 5.5), with 5 mM CoCl2
592.9
-
after 3fold purification, using gamma-cyclodextrin as substrate, at 90°C 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 90°C 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 90°C in 50 mM sodium acetate buffer (pH 5.5), with 5 mM MgCl2
728.8
-
after 3fold purification, using gamma-cyclodextrin as substrate, at 90°C in 50 mM sodium acetate buffer (pH 5.5), with 5 mM CaCl2
785.5
-
after 3fold purification, using gamma-cyclodextrin as substrate, at 90°C in 50 mM sodium acetate buffer (pH 5.5), with 5 mM AlCl3
additional information
additional information
kinetic mechanism for recombinant enzyme MAUS149 shown, determined by measuring the amount of reducing sugars released during incubation with starch using the dinitrosalicylic acid method, substrate concentrations ranging from 1 to 7.5 g/ml for starch and from 0.5 to 4 g/l for cyclodextrin and pullulan
additional information
kinetic properties determined in wild-type and mutant enzymes, relative activity indicated for several mutants from different lineages of DNA shuffling
additional information
kinetic properties determined by amount of reducing sugars, assayed according to the dinitrosalicylic acid method
additional information
-
catalytic activity of wild-type LGMA and recombinant rLGMA tested in liquid and solid media, kinetic properties determined by amount of reducing sugars, assayed according to the dinitrosalicylic acid method, reaction at pH 6.5 for 13.5 h at 50°C, yields of 53.1% obtained
additional information
-
specific activity of starch hydrolysis estimated for the high oligomer conformation is about 7 U/mg and for monomeric state about 110 U/mg
additional information
-
kinetic properties determined towards amylose and amylopectin in wild-type, double and triple mutant enzymes, bicinchonimate method, determination of reducing sugars
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
6.5
6.5
maximal activity, effect of pH on activity studied at 40°C using 50 mM buffers with different pH values ranging from 3.5 to 10
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
3.5 - 7
-
about 65% of activity maximum at pH 3.5, about 35% of activity maximum at pH 7.3
4 - 6
4 - 8
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 - 9
5 - 6.5
-
more than 50% of the maximum activity is retained in the range between pH 5.0 and pH 6.5
4 - 6
-
pH 4.0: less than 10% of maximal activity, pH 6.0: about 40% of maximal activity
4 - 6
pH 4.0: about 85% of maximal activity, pH 6.0: about 60% of maximal activity, alpha-1,4-glycosidic linkage hydrolysis of maltotriose
4 - 8
pH 4.0: about 50% of maximal activity, pH 8.0: about 70% of maximal activity, alpha-1,6-glycosidic linkage hydrolysis of 6-O-maltotetraosyl-beta-cyclodextrin
4 - 8
-
more than 70% activity between pH 4.0 and 7.0, about 40% activity at pH 8.0
5 - 9
about 35% activity at pH 5.0, 100% activity at pH 6.0, about 85% activity at pH 7.0, about 60% activity at pH 8.0, about 40% activity at pH 9.0
5 - 9
the enzyme is active over a wide range of pH between 5.0 and 9.0 (more than 90% activity) but the activity drastically declines at pH 4.0 and 10.0 to 20 and less than 5%, respectively
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
40
50
55
60
70
75
80
40
maximal activity, effect of temperature on activity investigated in a 50 mM sodium phosphate buffer at pH 6.5 using different temperatures ranging from 10°C to 60°C
55
synthesis reaction performed for 12 h using 500 units/g substrate for transgylcosylation
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
10 - 60
kinetics shown, more than 50% of activity retained at the temperature range of 10–50°C, rapid decrease in activity above 60°C
20 - 55
-
more than 50% activity between 20 and 50°C, at 55°C the enzyme retains 38% of activity
20 - 80
-
about 20% of activity maximum at 30°C, about 60% of activity maximum at 80°C
30 - 60
-
30°C: about 20% of maximal activity with starch, about 25% of maximal activity with beta-cyclodextrin and about 35% of maximal activity with pullulan, 60°C: 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 40°C, about 80% activity at 50°C, 100% activity at 60°C, about 30% activity at 70°C
40 - 100
about 35% activity at 40°C, about 60% activity at 50°C, about 80% activity at 90°C, about 35% activity at 70°C, 100% activity at 80°C, about 97% activity at 90°C, and about 75% activity at 100°C
60 - 95
60°C; about 50% of maximal activity, 60°C: 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 75°C and 80°C for beta-cyclodextrin and soluble starch, half-lifes of 61 min at 75°C and of 24 min at 80°C
80 - 98
activity at 80°C is about 50% compared to the activity at 98°C, alpha-1,6-glycosidic linkage hydrolysis of 6-O-maltotetraosyl-beta-cyclodextrin
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pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
additional information
additional information
the enzyme sequence contains no signal sequence
-
additional information
-
the enzyme sequence contains no signal sequence
-
-
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PDB
SCOP
CATH
ORGANISM
UNIPROT
Staphylothermus marinus (strain ATCC 43588 / DSM 3639 / JCM 9404 / F1)
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
65000
67500
68000
70000
65000
-
SDS-PAGE, difference in molecular masses due to the presence of the six-histidine tag and two additional amino acids
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
homodimer
homotetramer
monomer
oligomer
?
-
x * 74000, SDS-PAGE; x * 74252, calculated from amino acid sequence
-
dimer
-
monomer:dimer equilibrium with a molar ratio of 54:46 in 50 mM glycine-NaOH buffer pH 8.0
dimer
-
monomer:dimer equilibrium with a molar ratio of 54:46 in 50 mM glycine-NaOH buffer pH 8.0
-
monomer
-
monomer:dimer equilibrium with a molar ratio of 54:46 in 50 mM glycine-NaOH buffer pH 8.0
monomer
-
monomer:dimer equilibrium with a molar ratio of 54:46 in 50 mM glycine-NaOH buffer pH 8.0
-
monomer
-
1 * 68000, monomer/dimer equilibrium, ratio 48:52, ultracentrifugation; truncated mutant ThMAdelta124 exists only as a monomeric form
oligomer
-
recombinant protein, high oligomer in solution, dissociation into dimer and monomer mixture by a high concentration of NaCl
oligomer
-
recombinant protein, high oligomer in solution, dissociation into dimer and monomer mixture by a high concentration of NaCl
-
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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
crystals obtained by vapor diffusion, space group P6(1), cell dimensions of a = b = 118.04 A, c = 266.88 A
-
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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 4°C followed by activity assay
681861
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
-20 - 40
the enzyme retains more than 50% of its activity at 40°C and 30°C for 10 min. The half-lives of the enzyme are 239.02 h, 126.03 h and 108.30 h at -20°C, 4°C and 20°C, respectively. However, the half-lives are dramatically reduced to 0.37 h and 0.04 h at 30°C and 40°C, 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 20-70°C
40 - 70
-
the enzyme is stable up to 40°C at atmospheric pressure, but the activity of the maltogenic alpha-amylase is drastically reduced above 40°C. Hhigher temperatures (60 and 70°C) are destructive to the enzyme even in the presence of substrate starch
50 - 55
55 - 75
the enzyme remains stable for up to 60 min at 55°C, after 60 min at 60 and 65°C the enzyme shows 75% activity, while after 20 min at 70 and 75°C the enzyme is inactive
60 - 70
-
stable at pH 5.5 at 60°C, 25% loss of activity at 70°C
60
75
78
half-life of mutant III-1 about 20 times greater than half-life of the wild-type at 78°C
80
85
85 - 100
-
purified enzyme is extremely thermostable with a half-life of 60 min at an optimal temperature of 95°C. The enzyme retains about 80% relative activity after 60 min of incubation at 85°C and 90°C, about 50% relative activity after 60 min of incubation at 95°C, and about 25% relative activity after 60 min of incubation at 100°C
85
-
Tm-value of mutant enzyme R26Q/I152N/S153N/S169N/I333V/A398V/Q411L/P453L, Tm-value decreases to 79.9°C in presence of EDTA. Tm-value increases to 87.4°C in presence of CaCl2
88
-
TD of variant NM447 at pH 4.0, an increase of 10°C relative to the wild-type Novamyl
90
the half-life values of the monomeric enzyme at 80, 90 and 100°C are 35.7, 8.2 and 3.12 h, respectively. The half-life values of the dimeric enzyme at 80, 90 and 100°C are 55.7, 12.63 and 3.58 h, respectively
109
109°C melting temperature with enzymatic activity under acidic conditions (pH 3.5-5.0)
additional information
50 - 55
-
after incubation for 2 h at 50°C, the wild type enzyme retains 35% of its initial activity. The wild type enzyme has a half-life of 15 min at 55°C
50 - 55
-
the half-life times for the wild type enzyme at 50°C and 55°C are 30 min and 13 min, respectively
60
-
the enzyme is rapidly inactivated at temperature above 60°C and is totally inactivated at 60°C within 60 min in the absence of the substrate. However, in the presence of 1% (w/v) of soluble starch, the half time rises from approximately 10 to 60 min at 60°C
80
-
the variants NM447 and NM319 retain 67 and 40% specific activity following a 25-min incubation, respectively
80
half-life of mutant III-2 is 568 min, half-life of the wild-type is below 1 min at 80°C
80
-
ThMA-DM, highly thermostable mutant enzyme, half-life 172 min, wild type enzyme completely inactivated in less than 1 min
85
the half-life is 69 min, 225 min, and 255 min at pH 5.0, pH 6.0, and pH 7.0
additional information
kinetics shown, stable at 45°C for 90 min, half-life duration of 18 min at 55°C
additional information
-
NM404 and NM398 has modest improvements in thermal stability, whereas NM326 shows no significant improvement in thermal stability
additional information
half-lives determined at 75°C, 78°C, 80°C, and 85°C, melting temperatures of mutants III-1 and III-2 determined by differential scanning calorimetry increased by 6.1°C and 11.4°C, respectively, hydrogen bonding, hydrophobic interaction, electrostatic interaction, proper packing, and deamidation predicted as mechanisms for enhanced thermostability in the mutant enzymes
additional information
-
high thermostability, half-lives of thermal inactivation and melting temperature tested
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
at atmospheric pressure the enzyme is unstable at 60 and 70°C, while under negative pressure (_-200 mbar) the enzyme shows its activity on the starch at up to 70°C
-
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ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
DMSO
Ethanol
Methanol
DMSO
-
enzyme activity increases to 136% in the presence of 10% (v/v) DMSO
DMSO
-
enzyme activity increases to 136% in the presence of 10% (v/v) DMSO
-
Ethanol
-
enzyme activity increases to 113% in the presence of 10% (v/v) ethanol
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
Methanol
-
enzyme activity increases to 104.4% in the presence of 10% (v/v) methanol
-
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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
-
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
-
Ni-NTA column chromatography
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 protein, from cell crude extracts with a yield of 23%
wild-type and mutant enzymes
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
amplified in Escherichia coli DH5alpha, heterogenous expression in Lactococcus lactis MG1363, P170 expression system for production of recombinant LGMA
-
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 BL21(DE3) 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, His-tagged, wild-type and mutant protein
expressed in the periplasm of Escherichia coli BL21(DE3) cells
-
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
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D30A/K40R/D261G
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Novamyl variant NM398, similar to wild-type at pH 4.0; variant NM398, medium thermotolerance due to D261G mutation
D46G
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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
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the mutant behaves similar to the wild type enzyme regarding kinetic parameters, thermoactivity, thermostability and pH profile
D46V
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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
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the half-life times for the mutant at 50°C and 55°C are 70 min and 25 min, compared to 30 min and 13 min for the wild type, respectively
F188L/D261G/T288P
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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
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the mutant has an optimal temperature of 45°C instead of the 40°C for the wild type enzyme
G312A/K436R
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the half-life time at 55°C increase from 15 to 25 min for the double mutant
K436R
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the mutant has an optimal temperature of 45°C instead of the 40°C for the wild type enzyme
N115D/F188L
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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
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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
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variant NM404, medium thermotolerance due to D261G mutation
T142A/D261G/N327S/K425E/K520R/N595I
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Novamyl variant NM404, similar to wild-type at pH 4.0
F188L/D261G/T288P
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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
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T142A
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Novamyl variant NM326, similar to wild-type at pH 4.0; variant NM326, inducing 20% activity increase and 50% thermal stability increase
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D46G
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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
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D46N
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the mutant behaves similar to the wild type enzyme regarding kinetic parameters, thermoactivity, thermostability and pH profile
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D46V
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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
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D46V/P78L/V145A/K548E
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the half-life times for the mutant at 50°C and 55°C are 70 min and 25 min, compared to 30 min and 13 min for the wild type, respectively
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G312A
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the mutant has an optimal temperature of 45°C instead of the 40°C for the wild type enzyme
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G312A/K436R
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the half-life time at 55°C increase from 15 to 25 min for the double mutant
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K436R
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the mutant has an optimal temperature of 45°C instead of the 40°C for the wild type enzyme
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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
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mutant III-1, seven mutations, generated by random mutagenesis after three rounds of DNA shuffling and recombination, lineage of shuffling mutants indicated
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N147D/F195L/N263S/D311G/A344V/F397S/N508D/M375T
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additional exchange M375T of mutant III-2 responsible for decreased specific activity, lineage of shuffling mutants shown
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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
F218A
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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
F218A
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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
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A290I
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
E332D
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site-directed mutagenesis, significantly decreased transglycosylation activity
E332H
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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
G50I/D109E
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double mutation, two main residues of the catalytic binding pocket, site-directed mutagenesis
G50I/D109E/V431I