Information on EC 3.2.1.141 - 4-alpha-D-{(1->4)-alpha-D-glucano}trehalose trehalohydrolase

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

EC NUMBER
COMMENTARY hide
3.2.1.141
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RECOMMENDED NAME
GeneOntology No.
4-alpha-D-{(1->4)-alpha-D-glucano}trehalose trehalohydrolase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
hydrolysis of (1->4)-alpha-D-glucosidic linkage in 4-alpha-D-[(1->4)-alpha-D-glucanosyl]n trehalose to yield trehalose and (1->4)-alpha-D-glucan
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis
hydrolysis of O-glycosyl bond
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of secondary metabolites
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Metabolic pathways
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Starch and sucrose metabolism
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trehalose biosynthesis V
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metabolism of disaccharids
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SYSTEMATIC NAME
IUBMB Comments
4-alpha-D-[(1->4)-alpha-D-glucano]trehalose glucanohydrolase (trehalose-producing)
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CAS REGISTRY NUMBER
COMMENTARY hide
170780-50-4
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176431-02-0
trehalosidase, maltooligosyltrehalose (Rhizobium strain M-11 clone pBMTU1 gene treZ reduced) /maltooligosyl trehalose trehalohydrolase (Rhizobium strain M-11 clone pMBTU1 gene treZ reduced)
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
7120, facing NaCl stress, grown in BG-11 medium free from combined nitrogen sources, continuous tungsten plus fluorescent illumination for trehalose hyperproduction
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Manually annotated by BRENDA team
strain O36
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Manually annotated by BRENDA team
strain JMC8857 grown on JCM165 medium at 70°C
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Manually annotated by BRENDA team
strain IAM M-15
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Manually annotated by BRENDA team
strain IAM M-15
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
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biosynthesis of trehalose is mediated by maltooligosyltrehalose synthase and maltooligosyltrehalose trehalohydrolase. In this pathway, trehalose 6-phosphate is not generated as an intermediate
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
4'-O-(1,4-alpha-D-glucosyl)n-alpha,alpha'-trehalose + H2O
(1,4-alpha-D-glucosyl)n + alpha,alpha'-trehalose
show the reaction diagram
glucosyl trehalose + H2O
glucose + trehalose
show the reaction diagram
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maltoheptaose + H2O
?
show the reaction diagram
maltoheptaosyltrehalose + H2O
maltoheptaose + alpha,alpha'-trehalose
show the reaction diagram
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-
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?
maltohexaose + H2O
?
show the reaction diagram
maltooligosacccharide + H2O
D-glucose
show the reaction diagram
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-
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?
maltooligosyltrehalose + H2O
maltooligosaccharide + alpha,alpha'-trehalose
show the reaction diagram
maltooligosyltrehalose + H2O
trehalose + ?
show the reaction diagram
maltopentaose + H2O
?
show the reaction diagram
maltopentaosyltrehalose + H2O
maltopentaose + alpha,alpha'-trehalose
show the reaction diagram
maltosylpentaosyltrehalose + H2O
trehalose + maltopentaose
show the reaction diagram
maltosyltetraosyltrehalose + H2O
trehalose + maltotetraose
show the reaction diagram
maltosyltrehalose + H2O
maltose + alpha,alpha'-trehalose
show the reaction diagram
maltosyltrehalose + H2O
trehalose + maltose
show the reaction diagram
maltosyltriosyltrehalose + H2O
trehalose + maltotriose
show the reaction diagram
maltotetraose + H2O
?
show the reaction diagram
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maltotetraosyltrehalose + H2O
maltotetraose + alpha,alpha'-trehalose
show the reaction diagram
maltotriosyltrehalose + H2O
maltotriose + alpha,alpha'-trehalose
show the reaction diagram
maltotriosyltrehalose + H2O
trehalose + maltotriose
show the reaction diagram
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?
additional information
?
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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
4'-O-(1,4-alpha-D-glucosyl)n-alpha,alpha'-trehalose + H2O
(1,4-alpha-D-glucosyl)n + alpha,alpha'-trehalose
show the reaction diagram
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
Na+, K+, Mg2+, Ca2+, and Zn2+ do not activate
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
EDTA, Na+, K+, Mg2+, Ca2+, and Zn2+ do not inhibit
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
IPTG
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transcription induced by 1 mM
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.19 - 2.88
maltoheptaose
2.16 - 23.2
maltohexaose
4.18 - 39.2
maltooligosyltrehalose
5.08 - 7.74
maltopentaose
4.2 - 5.89
maltopentaosyl trehalose
5.26 - 7.15
maltopentaosyltrehalose
5.5 - 16.7
maltosyl trehalose
4.7
maltosylpentaosyltrehalose
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pH 5.5, 60°C
5.6
maltosyltetraosyltrehalose
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pH 5.5, 60°C
22.4
maltosyltrehalose
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pH 5.5, 60°C
5.8
maltosyltriosyltrehalose
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pH 5.5, 60°C
10.9
maltotetraose
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3.7 - 7
maltotetraosyl trehalose
5.03 - 7.91
maltotetraosyltrehalose
2.7 - 7.22
maltotriosyl trehalose
7.22 - 13.6
maltotriosyltrehalose
additional information
additional information
kinetics of wild-type and mutant enzymes, overview
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5 - 6.49
maltoheptaose
0.36 - 18.2
maltohexaose
2.15 - 1070
maltooligosyltrehalose
14.4 - 19.1
maltopentaose
1230
maltopentaosyl trehalose
Sulfolobus solfataricus
P95867
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675 - 1282
maltopentaosyltrehalose
193
maltosyl trehalose
Sulfolobus solfataricus
P95867
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5.49
maltotetraose
Sulfolobus solfataricus
P95867
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1190
maltotetraosyl trehalose
Sulfolobus solfataricus
P95867
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16 - 1192
maltotetraosyltrehalose
1030
maltotriosyl trehalose
Sulfolobus solfataricus
P95867
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572 - 1030
maltotriosyltrehalose
additional information
maltoheptaose
Sulfolobus solfataricus
P95867
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.51 - 182
maltooligosyltrehalose
4740
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8 - 12
wild-type, 12fold purified
21.1
His-tagged enzyme, crude extract
67.5
wild-type, crude extract
310
His-tagged enzyme, 14.7fold purified
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 9
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pH 4: about 70% activity, pH 5-6: about 100% activity, pH 7: about 40% activity, pH 8-9: between 20-30%, pH 4-5: 0.1 M McIlvaine buffer, pH 5-8: 0.1 M sodium phosphate buffer, pH 8-9: 0.1 M sodium bicarbonate buffer, 70°C, 30 min
5 - 8
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pH 5: about 50% of maximal activity, pH 8: about 40% of maximal activity
5 - 7
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pH 5: about 80% of maximal activity, pH 7: about 65% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 55
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30°C: about 75% of maximal activity, 55°C: about 40% of maximal activity
40 - 90
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.8
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isoelectric focusing
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Deinococcus radiodurans (strain ATCC 13939 / DSM 20539 / JCM 16871 / LMG 4051 / NBRC 15346 / NCIMB 9279 / R1 / VKM B-1422)
Deinococcus radiodurans (strain ATCC 13939 / DSM 20539 / JCM 16871 / LMG 4051 / NBRC 15346 / NCIMB 9279 / R1 / VKM B-1422)
Deinococcus radiodurans (strain ATCC 13939 / DSM 20539 / JCM 16871 / LMG 4051 / NBRC 15346 / NCIMB 9279 / R1 / VKM B-1422)
Deinococcus radiodurans (strain ATCC 13939 / DSM 20539 / JCM 16871 / LMG 4051 / NBRC 15346 / NCIMB 9279 / R1 / VKM B-1422)
Deinococcus radiodurans (strain ATCC 13939 / DSM 20539 / JCM 16871 / LMG 4051 / NBRC 15346 / NCIMB 9279 / R1 / VKM B-1422)
Deinococcus radiodurans (strain ATCC 13939 / DSM 20539 / JCM 16871 / LMG 4051 / NBRC 15346 / NCIMB 9279 / R1 / VKM B-1422)
Deinococcus radiodurans (strain ATCC 13939 / DSM 20539 / JCM 16871 / LMG 4051 / NBRC 15346 / NCIMB 9279 / R1 / VKM B-1422)
Deinococcus radiodurans (strain ATCC 13939 / DSM 20539 / JCM 16871 / LMG 4051 / NBRC 15346 / NCIMB 9279 / R1 / VKM B-1422)
Deinococcus radiodurans (strain ATCC 13939 / DSM 20539 / JCM 16871 / LMG 4051 / NBRC 15346 / NCIMB 9279 / R1 / VKM B-1422)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
59000
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x * 59000, SDS-PAGE
61000
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x * 61000, SDS-PAGE
63700
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sequence analysis
64000
2 * 64000, SDS-PAGE, 2 * 64240, electrospray ionization mass spectrometry, wild-type enzyme
64240
2 * 64000, SDS-PAGE, 2 * 64240, electrospray ionization mass spectrometry, wild-type enzyme
64790
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x * 64790, calculated from sequence
65000
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x * 65000, calculated from sequence
66000
1 * 66000, SDS-PAGE, His-tagged enzyme
68700
gel filtration, His-tagged enzyme
70000
x * 70000, deduced from amino acid sequence
75000
x * 75000, including the hexahistidine domain from the pET22bZ vector, SDS-PAGE
128800
gel filtration, wild-type enzyme
129000
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mass of wild-type and mutant enzymes, BioSep-SEC-S4000 gel filtration column
150000
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SDS-PAGE, in accordance with the calculated value from the amino acid sequence, fusion enzyme including the hexahistidine domain from the vector
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
covalently linked by an intermolecular disulfide bond at residue C298
homodimer
2 * 64000, SDS-PAGE, 2 * 64240, electrospray ionization mass spectrometry, wild-type enzyme
monomer
1 * 66000, SDS-PAGE, His-tagged enzyme
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging-drop vapour-diffusion method. 1.1A resolution crystal structure of MTHase, structures of MTHase in complex with maltose and trehalose are obtained at 1.2 A and 1.5 A resolution
hanging drop vapor diffusion method at 4°C. The crystal structure of the E283Q mutant enzyme in complex with the substrate, maltotriosyltrehalose is determined to 2.6 A resolution. The structure with maltotriosyltrehalose indicates that the enzyme has at least five substrate binding subsites and that Glu283 is the catalytic acid, and Asp252 is the nucleophile that attacks the C1 carbon in the glycosidic linkage of maltotriosyltrehalose
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hanging-drop vapor diffusion method, space group determined by precession photography: P3(2)21 or P3(1)21, cell dimensions: a = b = 80.2 A and c 0 281.9 A
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3.5 - 10
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stable
722996
5 - 10
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4°C, 24 h, stable
26156
5.5 - 6
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4°C, 24 h, stable
26155
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40 - 90
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80% activity retained after 48 h pre-incubation at 70°C, activities after pre-incubation at set temperatures for 30 min: 90-100% at 40-70°C, less than 80% at 80°C, quickly abolished beyond 80°C
45
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60 min, stable up to
70
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retainis 80% of the activity for 48 h, activity is gradually abolished at above 85°C
80
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all enzyme activity retained after 2 hours at 80°C, wild-type, W218A, W218F, A259S, Y328F, F355Y, and R356K MTHases
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
active in the presence of Tris
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by Ni2+-NTA-agarose affinity chromatography
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Ni2+-NTA-agarose column chromatography, gel filtration
Q-Sepharose anion exchange column chromatography
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recombinant wild-type and mutant enzymes from Escherichia coli by anion exchange chromatography and gel filtration
wild-type enzyme purified by heat treatment, nucleic acid precipitation, and ion-exchange chromatography, 12fold. His-tagged MTHase partially purified from the cell-free extract directly by metal chelating chromatography, 14.7fold
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA construct pRBMhMTH expressed in Escherichia coli BL21(DE3)pLysS
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Escherichia coli BL21(DE3)pLysS, LB medium, vector pRSET-B
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Escherichia coli Rosetta (DE3) with mutated vector pET-15b-deltaH-treZ, Terrific broth, 37°C
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expressed in Escherichia coli BL21 (DE3) pLysS cells
expression in Escherichia coli
expression of wild-type and mutant enzymes in Escherichia coli
gene treZ, expression in fusion with maltooligosyltrehalose synthase in Oryzsa sativa subsp. japonica cv. Nakdong using Agrobacterium tumefaciens LBA4404 transfection method
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overexpression of the treZ gene and the treY/treZ synthetic operon
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pET-15b-deltaH-treZ vector expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL
the artifical gene is cloned into yeast expression vector pPICZalphaA, and then overexpressed in Pichia pastoris. The yeast strains possess a highly efficient and stably expression for enzyme production. The enzyme activity level from the secretion of recombinant protein reaches 800 U/mg protein
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
the expressions of maltooligosyltrehalose trehalohydrolase in the filaments of Nostoc flagelliforme are upregulated significantly under dehydration stress (exposition to air for 24 h), NaCl stress (exposition to 24-175 mM NaCl for 4 h), and high temperature-drought stress (exposition to air at various 4°C, 10°C, 25°C, 37°C, and 42°C, respectively, for 4 h)
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D156A
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kcat/Km for maltooligosyltrehalose is 3.8fold lower than wild-type value
D252E
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mutant shows 0.03% of enzymatic activity compared to the wild-type
D252S
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mutant shows 0.6% of enzymatic activity compared to the wild-type
E283Q
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mutant shows 0.04% of enzymatic activity compared to the wild-type
E450A
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kcat/Km for maltooligosyltrehalose is 1.2fold lower than wild-type value
H195A
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kcat/Km for maltooligosyltrehalose is 357fold lower than wild-type value
R447A
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kcat/Km for maltooligosyltrehalose is 6.5fold lower than wild-type value
Y155A
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kcat/Km for maltooligosyltrehalose is 160fold lower than wild-type value
Y155F
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kcat/Km for maltooligosyltrehalose is 33fold lower than wild-type value
A259S
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site-directed mutagenesis, the mutant shows increased selectivity ratios, i.e. ratios of the catalytic efficiencies for glucose formation to those for trehalose formation in the hydrolysis of maltooligosaccharides and maltooligosyltrehaloses, respectively
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D156A
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kcat/Km for maltooligosyltrehalose is 3.8fold lower than wild-type value
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D252E
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mutant shows 0.03% of enzymatic activity compared to the wild-type
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D252S
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mutant shows 0.6% of enzymatic activity compared to the wild-type
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D255A
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site-directed mutagenesis, the mutant shows highly reduced catalytic activity compared to the wild-type MTHase
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D380A
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site-directed mutagenesis, the mutant shows highly reduced catalytic activity compared to the wild-type MTHase
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E283Q
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mutant shows 0.04% of enzymatic activity compared to the wild-type
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E286A
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site-directed mutagenesis, the mutant shows highly reduced catalytic activity compared to the wild-type MTHase
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E450A
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kcat/Km for maltooligosyltrehalose is 1.2fold lower than wild-type value
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H195A
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kcat/Km for maltooligosyltrehalose is 357fold lower than wild-type value
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W218A
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site-directed mutagenesis, the mutant shows decreased selectivity ratios, i.e. ratios of the catalytic efficiencies for glucose formation to those for trehalose formation in the hydrolysis of maltooligosaccharides and maltooligosyltrehaloses, respectively
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Y155A
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kcat/Km for maltooligosyltrehalose is 160fold lower than wild-type value
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Y155F
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kcat/Km for maltooligosyltrehalose is 33fold lower than wild-type value
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additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
food industry
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trehalose has many potential applications in biotechnology and the food industry due to its protective effect against environmental stress. Raising trehalose productivity can be achieved through homologous overexpression of maltooligosyltrehalose synthase and maltooligosyltrehalose trehalohydrolase. Overexpression of the treY gene and the treY/treZ synthetic operon significantly increases maltooligosyltrehalose synthase activity, the rate-limiting step, and improves the specific productivity and the final titer of trehalose. Furthermore, a strong decrease is noted in glycogen accumulation. Expression of galU/treY and galU/treYZ synthetic operons show a partial recovery in the intracellular glycogen levels and a significant improvement in both intra- and extracellular trehalose content
synthesis
additional information
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trehalose is gaining applications as sweetener component, preservative or stabilizer of food, cosmetics, vaccines, medicines, cells, and organs
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