Information on EC 5.4.99.16 - maltose alpha-D-glucosyltransferase

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

EC NUMBER
COMMENTARY hide
5.4.99.16
-
RECOMMENDED NAME
GeneOntology No.
maltose alpha-D-glucosyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
maltose = alpha,alpha-trehalose
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycosyl bond isomerization
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Metabolic pathways
-
-
metabolism of disaccharids
-
-
Starch and sucrose metabolism
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trehalose biosynthesis IV
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SYSTEMATIC NAME
IUBMB Comments
maltose alpha-D-glucosylmutase
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CAS REGISTRY NUMBER
COMMENTARY hide
147994-22-7
protein (Saccharomyces cerevisiae clone pMB14 gene CIF reduced) /57-KDa trehalose synthase (Saccharomyces cerevisiae gene CIF1) /protein (Saccharomyces cerevisiae gene CIF1 reduced) /synthase, trehalose (Saccharomyces cerevisiae gene TPS1 subunit)
178604-93-8
synthase, trehalose (Pimelobacter strain R48 clone pBRM8 gene treS precursor reduced) /trehalose synthase (Pimelobacter strain R48 clone pBRM8 gene treS precursor reduced)
187285-67-2
synthase, trehalose (Thermus aquaticus strain ATCC33923 clone pBTM5)
211621-92-0
synthase, trehalose (Saccharomyces cerevisiae gene TSL1 subunit)
395644-91-4
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain SN223/29
UniProt
Manually annotated by BRENDA team
strain SN223/29
UniProt
Manually annotated by BRENDA team
Arthrobacter aurescens
strain CGMCC 1.1892
UniProt
Manually annotated by BRENDA team
Arthrobacter aurescens CGMCC 1.1892
strain CGMCC 1.1892
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
locus Dgeo-0537
UniProt
Manually annotated by BRENDA team
locus Dgeo-0537
UniProt
Manually annotated by BRENDA team
locus DR-2036
UniProt
Manually annotated by BRENDA team
-
UniProt
Manually annotated by BRENDA team
strain R48
-
-
Manually annotated by BRENDA team
strain R48
-
-
Manually annotated by BRENDA team
gene treS
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-
Manually annotated by BRENDA team
strain F1
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-
Manually annotated by BRENDA team
strain F1
-
-
Manually annotated by BRENDA team
strain HJ6
-
-
Manually annotated by BRENDA team
isolated from saline-alkali soil, gene treS
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-
Manually annotated by BRENDA team
additional information
no activity in Thermoproteus tenax strain Kra1
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-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
metabolism
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
alpha,alpha-trehalose
maltose
show the reaction diagram
high-maltose rice syrup
alpha,alpha-trehalose
show the reaction diagram
-
-
the highest alpha,alpha-trehalose yield (54.6%) can be obtained by 3.5 units/maltose (g) of trehalose synthase from the maltose syrup at 50C for 20-24 h
-
?
maltose
alpha,alpha-trehalose
show the reaction diagram
maltose
alpha,alpha-trehalose + D-glucose + maltose
show the reaction diagram
starch
?
show the reaction diagram
-
-
-
-
?
starch
alpha,alpha-trehalose
show the reaction diagram
-
substrate of recombinant fusion protein with N-terminal beta-amylase of Clostridium thermofluorogenes and C-terminal trehalose synthase or vice versa. Catalytic efficiency of fusion protein is higher than that of a mixture of individual enzymes
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-
?
sucrose
D-glucose + D-fructose + [alpha-D-glucopyranosyl-(1,1)-D-fructofuranose]
show the reaction diagram
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low activity on sucrose
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-
?
Sucrose
Trehalulose
show the reaction diagram
-
activity is very low compared to that with maltose
i.e. 1-O-alpha-D-glucopyranosyl-D-fructose
-
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
maltose
alpha,alpha-trehalose
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Cl-
enzyme binding structure of 4 Cl- ions, overview
Fe2+
-
114% activity at 1 mM
K+
-
10 mM, 22% increase in activity
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5-fluoro-alpha-D-glucosyl fluoride
-
behaves like a reversible inhibitor
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acarbose
competitively inhibited by the potent alpha-glucosidase inhibitor acarbose, acarbose-binding site structure, overview
beta-mercaptoethanol
complete inhibition at 1 mM
Cd2+
-
slight inhibition
D-gluconohydroximino-1,5-lactam
-
-
-
D-glucose
in the presence of 10 mM D-glucose, TreS shows a 3.6fold increase in Km and a nearly unchanged Vmax for maltose, implying that D-glucose is a competitive inhibitor of TreS
deoxynojirimycin
-
-
dithiotreitol
9% inhibition at 5 mM, 12% at 10 mM
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Fe3+
1 mM, 60% residual activity
Guanidine-HCl
-
complete inhibition at 1 M, 51% inhibition of aggregated enzyme
Na+
90% residual activity at 1 mM
sodium dodecylsulfate
-
1 mM, complete inhibition
Sucrose
-
competitive inhibition of the interconversion between maltose and trehalose
Urea
-
90% inhibition at 2 M, 1% inhibition of aggregated enzyme
validoxylamine
xylodeoxynojirimycin
-
-
-
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
dithiothreitol
1 mM, 110% of initial activity
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.087 - 154
alpha,alpha-trehalose
0.008 - 290.7
maltose
96.5
Sucrose
-
-
26 - 210.3
trehalose
additional information
additional information
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Michaelis-Menten steady-state kinetics with 5-fluoroglycosyl fluorides, alpha-aryl glucosides, and alpha-glucosyl fluoride
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
61 - 202
alpha,alpha-trehalose
19 - 227
maltose
187.6
trehalose
Picrophilus torridus
-
45C, pH 6.0
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.697 - 1.33
alpha,alpha-trehalose
1.16 - 2.7
maltose
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0025
casuarine
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pH 6.8, 37C
0.0021
D-gluconohydroximino-1,5-lactam
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pH 6.8, 37C
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3.76
D-glucose
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0.00025
deoxynojirimycin
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pH 6.8, 37C
0.14
isofagomine
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pH 6.8, 37C
0.000025
validoxylamine
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in 40 mM potassium phosphate buffer (pH 6.8), at 37C
0.3
xylodeoxynojirimycin
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pH 6.8, 37C
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.05
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cell-free extract, at pH 6.0 and 45C for 25 min using maltose as substrate
0.25
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after 5.3fold purification, at pH 6.0 and 45C for 25 min using maltose as substrate
0.96
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Mn2+-loaded, immobilized enzyme at pH 6.0 and 40C
1.17
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Fe3+-loaded, immobilized enzyme at pH 6.0 and 40C
1.55
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Cu2+-loaded, immobilized enzyme at pH 6.0 and 40C
2.84
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Ni2+-loaded, immobilized enzyme at pH 6.0 and 40C
3.3
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Zn2+-loaded, immobilized enzyme at pH 6.0 and 40C
3.98
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Co2+-loaded, immobilized enzyme at pH 6.0 and 40C
18.5
purified enzyme, at 37C
80
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45C, pH 6.0
133.5
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purified recombinant enzyme, pH 9.0, 45C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8 - 9
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-
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5 - 9.5
activity range, profile, overview
5 - 8
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free and spherical aggregated enzyme
5 - 7.5
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more than 80% of maximum activity within this range
5 - 7
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pH 5: about 25% of maximal activity, pH 7: about 55% of maximal activity
5 - 10
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over 50% of maximal activity within this range
6.5 - 9
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more than 80% of activity is maintained from pH 6.5 and 7.5, the activities of the enzyme markedly decrease in 50 mM Tris-HCl buffer from pH 7.0-9.0
6.8 - 8.8
more than 80% of maximum activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
53
-
free enzyme
60
recombinant wild-type enzyme and mutant TSTtMr
70
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spherical aggregated enzyme
70 - 80
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both free and chitosan-immobilized enzymes
70
-
temperature optimum for enzyme and for recombinant fusion protein with N-terminal beta-amylase of Clostridium thermofluorogenes and C-terminal trehalose synthase or vice versa
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 50
activity range, profile, overview
15 - 55
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over 30% of maximal activity within this range
25 - 55
more than 80% of maximum activity
40 - 70
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free enzyme
50 - 80
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spherical aggregated enzyme
50
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at 50C a higher relative activity, 88.8%, is observed for the immobilized enzyme, compared to 76.92% for the free enzyme
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.94
sequence calculation
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)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
61800
predicted from amino acid sequence
65000
His-tagged enzyme, SDS-PAGE
68000
Arthrobacter aurescens
SDS-PAGE
110000
SDS-PAGE
126900
recombinant His6-tagged enzyme, gel filtration
132300
gel filtration, recombinant protein
250000
-
gel filtration
390000
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
homohexamer
tetramer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
free enzyme and enzyme in complex with inhibitor acarbose, hanging drop vapor diffusion technique, mixing of 0.002 ml of 20 mg/ml protein in 40 mM sodium phosphate buffer, pH 6.0, with 0.002 ml of reservoir solution containing 0.1 M sodium cacodylate, pH 6.5, 0.2 M MgCl2, and 10-14% PEG 1000, 20C, 1 day to 1 week, X-ray diffraction structure determination and analysis at 1.84 A resolution, molecular replacement
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4
purified recombinant His6-tagged enzyme, 2 h, 30C, inactivation
726558
4 - 8
the enzyme can maintain very high activity (above 90%) at pH 4.0-8.0 for 5 h, below pH 5.0 the enzyme shows no activity
703606
5 - 8.5
-
-
715078
5 - 7
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both enzyme and recombinant fusion protein with N-terminal beta-amylase of Clostridium thermofluorogenes and C-terminal trehalose synthase or vice versa, stable
680294
5.5 - 7.5
Arthrobacter aurescens
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701844
5.5 - 8
-
wild-type
680304
5.5
purified recombinant His6-tagged enzyme, 2 h, 30C, 50% activity remaining
726558
5.5 - 9.5
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60C, 60 min, stable
3489
6.5 - 8
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the enzyme activity declines significantly at pH above 6.5. At pH 8.0 a complete loss in enzyme activity is observed for both the free and the immobilized enzyme. At pH5.0, the immobilized enzyme exhibits a relative activity, 82.13%, higher than the free enzyme, 50.57%.
716805
6.6 - 7.4
-
30 min, more than 80% residual activity
681153
7 - 9
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37C, 1 h, stable
3493
7.6
purified recombinant His6-tagged enzyme, 2 h, 45C and pH 7.6, stable
726558
9.5
purified recombinant His6-tagged enzyme, 2 h, 30C, 50% activity remaining
726558
10
purified recombinant His6-tagged enzyme, 2 h, 30C, inactivation
726558
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
-
pH 7.0, 60 min, stable up to
35
-
60 min, more than 80% residual activity
40 - 60
Arthrobacter aurescens
the enzyme remains stable up to 40C, and shows complete loss of activity at 60C
45
purified recombinant His6-tagged enzyme, 2 h, 45C and pH 7.6, stable up to
50
purified recombinant His6-tagged enzyme, 2 h, pH 7.6, inactivation; purified recombinant His6-tagged enzyme, 8 h, pH 7.6, 56% activity remaining
65
-
the wild type enzyme retains 40% of activity after 120 min at 65C
70
half-lives of purified wild-type enzyme: 48.5 min, purified mutant TSMrTt : 47.6 min, and purified mutant TSTtMr: over 480 min, mutant R392A: 42.2 min
70 - 80
-
the half-life of heat inactivation for free and chitosan-immobilized enzymes is 5.7 and 6.3 days at 70C, respectively. The free enzyme displays complete loss of activity after 8 days at 80C, whereas the chitosan-immobilized enzyme still retains about 25% of the initial activity
70
half-lives of purified wild-type enzyme: over 480 min, purified mutant TSMrTt : 47.6 min, and purified mutant TSTtMr: over 480 min
100
purified enzyme, 10 min, inactivation
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
the enzyme immobilized with highly porous cross-linked polystyrene divinylbezene-based metal chelator maintains a residual activity of ca. 80% after 24 cycles
-
when tested in batch reaction, the immobilized enzyme retains its relative activity of 53% after 30 reuses of reaction within 12 days
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4C, 150 days, retains 82% of its initial activity
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
HiTrap Chelating HP column chromatography
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Ni column chromatography
-
Ni column chromatography, DEAE-cellulose column chromatography, hydroxyapatite column chromatography, aminohexylagarose column chromatography, phenyl-Sepharose column chromatography, and Sephacryl S-200 gel filtration
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Ni-NTA column chromatography
Ni-NTAcolumn chromatography
recombinant C-terminally His6-tagged enzyme from Escherichia coli strain (DE3) Rosetta pLysS by cobalt affinity chromatography and ultrafiltration
recombinant enzyme
recombinant enzyme from Escherichia coli strain BL21(DE3)
-
recombinant fusion protein with N-terminal beta-amylase of Clostridium thermofluorogenes and C-terminal trehalose synthase and vice versa having both activities
-
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
-
recombinant His6-tagged wild-type and chimeric mutant enzymes from Escherichia coli strain Rosetta-gami (DE3) by nickel affinity chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
construction of fusion genes from beta-amylase gene of Clostridium thermofluorogenes and trehalose synthase and overexpression in Escherichia coli
-
expressed in Escherichia coli
expressed in Escherichia coli BL21(DE3) cells
expressed in Escherichia coli BL21(DE3)pLysS cells
expressed in Escherichia coli MC1061 cells
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expressed in Escherichia coli Rosetta gami (DE3) cells
expressed in Escherichia coli Rosetta-gami B cells
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expression in Escherichia coli
gene treS, DNA and amino acid sequence determination and analysis, sequence comparisons, and phylogenetic tree, recombinant overexression in Escherichia coli strain BL21(DE3), cloning in Escherichia coli strain D5alpha
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gene treS, overexpression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
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gene treS, sequence comparison, expression of His6-tagged wild-type enzyme and chimeric mutant in Escherichia coli strain Rosetta-gami (DE3), subcloning in Escherichia coli strain DH5alpha
gene treS, sequence comparison, expression of His6-tagged wild-type enzyme and the chimeric mutant in Escherichia coli strain Rosetta-gami (DE3), subcloning in Escherichia coli strain DH5alpha
locus DR-2036, DNA and amino acid sequence determination and analysis, sequence comparisons, expression of C-terminally His6-tagged enzyme in Escherichia coli strain (DE3) Rosetta pLysS from pET30Ek/LIC vector
recombinant expression in Escherichia coli
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recombinant expression in two plasmid-bearing Escherichia coli strains BL21(DE3)/pET23a(+)-PTTS and Rosetta(DE3)/pET23a(+)-PTTS, optimized expression in Escherichia coli strains BT04-BT06 with deleted genes treA or treF or both, which encode for trehalases, overview; strategy for stable and high-level expression of recombinant trehalose synthase in Escherichia coli
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
expression induction with 0.4 mM IPTG at 25C for 5 h is the best condition for the expression of soluble TreS, higher temperature or IPTG concentrations lead to a higher rate of inclusion bodies, whereas a temperature below 20C and lower IPTG concentrations decreases the total amount of TreS
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
R392A
site-directed mutagenesis, the mutant shows a sharp decrease in activity compared to the wild-type enzyme
R392F
site-directed mutagenesis, the mutation leads to a complete loss in activity
R392A
-
site-directed mutagenesis, the mutant shows a sharp decrease in activity compared to the wild-type enzyme
-
R392F
-
site-directed mutagenesis, the mutation leads to a complete loss in activity
-
A255P
-
2.0% activity compared to the wild type enzyme
D411P
-
3.2% activity compared to the wild type enzyme
D41P
-
99.6% activity compared to the wild type enzyme
E469P
-
50% activity compared to the wild type enzyme
I121P
-
21.3% activity compared to the wild type enzyme
I385P
-
5.1% activity compared to the wild type enzyme
K332P
-
96.7% activity compared to the wild type enzyme
N503P
-
the mutant shows about 39% higher relative activity than that of the wild type at 65C for 120 min. The trehalose yield of the mutant is 1.3fold higher than that of the wild type with sweet potato starch as substrate at 50C for 4 h
R523P
-
90.8% activity compared to the wild type enzyme
S439P
-
20.8% activity compared to the wild type enzyme
D112A
-
site-directed mutagenesis, inactive mutant
D294A
-
site-directed mutagenesis, inactive mutant
D403A
-
site-directed mutagenesis, inactive mutant
E338A
-
site-directed mutagenesis, inactive mutant
F115A
-
site-directed mutagenesis, inactive mutant
F223A
-
site-directed mutagenesis, the mutation only modestly affects the enzymatic activity
F255A
-
site-directed mutagenesis, inactive mutant
Q259A
-
site-directed mutagenesis, the mutant shows 70% reduced activity compared to the wild-type enzyme
R535A
-
site-directed mutagenesis, the mutation only modestly affects the enzymatic activity
D112A
-
site-directed mutagenesis, inactive mutant
-
D403A
-
site-directed mutagenesis, inactive mutant
-
E338A
-
site-directed mutagenesis, inactive mutant
-
F115A
-
site-directed mutagenesis, inactive mutant
-
F223A
-
site-directed mutagenesis, the mutation only modestly affects the enzymatic activity
-
synthesis
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
-
trehalose synthase from Pyrococcus horikoshii can be applied to the sugar nucleotide cycling process for the synthesis of functional alpha-galactosyl oligosaccharides, alpha-galactose epitopes and globotriose, using the effective regeneration of UDP-Gal. The alpha-Gal epitope III with galactulose acceptor shows the most inhibitory activity of anti-adhesion of Escherichia coli cells to human Caco-2 cells. The alpha-galactosyl oligosaccharides may be alternative anti-adhesion molecules that overcome antibiotic resistance
synthesis
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