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Information on EC 2.4.1.10 - levansucrase and Organism(s) Bacillus subtilis and UniProt Accession P05655
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2.4.1.10 levansucraseIUBMB Comments
Some other sugars can act as D-fructosyl acceptors.
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Word Map
- 2.4.1.10
-
fructosylation
- fructooligosaccharides
-
transfructosylation
- mobilis
-
zymomonas
- exopolysaccharide
- erwinia
- raffinose
-
prebiotics
- inulin
-
levan-type
- diazotrophicus
- fructosyltransferases
- leuconostoc
- reuteri
- inulosucrase
-
gluconacetobacter
- mesenteroides
- dextransucrase
-
sucrose:sucrose
- synthesis
-
gluconobacter
- 6-kestose
- aquatilis
-
rahnella
-
exocellular
- invertases
-
hpaec-pad
-
sourdough
-
amylovoran
-
antitermination
- oxydans
- food industry
- agriculture
The taxonomic range for the selected organisms is: Bacillus subtilis
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Synonyms
levansucrase, 6-sft, sucrose:fructan 6-fructosyltransferase, fructansucrase, sucrose 6-fructosyltransferase, lsc-3, t2-ls, t1-ls, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
beta-2,6-fructan:D-glucose 1-fructosyltransferase
-
-
-
-
beta-2,6-fructosyltransferase
-
-
-
-
fructosyltransferase, sucrose 6-
-
-
-
-
sucrose 6-fructosyltransferase
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
sucrose + [6)-beta-D-fructofuranosyl-(2->]n alpha-D-glucopyranoside = D-glucose + [6)-beta-D-fructofuranosyl-(2->]n+1 alpha-D-glucopyranoside
sucrose + [6)-beta-D-fructofuranosyl-(2->]n alpha-D-glucopyranoside = D-glucose + [6)-beta-D-fructofuranosyl-(2->]n+1 alpha-D-glucopyranoside
mechanism
-
sucrose + [6)-beta-D-fructofuranosyl-(2->]n alpha-D-glucopyranoside = D-glucose + [6)-beta-D-fructofuranosyl-(2->]n+1 alpha-D-glucopyranoside
ping-pong mechanism
-
sucrose + [6)-beta-D-fructofuranosyl-(2->]n alpha-D-glucopyranoside = D-glucose + [6)-beta-D-fructofuranosyl-(2->]n+1 alpha-D-glucopyranoside
reaction kinetics are dependent on sucrose concentration
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hexosyl group transfer
hexosyl group transfer
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
sucrose:[6)-beta-D-fructofuranosyl-(2->]n alpha-D-glucopyranoside 6-beta-D-fructosyltransferase
Some other sugars can act as D-fructosyl acceptors.
CAS REGISTRY NUMBER
COMMENTARY
9030-17-5
-
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
sucrose + (2,6-beta-D-fructosyl)n
D-glucose + (2,6-beta-D-fructosyl)n+1
-
-
-
?
sucrose + cellobiose
beta-D-glucopyranosyl-(1,4)-alpha-D-glucopyranosyl-(1,2)-beta-D-fructofuranoside + D-glucose
-
-
-
?
sucrose + D-fucose
beta-D-fructofuranosyl-alpha-D-fucopyranoside + D-glucose
-
-
-
?
sucrose + D-galactose
beta-D-fructofuranosyl-alpha-D-galactopyranoside + D-glucose
-
-
-
?
sucrose + D-xylose
beta-D-fructofuranosyl-alpha-D-xylopyranoside + D-glucose
-
-
-
?
sucrose + D-xylose
beta-D-fructofuranosyl-beta-D-xylopyranoside + D-glucose
-
-
-
?
sucrose + L-galactose
beta-D-fructofuranosyl-beta-L-galactopyranoside + D-glucose
-
-
-
?
sucrose + L-glucose
beta-D-fructofuranosyl-beta-L-glucopyranoside + D-glucose
-
-
-
?
sucrose + L-xylose
beta-D-fructofuranosyl-beta-L-xylopyranoside + D-glucose
-
-
-
?
sucrose + lactose
beta-D-galactopyranosyl-(1,4)-alpha-D-glucopyranosyl-(1,2)-beta-D-fructofuranoside + D-glucose
-
-
-
?
sucrose + levan
?
mutant enzymes H243L and S164A synthesize either high or low levan molecular weight
-
-
?
sucrose + [beta-D-fructofuranosyl-(2->6)]n alpha-D-glucopyranoside
D-glucose + [beta-D-fructofuranosyl-(2->6)]n+1 alpha-D-glucopyranoside
-
-
-
?
2 sucrose
D-glucose + beta-D-fructofuranosyl-(2,6)-beta-D-fructofuranosyl-(2,1)-alpha-D-glucopyranoside
-
-
-
-
?
raffinose + (2,6-beta-D-fructosyl)n
galactose + (2,6-beta-D-fructosyl)n+1
-
-
the levan synthesized on raffinose contains one mol of galactosylglucose per mol as one of the 2 terminal glycosyl moieties
?
sucrose + (2,6-beta-D-fructosyl)n
D-glucose + (2,6-beta-D-fructosyl)n+1
-
reduction of glucose by yeasts (Candida cacaoi DSM 2226), introduced into a dialysing membrane, situated in the reaction medium, and the presence of Mn2+ results in an increase of levan synthesis efficiency to 64%
-
-
?
sucrose + D-xylose
D-glucose + beta-D-fructofuranosyl-(2,1)-alpha-D-xylopyranoside
-
-
NMR product analysis
-
?
sucrose + lactose
lactosucrose
-
Bacillus subtilis is the most effective producer of lactofructose
-
-
?
sucrose + sucrose
6-kestose + levanbiose + blastose + 1-kestose + ?
-
-
-
-
?
sucrose + [beta-D-fructofuranosyl-(2->6)]n alpha-D-glucopyranoside
D-glucose + [beta-D-fructofuranosyl-(2->6)]n+1 alpha-D-glucopyranoside
-
-
-
-
?
sucrose + (2,6-beta-D-fructosyl)n
glucose + (2,6-beta-D-fructosyl)n+1
-
-
-
?
sucrose + (2,6-beta-D-fructosyl)n
glucose + (2,6-beta-D-fructosyl)n+1
-
-
-
?
sucrose + (2,6-beta-D-fructosyl)n
glucose + (2,6-beta-D-fructosyl)n+1
-
-
-
?
sucrose + (2,6-beta-D-fructosyl)n
glucose + (2,6-beta-D-fructosyl)n+1
-
-
-
?
sucrose + (2,6-beta-D-fructosyl)n
glucose + (2,6-beta-D-fructosyl)n+1
-
-
-
?
sucrose + (2,6-beta-D-fructosyl)n
glucose + (2,6-beta-D-fructosyl)n+1
-
-
-
?
sucrose + (2,6-beta-D-fructosyl)n
glucose + (2,6-beta-D-fructosyl)n+1
-
-
-
?
sucrose + (2,6-beta-D-fructosyl)n
glucose + (2,6-beta-D-fructosyl)n+1
-
-
-
?
sucrose + (2,6-beta-D-fructosyl)n
glucose + (2,6-beta-D-fructosyl)n+1
-
-
-
r
sucrose + (2,6-beta-D-fructosyl)n
glucose + (2,6-beta-D-fructosyl)n+1
-
-
-
r
sucrose + (2,6-beta-D-fructosyl)n
glucose + (2,6-beta-D-fructosyl)n+1
-
-
-
r
sucrose + (2,6-beta-D-fructosyl)n
glucose + (2,6-beta-D-fructosyl)n+1
-
polymerase and hydrolase activity can be separately modulated by site-directed mutagenesis
-
r
sucrose + (2,6-beta-D-fructosyl)n
glucose + (2,6-beta-D-fructosyl)n+1
-
reaction kinetics are dependent on sucrose concentration
-
?
sucrose + (2,6-beta-D-fructosyl)n
glucose + (2,6-beta-D-fructosyl)n+1
-
activity is affected by sacU mutation
-
?
additional information
?
-
mutant enzymes Y429N and R433A no longer produce levan but exclusively oligosaccharides
-
-
?
additional information
?
-
-
mutant enzymes Y429N and R433A no longer produce levan but exclusively oligosaccharides
-
-
?
additional information
?
-
levan synthesis from sucrose, structure analysis by NMR spectrometry, overview. Levan size analysis by gel filtration. As sucrose concentration increases, the average molecular weight of the low dextran distribution decreases, at 4°C compared to 37°C, the low molecular weight distribution region increases in size at any given substrate concentration, and a maximum of 20.64 kDa is obtained at 200 g/l
-
-
?
additional information
?
-
measurement of sucrose hydrolysis, releasing D-glucose and D-fructose. As the reaction proceeds, SacB becomes less hydrolytic while transferase becomes preponderant to reach up to 80% at the end of the reaction
-
-
?
additional information
?
-
-
levansucrase catalyzes the synthesis of levan from sucrose, but it may also transfer the fructosyl moiety from sucrose to acceptor molecules included in the reaction medium
-
-
?
additional information
?
-
-
levansucrase catalyzes the synthesis of levan from sucrose, but it may also transfer the fructosyl moiety from sucrose to acceptor molecules. The wild-type enzyme synthesizes levan with molecular weight bimodal distribution, while the mutants R360K and Y429N synthesize only oligosaccharides, substrate specificities, overview
-
-
?
additional information
?
-
-
levansucrase catalyzes three distinct reactions depending on the fructosyl acceptor molecule, including polymerization, transfructosylation, and hydrolysis. Wide substrate specificity of levansucrase toward monosaccharides, disaccharides, and aromatic and alkyl alcohols, producing diverse sucrose analogues, hetero-oligosaccharides (especially lactosucrose), and fructosides, overview
-
-
?
additional information
?
-
-
levan synthesis, the first reaction of levan synthesis is formation of 6-kestose from two molecules of sucrose, one acting as a fructosyl donor and the other as an acceptor. 6-Kestose is further extended through numerous transfructosylation reactions
-
-
?
additional information
?
-
-
levansucrase catalyzes three distinct reactions depending on the fructosyl acceptor molecule: polymerization (using the growing fructan chain as an acceptor), transfructosylation (using monosaccharides, disaccharides, or oligosaccharides as acceptors), and hydrolysis (using water as an acceptor), overview
-
-
?
NATURAL SUBSTRATE
NATURAL 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
sucrose + (2,6-beta-D-fructosyl)n
D-glucose + (2,6-beta-D-fructosyl)n+1
-
-
-
?
sucrose + [beta-D-fructofuranosyl-(2->6)]n alpha-D-glucopyranoside
D-glucose + [beta-D-fructofuranosyl-(2->6)]n+1 alpha-D-glucopyranoside
-
-
-
?
2 sucrose
D-glucose + beta-D-fructofuranosyl-(2,6)-beta-D-fructofuranosyl-(2,1)-alpha-D-glucopyranoside
-
-
-
-
?
sucrose + (2,6-beta-D-fructosyl)n
glucose + (2,6-beta-D-fructosyl)n+1
-
activity is affected by sacU mutation
-
?
sucrose + [beta-D-fructofuranosyl-(2->6)]n alpha-D-glucopyranoside
D-glucose + [beta-D-fructofuranosyl-(2->6)]n+1 alpha-D-glucopyranoside
-
-
-
-
?
additional information
?
-
levan synthesis from sucrose, structure analysis by NMR spectrometry, overview. Levan size analysis by gel filtration. As sucrose concentration increases, the average molecular weight of the low dextran distribution decreases, at 4°C compared to 37°C, the low molecular weight distribution region increases in size at any given substrate concentration, and a maximum of 20.64 kDa is obtained at 200 g/l
-
-
?
additional information
?
-
-
levansucrase catalyzes the synthesis of levan from sucrose, but it may also transfer the fructosyl moiety from sucrose to acceptor molecules included in the reaction medium
-
-
?
additional information
?
-
-
levansucrase catalyzes three distinct reactions depending on the fructosyl acceptor molecule, including polymerization, transfructosylation, and hydrolysis. Wide substrate specificity of levansucrase toward monosaccharides, disaccharides, and aromatic and alkyl alcohols, producing diverse sucrose analogues, hetero-oligosaccharides (especially lactosucrose), and fructosides, overview
-
-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Ca2+
-
dependent on for tertiary structure, can partially be replaced by Sr2+, not Mg2+, Sr2+, Ba2+, and Mn2+
Fe3+
-
dependent on for tertiary structure, can partially be replaced by Sr2+, not Mg2+, Ba2+, and Mn2+
Mn2+
-
The transferase activity of levansucrase in the reaction mixture supplemented with Mn2+ is 100% higher than the enzyme activity in medium without metal ions, the hydrolytic activity of the levansucrase is lowered by 80%
MnCl2
-
22% activation of the immobilized enzyme at 0.1 mM, 30% activation at 0.1 mM of the free enzyme
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
AgCl
-
immobilized enzyme: 38% inhibition at 0.1 mM, 67% inhibition at 1 mM, complete inhibition at 10 mM. Free enzyme: 49% inhibition at 0.1 mM, 71% inhibition at 1 mM, 95% inhibition at 10 mM
CuSO4
-
immobilized enzyme: 25% inhibition at 0.1 mM, 37% inhibition at 1 mM, 65% inhibition at 10 mM. Free enzyme: 30% inhibition at 0.1 mM, 42% inhibition at 1 mM, 79% inhibition at 10 mM
FeCl3
-
immobilized enzyme: 8% inhibition at 0.1 mM, 21% inhibition at 1 mM, 35% inhibition at 10 mM. Free enzyme: 13% inhibition at 0.1 mM, 29% inhibition at 1 mM, 40% inhibition at 10 mM
HgCl2
-
immobilized enzyme: 54% inhibition at 0.1 mM, 61% inhibition at 1 mM, 79% inhibition at 10 mM. Free enzyme: 61% inhibition at 0.1 mM, 75% inhibition at 1 mM, 91% inhibition at 10 mM
MgSO4
-
11% inhibition of the immobilized enzyme at 10 mM, 21% inhibition of the free enzyme at 10 mM
NaCl
-
immobilized enzyme: 20% inhibition at 0.1 mM, 29% inhibition at 1 mM, 50% inhibition at 10 mM. Free enzyme: 26% inhibition at 0.1 mM, 40% inhibition at 1 mM, 55% inhibition at 10 mM
SDS
-
immobilized enzyme: 11% inhibition at 0.1 mM, 21% inhibition at 1 mM, 48% inhibition at 10 mM. Free enzyme: 14% inhibition at 0.1 mM, 27% inhibition at 1 mM, 52% inhibition at 10 mM
D-glucose
-
D-glucose acts as an inhibitor of the transfructosylation reaction, the decrease of glucose concentration by yeast in the medium by 16-19% results in the increased degree of levan polymerisation (by 6 to 9%) and efficiency of levan synthesis (by 9 to 11%)
high ionic strength
-
suppresses transfer of fructosyl residues, especially in synthesis of high molecular weight levan, suppresses hydrolysis of levan, effect occurs with almost the same degree in hydrolysis of both low and high molecular weight levans
-
high ionic strength
-
under conditions of high ionic strength, only levan with an average degree of polymerization of 120 is synthesized with a reasonable yield
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
levan
-
accelerates the rate of polymerization of levan, effective only under conditions of low ionic strength
levan
1.4fold increase in Vmax of wild-type in the presence of 15 g/l of levan is observed
levan
3.7fold increase in Vmax of S164A in the presence of 15 g/l of levan is observed
additional information
levan has no effect on Y429N, a mutant that has lost the fructan-synthesizing activity
-
additional information
-
levan has no effect on Y429N, a mutant that has lost the fructan-synthesizing activity
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2.5
sucrose
mutant enzyme S164A, 0.5 U/ml of enzyme activity at 37°C and pH 6.0
7.4
sucrose
mutant enzyme I341V, 0.5 U/ml of enzyme activity at 37°C and pH 6.0
10.5
sucrose
mutant enzyme H243L, 0.5 U/ml of enzyme activity at 37°C and pH 6.0
29.3
sucrose
mutant enzyme R433A, 0.5 U/ml of enzyme activity at 37°C and pH 6.0
30
sucrose
mutant enzyme R369K, 0.5 U/ml of enzyme activity at 37°C and pH 6.0
154
sucrose
mutant enzyme R360S, 0.5 U/ml of enzyme activity at 37°C and pH 6.0
297.3
sucrose
mutant enzyme G361F, 0.5 U/ml of enzyme activity at 37°C and pH 6.0
319.4
sucrose
mutant enzyme Y429N, 0.5 U/ml of enzyme activity at 37°C and pH 6.0
additional information
additional information
Michaelis-Menten kinetics
-
additional information
additional information
mutant enzyme S164K is inactive
-
additional information
additional information
-
mutant enzyme S164K is inactive
-
additional information
additional information
mutants A344P and F414W have the same behavior like the wild-type
-
additional information
additional information
-
mutants A344P and F414W have the same behavior like the wild-type
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
108
sucrose
-
levansucrase-endolevanase fusion enzyme, pH and temperature not specified in the publication
164
sucrose
-
levansucrase, pH and temperature not specified in the publication
additional information
additional information
mutants A344P and F414W have the same behavior like the wild-type
-
additional information
additional information
-
mutants A344P and F414W have the same behavior like the wild-type
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
4.2 - 9.2
-
26% and 18% decrease in levansucrase activity is noticed at pH 4.2 and 9.2, respectively
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0
-
at 0°C the enzyme is more productive over a peroid of 10 h, but the enzyme activity is higher at 37°C
30
37
-
at 0°C the enzyme is more productive over a peroid of 10 h, but the enzyme activity is higher at 37°C
additional information
-
at 0°C the enzyme is more productive over a period of 10 h, but the enzyme activity is higher at 37°C
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
-
-
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
metabolism
-
the enzyme is a key biocatalyst in the synthesis of levan and levan-type fructooligosaccharides
physiological function
physiological function
-
in addition to fructooligosaccharides, levansucrases produce polymeric levan, degree of polymerization of which can be very high
physiological function
-
transfructosylation reaction is an important method for producing lactosucrose, in which sucrose and lactose serve as the fructosyl donor and acceptor, respectively
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
50000 - 52000
-
extracellular and membrane-associated enzyme forms, SDS-PAGE, gel filtration
additional information
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystal structures of the inactive single site mutants D86A, D247A and E342A of Bacillus subtilis levansucrase and from the sucrose- and raffinose-bound complexes of the E342A mutant, minimal structural consequences of the D247A, D86A mutations
mutant enzyme S164A, 12 days of micro-dialysis of the purified protein (8 g/l) against deionized water, PDB accession code 2VDT
3 different crystal types I, II and III, 3 methods for crystallization of the protein from NaH2PO4, 0.2 M, pH 4.3, structure analysis, overview
-
crystallization by precipitation with ethanol and ammonium sulfate and dialysis against distilled water or from 20 mM phosphate buffer, pH 5.0, + 0.5 mM ammonium sulfate and 5% v/v 2-methyl-2,4-pentanediol, multiple isomorphous replacements with 3 heavy atom derivatives for x-ray tertiary structure analysis
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D247A
D86A
E342A
G361F
site-directed mutagenesis, less stable than the wild-type, synthesizes mainly oligosaccharides, still catalyzes the synthesis of low amounts of polymer, pH-optimum 6, affinity for sucrose is reduced, shift of reaction specificity (hydrolysis/transfer)
H243L
I341V
N242H
the mutant shows 31fold decrease in catalytic efficiency compared to the wild type enzyme
R331K
mutant enzyme loses the ability to synthesize levan and is only able to produce the trisaccharide kestose
R331L
mutant enzyme loses the ability to synthesize levan and is only able to produce the trisaccharide kestose
R331S
mutant enzyme loses the ability to synthesize levan and is only able to produce the trisaccharide kestose
R360H
the mutant shows 5fold decrease in catalytic efficiency compared to the wild type enzyme. The mutant still can produce levan, but has 60% less transfructosylation activity
R360K
R360S
R433A
site-directed mutagenesis, synthesizes only oligosaccharides, pH-optimum 6-7, affinity for sucrose is reduced, shift of reaction specificity (hydrolysis/transfer)
S164A
Y429
site-directed mutagenesis, Y429 plays an indirect but important role in catalysis and acceptor specificity, as this is a key residue coordinating the sucrose position in the levansucrase binding pocket through a complex water network
Y429N
H331R
-
invers directed mutation of natural point mutation R331H to H331R restores the wild-type enzyme properties
R331H
-
natural point mutation, low polymerase activity, invers mutation H331R restores the wild-type enzyme properties
R331K
-
site-directed mutagenesis, loss of ability to perform the whole production of levan from sucrose, only capable to perform the first reaction step, the formation of the trisaccharide kestose, higher kcat than the wild-type for sucrose hydrolysis
R331L
-
site-directed mutagenesis, loss of ability to perform the whole production of levan from sucrose, only capable to perform the first reaction step, the formation of the trisaccharide kestose, reduced fructosyl-enzyme intermediate formation
R331S
-
site-directed mutagenesis, loss of ability to perform the whole production of levan from sucrose, only capable to perform the first reaction step, the formation of the trisaccharide kestose, reduced fructosyl-enzyme intermediate formation
R360K
-
the immobilized mutant enzyme shows increased activity and is improved for fructosyl-xyloside synthesis compared to the wild-type enzyme
Y429N
-
the immobilized mutant enzyme shows increased activity and is improved for fructosyl-xyloside synthesis compared to the wild-type enzyme
additional information
D247A
single site mutant, substitutions have little effect on the active site geometry, catalytically inactive
D86A
single site mutant, substitutions have little effect on the active site geometry, catalytically inactive
E342A
single site mutant, catalytically inactive, the E342A mutant reveals conformational flexibility of functionally relevant side chains in the vicinity of the general acid Glu342, including Arg360, a residue required for levan polymerisation
H243L
site-directed mutagenesis, less stable than the wild-type, pH-optimum 6, shift of reaction specificity (hydrolysis/transfer)
H243L
the mutant shows 2fold decrease in catalytic efficiency compared to the wild type enzyme
R360K
site-directed mutagenesis, pH-optimum 6, affinity for sucrose is reduced, shift of reaction specificity (hydrolysis/transfer)
R360K
the mutant shows 1-4fold decrease in catalytic efficiency compared to the wild type enzyme
R360S
site-directed mutagenesis, pH-optimum 6, decrease in activity, affinity for sucrose is reduced, shift of reaction specificity (hydrolysis/transfer)
R360S
the mutant shows 98-226fold decrease in catalytic efficiency compared to the wild type enzyme
S164A
site-directed mutagenesis, S164A is catalytically important, as it maintains the nucleophile in an appropriate position regarding the sucrose molecule. S164A results in a 12fold more stable and less hydrolytic enzyme than the wild-type, with a half-life of 628.0 (+51.0) min, pH-optimum 6, decrease in activity, slightly higher affinity for sucrose
S164A
the mutant shows 8fold decrease in catalytic efficiency compared to the wild type enzyme
Y429N
site-directed mutagenesis, synthesizes only oligosaccharides, pH-optimum 5-6, decrease in activity, affinity for sucrose is reduced, shift of reaction specificity (hydrolysis/transfer)
Y429N
the mutant shows 1015fold decrease in catalytic efficiency compared to the wild type enzyme
additional information
-
covalent immobilization of recombinant purified wild-type and mutant enzymes by glutaraldehyde to give cross-linked enzyme aggregates, CLEAs. Usage of 60% ammonium sulfate, 0.2% glutaraldehyde and 4 mg protein/ml. All CLEAs show higher thermal stability than corresponding soluble enzymes, but in the long term, the operational stability is affected by levan synthesis
additional information
-
partially purified enzyme immobilization on Ca-alginate beads, or entrapment in agar and agarose, or crosslinking on chitosan by glutaraldehyde, activity of the immobilized enzyme is relatively lower than the activity of the free enzyme. The immobilized levansucrase shows a slight increase in activity compared with the free enzyme above 35°C. The activation energies are 6.62 and 9.27 kcal/mol for free and immobilized enzyme, respectively. The immobilized enzyme shows an increased thermal stability and reduced deactivation energy, as well as increased pH stability at acidic pH
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
4.2 - 9.2
-
the enzyme is stable at pH 5.2-9.2. At pH 4.2 the activity decreases gradually to 85% after 2 h
757108
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
40
35 - 50
-
the enzyme is stable at temperatures (35°C and 37°C) in all incubation periods. A decrease in the enzyme activity is noticed at 40°C and 45°C after 2 h 12% and 19%, respectively. At 50°C there is about 40% loss in activity after 2 h
50
additional information
40
t1/2: 16.0 min mutant enzym R433A, stability is determined following the loss of activity after incubation of 1 mg/ml of protein
40
t1/2: 17.21 min mutant enzym R360S, stability is determined following the loss of activity after incubation of 1 mg/ml of protein
40
t1/2: 31.4 min mutant enzym I341V, stability is determined following the loss of activity after incubation of 1 mg/ml of protein
40
t1/2: 42.11 min mutant enzym R360K, stability is determined following the loss of activity after incubation of 1 mg/ml of protein
40
t1/2: 47.04 min mutant enzym Y429N, stability is determined following the loss of activity after incubation of 1 mg/ml of protein
40
t1/2: 52.0 min wild-type, stability is determined following the loss of activity after incubation of 1 mg/ml of protein
40
t1/2: 628.0 min mutant enzym S164A, stability is determined following the loss of activity after incubation of 1 mg/ml of protein
40
t1/2: 7.9 min mutant enzym H243L, stability is determined following the loss of activity after incubation of 1 mg/ml of protein
40
t1/2: less than 1min mutant enzym G361F, stability is determined following the loss of activity after incubation of 1 mg/ml of protein
50
-
although the thermal stability of the immobilised levansucrase is significantly improved in comparison to the free form, the immobilised enzyme at 50°C loses the activity faster than the free form, inactivation kinetics, overview
additional information
mutants A344P and F414W have the same behavior like the wild-type
additional information
-
mutants A344P and F414W have the same behavior like the wild-type
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
enzyme possesses a tertiary structure wholly dependent on the presence of Fe3+ or Ca2+
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme from Escherichia coli strain BL21(DE3) by cation exchange chromatography
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
gene sacB from mutant strain QB252, DNA sequencing reveals an amino acid exchange R331H, expression of mutants in Escherichia coli strain XL-1 B
-
gene sacB, overexpression in Bacillus subtilis mutant degU32(hy), deficient in secA translocase, enzyme protein content is directly correlated with translocase content, decrease of translocase leads to accumulation of unprocessed levansucrase precursor and vice versa
-
insertion of Shine Dalgarno like sequences in the 5'-untranslated sacR region controlling the expression of sacB. Depending on the number of stabilizing sequences inserted and the position of these sequences with respect to the translation start codon, it is observed that the mRNA stability and the final protein production could be increased or decreased
-
RENATURED/Commentary
ORGANISM
UNIPROT
LITERATURE
at 36-41°C, 0.1 mM EDTA, pH 7.0, unfolding of the enzyme, refolds at 20°C
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
food industry
-
the enzyme is interesting in food and pharmaceutical industries for synthesis of diverse sucrose analogues, hetero-oligosaccharides (especially lactosucrose), and interesting fructosides from a wide range of substrates, i.e. monosaccharides, disaccharides, and aromatic and alkyl alcohols
synthesis
synthesis
-
D-glucose acts as an inhibitor of the transfructosylation reaction, Candida cacaoi selectively removes glucose from the reaction medium, the decrease of glucose concentration by yeast in the medium by 16-19% results in the increased degree of levan polymerisation (by 6 to 9%) and efficiency of levan synthesis (by 9 to 11%)
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Chambert, R.; Petit-Glatron, M.F.
Reversible thermal unfolding of Bacillus subtilis levansucrase is modulated by Fe3+ and Ca2+
FEBS Lett.
275
61-64
1990
Bacillus subtilis, Bacillus subtilis QB112
Chambert, R.; Treboul, G.; Dedonder, R.
Kinetic studies of levansucrase of Bacillus subtilis
Eur. J. Biochem.
41
285-300
1974
Bacillus subtilis
Berthou, J.; Laurent, A.; Lebrun, E.; van Rapenbusch, R.
Letter: Crystallography of Bacillus subtilis levansucrase
J. Mol. Biol.
82
111-113
1974
Bacillus subtilis, Bacillus subtilis BS5C4
Yamamoto, S.; Iizuka, M.; Tanaka, T.; Yamamoto, T.
The mode of synthesis of levan by Bacillus subtilis levansucrase
Agric. Biol. Chem.
49
343-349
1985
Bacillus subtilis
-
Fouet, A.; Arnaud, M.; Klier, A.; Rapoport, G.
Characterization of the precursor form of the exocellular levansucrase from Bacillus subtilis
Biochem. Biophys. Res. Commun.
119
795-800
1984
Bacillus subtilis
LeBrun, E.; van Rapenbusch, R.
The structure of Bacillus subtilis levansucrase at 3.8 A resolution
J. Biol. Chem.
255
12034-12036
1980
Bacillus subtilis
Tanaka, T.; Oi, S.; Iizuka, M.; Yamamoto, T.
Levansucrase of Bacillus subtilis
Agric. Biol. Chem.
42
323-326
1978
Bacillus subtilis
-
Tanaka, T.; Oi, S.; Yamamoto, T.
Synthesis of levan by levansucrase. Some factors affecting the rate of synthesis and degree of polymerization of levan
J. Biochem.
85
287-293
1979
Bacillus subtilis
Mntsl, P.; Puntala, M.
Comparison of levansucrase from Bacillus subtilis and from Bacillus amyloliquefaciens
FEMS Microbiol. Lett.
13
395-399
1982
Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus subtilis QB127
-
Chambert, R.; Petit-Glatron, M.F.
Polymerase and hydrolase activities of Bacillus subtilis levansucrase can be separately modulated by site-directed mutagenesis
Biochem. J.
279
35-41
1991
Bacillus subtilis
-
Hettwer, U.; Gross, M.; Rudolph, K.
Purification and characterization of an extracellular levansucrase from Pseudomonas syringae pv. phaseolicola
J. Bacteriol.
177
2834-2839
1995
Bacillus subtilis, Erwinia amylovora, Pseudomonas savastanoi pv. phaseolicola, Streptococcus mutans, Zymomonas mobilis
Euzenat, O.; Guibert, A.; Combes, D.
Production and purification of Bacillus subtilis C4 levansucrase: kinetic characterization of the enzyme
Ann. N. Y. Acad. Sci.
864
288-294
1998
Bacillus subtilis
-
Leloup, L.; Driessen, A.J.; Freudl, R.; Chambert, R.; Petit-Glatron, M.F.
Differential dependence of levansucrase and alpha-amylase secretion on SecA (Div) during the exponential phase of growth of Bacillus subtilis
J. Bacteriol.
181
1820-1826
1999
Bacillus subtilis
Park, N.H.; Choi, H.J.; Oh, D.K.
Lactosucrose production by various microorganisms harboring levansucrase activity
Biotechnol. Lett.
27
495-497
2005
Geobacillus stearothermophilus, Bacillus amyloliquefaciens, Bacillus subtilis, Paenibacillus polymyxa, Pseudomonas syringae, Rahnella aquatilis, Sterigmatomyces elviae, Sterigmatomyces elviae ATCC 18894, Geobacillus stearothermophilus ATCC 12980, Pseudomonas syringae IFO14086, Bacillus amyloliquefaciens IFO15535, Rahnella aquatilis KCTC2858, Paenibacillus polymyxa KCCM35411
Seibel, J.; Moraru, R.; Goetze, S.; Buchholz, K.; Naamnieh, S.; Pawlowski, A.; Hecht, H.J.
Synthesis of sucrose analogues and the mechanism of action of Bacillus subtilis fructosyltransferase (levansucrase)
Carbohydr. Res.
341
2335-2349
2006
Bacillus subtilis (P05655), Bacillus subtilis, Bacillus subtilis 168 (P05655)
Abdel-Fattah, A.F.; Mahmoud, D.A.; Esawy, M.A.
Production of levansucrase from Bacillus subtilis NRC 33a and enzymic synthesis of levan and Fructo-Oligosaccharides
Curr. Microbiol.
51
402-407
2005
Bacillus subtilis, Bacillus subtilis NRC 33a
Daguer, J.P.; Chambert, R.; Petit-Glatron, M.F.
Increasing the stability of sacB transcript improves levansucrase production in Bacillus subtilis
Lett. Appl. Microbiol.
41
221-226
2005
Bacillus subtilis
van Hijum, S.A.; Kralj, S.; Ozimek, L.K.; Dijkhuizen, L.; van Geel-Schutten, I.G.
Structure-function relationships of glucansucrase and fructansucrase enzymes from lactic acid bacteria
Microbiol. Mol. Biol. Rev.
70
157-176
2006
Gluconacetobacter diazotrophicus, Lactobacillus sp., Streptococcus salivarius, Streptococcus sp., Zymomonas mobilis, Bacillus subtilis (P05655)
Meng, G.; Fuetterer, K.
Donor substrate recognition in the raffinose-bound E342A mutant of fructosyltransferase Bacillus subtilis levansucrase
BMC Struct. Biol.
8
16
2008
Bacillus subtilis (P05655), Bacillus subtilis
Szwengiel, A.; Czarnecka, M.; Czarnecki, Z.
Levan synthesis during associated action of levansucrase and Candida cacaoi DSM 2226 yeast
Pol. J. Food Nutr. Sci.
57
433-440
2007
Bacillus subtilis
-
Ortiz-Soto, M.E.; Rivera, M.; Rudino-Pinera, E.; Olvera, C.; Lopez-Munguia, A.
Selected mutations in Bacillus subtilis levansucrase semi-conserved regions affecting its biochemical properties
Protein Eng. Des. Sel.
21
589-595
2008
Bacillus subtilis (P05655), Bacillus subtilis
Ortiz-Soto, M.; Rudino-Pinera, E.; Rodriguez-Alegria, M.; Munguia, A.
Evaluation of cross-linked aggregates from purified Bacillus subtilis levansucrase mutants for transfructosylation reactions
BMC Biotechnol.
9
68-75
2009
Bacillus subtilis
Esawy, M.; Mahmoud, D.; Fattah, A.
Immobilisation of Bacillus subtilis NRC33a levansucrase and some studies on its properties
Braz. J. Chem. Eng.
25
237-246
2008
Bacillus subtilis, Bacillus subtilis NRC33a
-
Li, W.; Yu, S.; Zhang, T.; Jiang, B.; Mu, W.
Recent novel applications of levansucrases
Appl. Microbiol. Biotechnol.
99
6959-6969
2015
Bacillus subtilis, Bacillus licheniformis, Microbacterium laevaniformans
Porras-Dominguez, J.R.; Avila-Fernandez, A.; Miranda-Molina, A.; Rodriguez-Alegria, M.E.; Munguia, A.L.
Bacillus subtilis 168 levansucrase (SacB) activity affects average levan molecular weight
Carbohydr. Polym.
132
338-344
2015
Bacillus subtilis (P05655), Bacillus subtilis 168 (P05655)
Visnapuu, T.; Mardo, K.; Alamaee, T.
Levansucrases of a Pseudomonas syringae pathovar as catalysts for the synthesis of potentially prebiotic oligo- and polysaccharides
New Biotechnol.
32
597-605
2015
Priestia megaterium, Bacillus subtilis, Burkholderia cepacia, Dactylis glomerata, Erwinia amylovora, Lactobacillus gasseri, Limosilactobacillus reuteri, Fructilactobacillus sanfranciscensis, Zymomonas mobilis, Limosilactobacillus panis, Phleum pratense, Pseudomonas syringae (O68609), Gluconacetobacter diazotrophicus (Q43998), Pseudomonas syringae pv. tomato (Q883P5), Pseudomonas syringae pv. tomato (Q88BN6), Pseudomonas chlororaphis subsp. aurantiaca (Q93FU9), Bacillus licheniformis (W8GV60), Pseudomonas syringae pv. tomato DC3000 (Q883P5), Pseudomonas syringae pv. tomato DC3000 (Q88BN6)
Porras-Dominguez, J.R.; Rodriguez-Alegria, M.E.; Avila-Fernandez, A.; Montiel-Salgado, S.; Lopez-Munguia, A.
Levan-type fructooligosaccharides synthesis by a levansucrase-endolevanase fusion enzyme (LevB1SacB)
Carbohydr. Polym.
177
40-48
2017
Bacillus subtilis, Bacillus subtilis 168
Ortiz-Soto, M.E.; Porras-Dominguez, J.R.; Seibel, J.; Lopez-Munguia, A.
A close look at the structural features and reaction conditions that modulate the synthesis of low and high molecular weight fructans by levansucrases
Carbohydr. Polym.
219
130-142
2019
Pseudomonas syringae, Zymomonas mobilis, Priestia megaterium (D5DC07), Bacillus subtilis (P05655), Bacillus subtilis 168 (P05655), Priestia megaterium DSM 319 (D5DC07)
Ruiz-Aceituno, L.; Sanz, M.L.; de Las Rivas, B.; Munoz, R.; Kolida, S.; Jimeno, M.L.; Moreno, F.J.
Enzymatic synthesis and structural characterization of theanderose through transfructosylation reaction catalyzed by levansucrase from Bacillus subtilis CECT 39
J. Agric. Food Chem.
65
10505-10513
2017
Bacillus subtilis, Bacillus subtilis ATCC 6051, Bacillus subtilis CECT 3
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