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Literature summary for 2.4.1.10 extracted from

  • Homann, A.; Biedendieck, R.; Gtze, S.; Jahn, D.; Seibel, J.
    Insights into polymer versus oligosaccharide synthesis: mutagenesis and mechanistic studies of a novel levansucrase from Bacillus megaterium (2007), Biochem. J., 407, 189-198.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
expression in Escherichia coli Priestia megaterium
wild-type enzyme and 16 variants are expressed in Escherichia coli Priestia megaterium

Protein Variants

Protein Variants Comment Organism
D257A site-directed mutagenesis, no activity Priestia megaterium
D257A mutant shows almost no activity Priestia megaterium
D95A site-directed mutagenesis, no activity Priestia megaterium
E350A mutant is nearly inactive Priestia megaterium
E350A site-directed mutagenesis, nearly inactive Priestia megaterium
E352A site-directed mutagenesis, no measureable activity Priestia megaterium
L118A site-directed mutagenesis Priestia megaterium
L118A kcat/Km is 5.5% of wild-type value Priestia megaterium
N252A site-directed mutagenesis Priestia megaterium
N252A kcat/Km is 104% of wild-type value Priestia megaterium
R256A mutant is nearly inactive Priestia megaterium
R256A site-directed mutagenesis, nearly inactive Priestia megaterium
R370A site-directed mutagenesis, after a reaction time of 60 min an accumulation of neokestose (2,6-beta-Fru-betaGlc-1,2-beta-Fru, 32.7 mM) is determined, whereas after 19 h, blastose (2,6-beta-Fru-alpha,betaGlc) is the main reaction product (69.7 mM) Priestia megaterium
S173A site-directed mutagenesis Priestia megaterium
S173A kcat/Km is 46% of wild-type value Priestia megaterium
W172A kcat/Km is 1.4% of wild-type value Priestia megaterium
W172A site-directed mutagenesis, 72fold increase of the KM of the wild-type Priestia megaterium
W94A kcat/Km is 9% of wild-type value Priestia megaterium
W94A site-directed mutagenesis, 9% of the catalytic efficiency of the wild-type Priestia megaterium
Y421A kcat/Km is 1.9% of wild-type value Priestia megaterium
Y421A site-directed mutagenesis, 3%of the wild-type activity Priestia megaterium

General Stability

General Stability Organism
in standard reactions, long-term temperature treatment revealed a high protein stability at 37°C for at least 24h Priestia megaterium

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information 2.3 mM (sucrose) total, mutant enzyme S173A Priestia megaterium
additional information
-
additional information 29.2 mM (sucrose) total, mutant enzyme R370A, a 4fold KM value decrease compared with the wild-type enzyme supported the interactions of Arg 370 with the 2-OH and 3-OH of the glucosyl residue in the active site Priestia megaterium
additional information
-
additional information 31.9 mM (sucrose) total, mutant enzyme W94A, 9% of the catalytic efficiency of the wild-type Priestia megaterium
additional information
-
additional information 35.1 mM (sucrose) total, mutant enzyme N252H Priestia megaterium
additional information
-
additional information 4.1 mM (sucrose) total, mutant enzyme N252A Priestia megaterium
additional information
-
additional information 480.4 mM (sucrose) total, mutant enzyme W172A, 72fold increase of the KM of the wild-type Priestia megaterium
additional information
-
additional information 51.9 mM (sucrose) total, mutant enzyme Y421A, 3%of the wild-type activity Priestia megaterium
additional information
-
additional information 6.6 mM (sucrose) total, wild-type, pH 6.6, 37°C, 50 mM Sorensen’s phosphate buffer, 7.36 mg/l enzyme concentration, reaction time 60 min Priestia megaterium
additional information
-
additional information 66.6 mM (sucrose), mutant enzyme L118A Priestia megaterium
additional information
-
additional information 7.5 mM (sucrose) total, mutant enzyme N252G Priestia megaterium
additional information
-
additional information 8.4 mM (sucrose) total, mutant enzyme N252D Priestia megaterium
additional information
-
additional information mutant enzyme D257A, no activity Priestia megaterium
additional information
-
additional information mutant enzyme D95A, no activity Priestia megaterium
additional information
-
additional information mutant enzyme E350A, nearly inactive Priestia megaterium
additional information
-
additional information mutant enzyme E352A, no measureable activity Priestia megaterium
additional information
-
additional information mutant enzyme R256A, nearly inactive Priestia megaterium
2.3
-
sucrose pH 6.6, mutant enzyme S173A Priestia megaterium
4.1
-
sucrose pH 6.6, mutant enzyme N252A Priestia megaterium
6.6
-
sucrose pH 6.6, wild-type enzyme Priestia megaterium
31.9
-
sucrose pH 6.6, mutant enzyme W94A Priestia megaterium
51.9
-
sucrose pH 6.6, mutant enzyme Y421A Priestia megaterium
66.6
-
sucrose pH 6.6, mutant enzyme L118A Priestia megaterium
480.4
-
sucrose pH 6.6, mutant enzyme W172A Priestia megaterium

Localization

Localization Comment Organism GeneOntology No. Textmining
extracellular
-
Priestia megaterium
-
-

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
52000
-
MALDI-TOF Priestia megaterium
52000
-
FPLC, proteins are identified by peptide mass fingerprinting, as well as by using postsource decay fragmentation data recorded with an Ultraflex MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) mass spectrometer Priestia megaterium

Organism

Organism UniProt Comment Textmining
Priestia megaterium
-
DSM319
-
Priestia megaterium
-
strain DSM319, gene sacB, expression in Escherichia coli, 74% identity at the amino acid sequence level with SacB from Bacillus subtilis
-
Priestia megaterium DSM 319
-
DSM319
-
Priestia megaterium DSM 319
-
strain DSM319, gene sacB, expression in Escherichia coli, 74% identity at the amino acid sequence level with SacB from Bacillus subtilis
-

Purification (Commentary)

Purification (Comment) Organism
-
Priestia megaterium
by FPLC using a 15 ml CM-Sepharose column system Priestia megaterium

Source Tissue

Source Tissue Comment Organism Textmining
culture supernatant
-
Priestia megaterium
-

Storage Stability

Storage Stability Organism
-20°C, pH 6.6, stable for more than 6 months Priestia megaterium
20°C, more than 6 months, pH 6.6 Priestia megaterium

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
sucrose + (2,6-beta-D-fructosyl)n polyfructan produced by the levansucrase from Bacillus megaterium has a molecular mass of 2711 kDa and consisted mainly of beta(2,6) linkages. Besides the polyfructan formation, the wild-type levansucrase of Bacillus megaterium also synthesizes five different detectable oligosaccharides. Three products are identified: 1-kestose (isokestose), 6-kestose and nystose which are known acceptors for the transfer of fructosyl units Priestia megaterium D-glucose + (2,6-beta-D-fructosyl)n+1
-
?
sucrose + (2,6-beta-D-fructosyl)n polyfructan produced by the levansucrase from Bacillus megaterium has a molecular mass of 2711 kDa and consisted mainly of beta(2,6) linkages. Besides the polyfructan formation, the wild-type levansucrase of Bacillus megaterium also synthesizes five different detectable oligosaccharides. Three products are identified: 1-kestose (isokestose), 6-kestose and nystose which are known acceptors for the transfer of fructosyl units Priestia megaterium DSM 319 D-glucose + (2,6-beta-D-fructosyl)n+1
-
?
sucrose + ?
-
Priestia megaterium blastose + ? two-dimensional COSY, TOCSY, HMBC and HSQC experiments ?
sucrose + ?
-
Priestia megaterium DSM 319 blastose + ? two-dimensional COSY, TOCSY, HMBC and HSQC experiments ?
sucrose + ?
-
Priestia megaterium polyfructan + ? polyfructan, molecular mass of 2711 kDa and consisted mainly of beta(2-6) linkages ?
sucrose + ?
-
Priestia megaterium DSM 319 polyfructan + ? polyfructan, molecular mass of 2711 kDa and consisted mainly of beta(2-6) linkages ?
sucrose + sucrose
-
Priestia megaterium D-glucose + 1-kestose identified by HPAEC ?
sucrose + sucrose
-
Priestia megaterium DSM 319 D-glucose + 1-kestose identified by HPAEC ?
sucrose + sucrose
-
Priestia megaterium D-glucose + 6-kestose identified by HPAEC ?
sucrose + sucrose
-
Priestia megaterium DSM 319 D-glucose + 6-kestose identified by HPAEC ?
sucrose + sucrose
-
Priestia megaterium D-glucose + neokestose identified by one-dimensional and correlation spectroscopy (i.e. COSY, TOCSY, HMBC, DEPT and HSQC) ?
sucrose + sucrose
-
Priestia megaterium D-glucose + nystose identified by HPAEC ?

Synonyms

Synonyms Comment Organism
levansucrase
-
Priestia megaterium

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
45
-
-
Priestia megaterium
45
-
wild-type, pH6.6, 50 mM Sorensen’s buffer Priestia megaterium

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
37
-
high stability for at least 24 h Priestia megaterium

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
325
-
sucrose pH 6.6, mutant enzyme Y421A Priestia megaterium
335
-
sucrose mutant enzyme Y421A Priestia megaterium
363
-
sucrose pH 6.6, mutant enzyme S173A Priestia megaterium
363
-
sucrose mutant enzyme S173A Priestia megaterium
1000
-
sucrose pH 6.6, mutant enzyme W94A Priestia megaterium
1000
-
sucrose mutant enzyme W94A Priestia megaterium
1231
-
sucrose pH 6.6, mutant enzyme L118A Priestia megaterium
1231
-
sucrose mutant enzyme L118A Priestia megaterium
1480
-
sucrose pH 6.6, mutant enzyme N252A Priestia megaterium
1480
-
sucrose mutant enzyme N252A Priestia megaterium
1529
-
sucrose mutant enzyme N252H Priestia megaterium
2256
-
sucrose mutant enzyme N252G Priestia megaterium
2272
-
sucrose wild-type Priestia megaterium
2272
-
sucrose pH 6.6, wild-type enzyme Priestia megaterium
2396
-
sucrose pH 6.6, mutant enzyme W172A Priestia megaterium
2396
-
sucrose mutant enzyme W172A Priestia megaterium
3743
-
sucrose mutant enzyme N252D Priestia megaterium

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
6 7
-
Priestia megaterium
6 7 wild-type, 50 mM Sorensen’s phosphate buffer at 37°C, containing 500 mM sucrose using an enzyme concentration of 7.36 mg/l Priestia megaterium

pH Range

pH Minimum pH Maximum Comment Organism
5.6 7.6 the enzyme activity significantly decreases at pH values below 5.6 and above 7.6 Priestia megaterium
5.6 7.6 wild-type, 50 mM Sorensen’s phosphate buffer at 37°C, containing 500 mM sucrose using an enzyme concentration of 7.36 mg/l Priestia megaterium