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Literature summary 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)

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

Protein Variants

EC Number Protein Variants Comment Organism
2.4.1.10 D257A site-directed mutagenesis, no activity Priestia megaterium
2.4.1.10 D257A mutant shows almost no activity Priestia megaterium
2.4.1.10 D95A site-directed mutagenesis, no activity Priestia megaterium
2.4.1.10 E350A mutant is nearly inactive Priestia megaterium
2.4.1.10 E350A site-directed mutagenesis, nearly inactive Priestia megaterium
2.4.1.10 E352A site-directed mutagenesis, no measureable activity Priestia megaterium
2.4.1.10 L118A site-directed mutagenesis Priestia megaterium
2.4.1.10 L118A kcat/Km is 5.5% of wild-type value Priestia megaterium
2.4.1.10 N252A site-directed mutagenesis Priestia megaterium
2.4.1.10 N252A kcat/Km is 104% of wild-type value Priestia megaterium
2.4.1.10 R256A mutant is nearly inactive Priestia megaterium
2.4.1.10 R256A site-directed mutagenesis, nearly inactive Priestia megaterium
2.4.1.10 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
2.4.1.10 S173A site-directed mutagenesis Priestia megaterium
2.4.1.10 S173A kcat/Km is 46% of wild-type value Priestia megaterium
2.4.1.10 W172A kcat/Km is 1.4% of wild-type value Priestia megaterium
2.4.1.10 W172A site-directed mutagenesis, 72fold increase of the KM of the wild-type Priestia megaterium
2.4.1.10 W94A kcat/Km is 9% of wild-type value Priestia megaterium
2.4.1.10 W94A site-directed mutagenesis, 9% of the catalytic efficiency of the wild-type Priestia megaterium
2.4.1.10 Y421A kcat/Km is 1.9% of wild-type value Priestia megaterium
2.4.1.10 Y421A site-directed mutagenesis, 3%of the wild-type activity Priestia megaterium

General Stability

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

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.4.1.10 additional information
-
additional information 2.3 mM (sucrose) total, mutant enzyme S173A Priestia megaterium
2.4.1.10 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
2.4.1.10 additional information
-
additional information 31.9 mM (sucrose) total, mutant enzyme W94A, 9% of the catalytic efficiency of the wild-type Priestia megaterium
2.4.1.10 additional information
-
additional information 35.1 mM (sucrose) total, mutant enzyme N252H Priestia megaterium
2.4.1.10 additional information
-
additional information 4.1 mM (sucrose) total, mutant enzyme N252A Priestia megaterium
2.4.1.10 additional information
-
additional information 480.4 mM (sucrose) total, mutant enzyme W172A, 72fold increase of the KM of the wild-type Priestia megaterium
2.4.1.10 additional information
-
additional information 51.9 mM (sucrose) total, mutant enzyme Y421A, 3%of the wild-type activity Priestia megaterium
2.4.1.10 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
2.4.1.10 additional information
-
additional information 66.6 mM (sucrose), mutant enzyme L118A Priestia megaterium
2.4.1.10 additional information
-
additional information 7.5 mM (sucrose) total, mutant enzyme N252G Priestia megaterium
2.4.1.10 additional information
-
additional information 8.4 mM (sucrose) total, mutant enzyme N252D Priestia megaterium
2.4.1.10 additional information
-
additional information mutant enzyme D257A, no activity Priestia megaterium
2.4.1.10 additional information
-
additional information mutant enzyme D95A, no activity Priestia megaterium
2.4.1.10 additional information
-
additional information mutant enzyme E350A, nearly inactive Priestia megaterium
2.4.1.10 additional information
-
additional information mutant enzyme E352A, no measureable activity Priestia megaterium
2.4.1.10 additional information
-
additional information mutant enzyme R256A, nearly inactive Priestia megaterium
2.4.1.10 2.3
-
sucrose pH 6.6, mutant enzyme S173A Priestia megaterium
2.4.1.10 4.1
-
sucrose pH 6.6, mutant enzyme N252A Priestia megaterium
2.4.1.10 6.6
-
sucrose pH 6.6, wild-type enzyme Priestia megaterium
2.4.1.10 31.9
-
sucrose pH 6.6, mutant enzyme W94A Priestia megaterium
2.4.1.10 51.9
-
sucrose pH 6.6, mutant enzyme Y421A Priestia megaterium
2.4.1.10 66.6
-
sucrose pH 6.6, mutant enzyme L118A Priestia megaterium
2.4.1.10 480.4
-
sucrose pH 6.6, mutant enzyme W172A Priestia megaterium

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
2.4.1.10 extracellular
-
Priestia megaterium
-
-

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
2.4.1.10 52000
-
MALDI-TOF Priestia megaterium
2.4.1.10 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

EC Number Organism UniProt Comment Textmining
2.4.1.10 Priestia megaterium
-
DSM319
-
2.4.1.10 Priestia megaterium
-
strain DSM319, gene sacB, expression in Escherichia coli, 74% identity at the amino acid sequence level with SacB from Bacillus subtilis
-
2.4.1.10 Priestia megaterium DSM 319
-
DSM319
-
2.4.1.10 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)

EC Number Purification (Comment) Organism
2.4.1.10
-
Priestia megaterium
2.4.1.10 by FPLC using a 15 ml CM-Sepharose column system Priestia megaterium

Source Tissue

EC Number Source Tissue Comment Organism Textmining
2.4.1.10 culture supernatant
-
Priestia megaterium
-

Storage Stability

EC Number Storage Stability Organism
2.4.1.10 -20°C, pH 6.6, stable for more than 6 months Priestia megaterium
2.4.1.10 20°C, more than 6 months, pH 6.6 Priestia megaterium

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.4.1.10 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
-
?
2.4.1.10 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
-
?
2.4.1.10 sucrose + ?
-
Priestia megaterium blastose + ? two-dimensional COSY, TOCSY, HMBC and HSQC experiments ?
2.4.1.10 sucrose + ?
-
Priestia megaterium DSM 319 blastose + ? two-dimensional COSY, TOCSY, HMBC and HSQC experiments ?
2.4.1.10 sucrose + ?
-
Priestia megaterium polyfructan + ? polyfructan, molecular mass of 2711 kDa and consisted mainly of beta(2-6) linkages ?
2.4.1.10 sucrose + ?
-
Priestia megaterium DSM 319 polyfructan + ? polyfructan, molecular mass of 2711 kDa and consisted mainly of beta(2-6) linkages ?
2.4.1.10 sucrose + sucrose
-
Priestia megaterium D-glucose + 1-kestose identified by HPAEC ?
2.4.1.10 sucrose + sucrose
-
Priestia megaterium DSM 319 D-glucose + 1-kestose identified by HPAEC ?
2.4.1.10 sucrose + sucrose
-
Priestia megaterium D-glucose + 6-kestose identified by HPAEC ?
2.4.1.10 sucrose + sucrose
-
Priestia megaterium DSM 319 D-glucose + 6-kestose identified by HPAEC ?
2.4.1.10 sucrose + sucrose
-
Priestia megaterium D-glucose + neokestose identified by one-dimensional and correlation spectroscopy (i.e. COSY, TOCSY, HMBC, DEPT and HSQC) ?
2.4.1.10 sucrose + sucrose
-
Priestia megaterium D-glucose + nystose identified by HPAEC ?

Synonyms

EC Number Synonyms Comment Organism
2.4.1.10 levansucrase
-
Priestia megaterium

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.4.1.10 45
-
-
Priestia megaterium
2.4.1.10 45
-
wild-type, pH6.6, 50 mM Sorensen’s buffer Priestia megaterium

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
2.4.1.10 37
-
high stability for at least 24 h Priestia megaterium

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.4.1.10 325
-
sucrose pH 6.6, mutant enzyme Y421A Priestia megaterium
2.4.1.10 335
-
sucrose mutant enzyme Y421A Priestia megaterium
2.4.1.10 363
-
sucrose pH 6.6, mutant enzyme S173A Priestia megaterium
2.4.1.10 363
-
sucrose mutant enzyme S173A Priestia megaterium
2.4.1.10 1000
-
sucrose pH 6.6, mutant enzyme W94A Priestia megaterium
2.4.1.10 1000
-
sucrose mutant enzyme W94A Priestia megaterium
2.4.1.10 1231
-
sucrose pH 6.6, mutant enzyme L118A Priestia megaterium
2.4.1.10 1231
-
sucrose mutant enzyme L118A Priestia megaterium
2.4.1.10 1480
-
sucrose pH 6.6, mutant enzyme N252A Priestia megaterium
2.4.1.10 1480
-
sucrose mutant enzyme N252A Priestia megaterium
2.4.1.10 1529
-
sucrose mutant enzyme N252H Priestia megaterium
2.4.1.10 2256
-
sucrose mutant enzyme N252G Priestia megaterium
2.4.1.10 2272
-
sucrose wild-type Priestia megaterium
2.4.1.10 2272
-
sucrose pH 6.6, wild-type enzyme Priestia megaterium
2.4.1.10 2396
-
sucrose pH 6.6, mutant enzyme W172A Priestia megaterium
2.4.1.10 2396
-
sucrose mutant enzyme W172A Priestia megaterium
2.4.1.10 3743
-
sucrose mutant enzyme N252D Priestia megaterium

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.4.1.10 6 7
-
Priestia megaterium
2.4.1.10 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

EC Number pH Minimum pH Maximum Comment Organism
2.4.1.10 5.6 7.6 the enzyme activity significantly decreases at pH values below 5.6 and above 7.6 Priestia megaterium
2.4.1.10 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