Cloned (Comment) | Organism |
---|---|
expression in Escherichia coli | Priestia megaterium |
wild-type enzyme and 16 variants are expressed in Escherichia coli | Priestia megaterium |
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 | 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 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 Sorensens 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 | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
extracellular | - |
Priestia megaterium | - |
- |
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 | 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 (Comment) | Organism |
---|---|
- |
Priestia megaterium |
by FPLC using a 15 ml CM-Sepharose column system | Priestia megaterium |
Source Tissue | Comment | Organism | Textmining |
---|---|---|---|
culture supernatant | - |
Priestia megaterium | - |
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 | 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 | Comment | Organism |
---|---|---|
levansucrase | - |
Priestia megaterium |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
45 | - |
- |
Priestia megaterium |
45 | - |
wild-type, pH6.6, 50 mM Sorensens buffer | Priestia megaterium |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
high stability for at least 24 h | Priestia megaterium |
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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
6 | 7 | - |
Priestia megaterium |
6 | 7 | wild-type, 50 mM Sorensens phosphate buffer at 37°C, containing 500 mM sucrose using an enzyme concentration of 7.36 mg/l | Priestia megaterium |
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 Sorensens phosphate buffer at 37°C, containing 500 mM sucrose using an enzyme concentration of 7.36 mg/l | Priestia megaterium |