Cloned (Comment) | Organism |
---|---|
expression in Escherichia coli BL21(DE3) via the T7 RNA polymerase expression system with pET9d | Zymomonas mobilis |
Protein Variants | Comment | Organism |
---|---|---|
H296R | no ability of the enzyme to form microfibrils and to synthesize levan at pH 6.0 | Zymomonas mobilis |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
sucrose | substrate inhibition | Zymomonas mobilis |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | 32 mM (sucrose), 25°C, pH 5.0, total activity | Zymomonas mobilis | |
additional information | - |
additional information | at pHs higher than 7.0, Zymomonas mobilis levansucrase does not exhibit levan synthesis activity | Zymomonas mobilis | |
36 | - |
sucrose | 25°C, pH 5.0, transfructosylation activity | Zymomonas mobilis | |
40 | - |
sucrose | 25°C, pH 5.0, levan synthesis activity is monitored by following the increase in turbidity at 600 nm that occurrs due to the insoluble levan formation | Zymomonas mobilis |
Localization | Comment | Organism | GeneOntology No. | Textmining |
---|
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
additional information | - |
at pH 6.0 apparent MW greater than 1000000 Da | Zymomonas mobilis |
94000 | - |
at pH 7.4, purification procedure includes two steps, MnCl2 precipitation and gel filtration, resulting in a 98% pure protein based on SDS-PAGE, calculated MW is 46700 Da, the enzyme appears to be a dimer in solution | Zymomonas mobilis |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Zymomonas mobilis | Q60114 | sacB gene, AAA27695; ATCC10988, expression in Escherichia coli BL21(DE3) | - |
Purification (Comment) | Organism |
---|---|
The cell extract is centrifuged (14,000xg for 15 min) and the soluble supernatant is kept at 4°C. Following the procedure of Vigants, the enzyme is selectively precipitated by adding 0.1 M MnCl2. Next, the solution is centrifuged (14,000xg for 15 min), the pellet is re-suspended in buffer B (50 mM phosphate buffer pH 6.0, 100 mM NaCl and 0.02% sodium azide), and then the soluble protein is passed via a Sephacryl S300 26/60 gel filtration column, running at 1 ml/min with either buffer A at pH 7.4 or with buffer B at pH 6.0. | Zymomonas mobilis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | change in substrate specificity appears to be unique to Zymomonas mobilis levansucrase because the levansucrases from Bacillus subtilis, Rahnella aquatilis, and Lactobacillus reuteri are capable of synthesizing levan in their soluble form and are not reported to form fibril-like structures | Zymomonas mobilis | ? | - |
? | |
sucrose + sucrose | dimer form | Zymomonas mobilis | D-glucose + (2,6-beta-D-fructosyl)3 | degree of polymerisation 3 | ? | |
sucrose + [6)-beta-D-fructofuranosyl-(2->]n alpha-D-glucopyranoside | pH lower than 7.0, fibril form | Zymomonas mobilis | D-glucose + [6)-beta-D-fructofuranosyl-(2->]n+1 alpha-D-glucopyranoside | - |
? |
Subunits | Comment | Organism |
---|---|---|
dimer | structure depends on ionic strength and pHs, above 7.0, products are short fructo-saccharides (degree of polymerization 3) | Zymomonas mobilis |
More | microfibrils, structure depends on ionic strength and pHs, below 6.0, well-ordered, precipate out of the solution, synthesis of levan (degree of polymerization greater than 20000). The fibril structure decreases the diffusion of the enzyme away from the bacteria cells, which results in the close association of the cell to the high molecular polymer, levan. | Zymomonas mobilis |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
379 | - |
sucrose | transfructosylation activity | Zymomonas mobilis | |
411 | - |
sucrose | levan synthesis activity | Zymomonas mobilis | |
461 | - |
sucrose | total activity | Zymomonas mobilis |
pH Minimum | pH Maximum | Comment | Organism |
---|---|---|---|
additional information | - |
Zymomonas mobilis levansucrase exists in two active forms, depending on the pH and ionic strength | Zymomonas mobilis |
Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
---|---|---|---|---|---|
2000 | - |
sucrose | - |
Zymomonas mobilis | |
2100 | - |
sucrose | - |
Zymomonas mobilis | |
2800 | - |
sucrose | - |
Zymomonas mobilis |