Literature summary for 2.4.1.10 extracted from

Goldman, D.; Lavid, N.; Schwartz, A.; Shoham, G.; Danino, D.; Shoham, Y.
Two active forms of Zymomonas mobilis levansucrase: an ordered microfibril structure of the enzyme promotes levan polymerization (2008), J. Biol. Chem., 283, 32209-32217.

Cloned(Commentary)

Cloned (Comment)	Organism
expression in Escherichia coli BL21(DE3) via the T7 RNA polymerase expression system with pET9d	Zymomonas mobilis

Protein Variants

Protein Variants	Comment	Organism
H296R	no ability of the enzyme to form microfibrils and to synthesize levan at pH 6.0	Zymomonas mobilis

Inhibitors

Inhibitors	Comment	Organism	Structure
sucrose	substrate inhibition	Zymomonas mobilis

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
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

Localization	Comment	Organism	GeneOntology No.	Textmining

Molecular Weight [Da]

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

Organism	UniProt	Comment	Textmining
Zymomonas mobilis	Q60114	sacB gene, AAA27695; ATCC10988, expression in Escherichia coli BL21(DE3)	-

Purification (Commentary)

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

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 and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
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

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 [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
379	-	sucrose	transfructosylation activity	Zymomonas mobilis
411	-	sucrose	levan synthesis activity	Zymomonas mobilis
461	-	sucrose	total activity	Zymomonas mobilis

pH Range

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 [mM]	Ki Value maximum [mM]	Inhibitor	Comment	Organism
2000	-	sucrose	-	Zymomonas mobilis
2100	-	sucrose	-	Zymomonas mobilis
2800	-	sucrose	-	Zymomonas mobilis