Partial characterization of an extracellular beta-fructofuranosidase from Clavibacter michiganensis subspecies sepedonicus

Clavibacter michiganensis subsp. sepedonicus, a phytopathogenic bacteria is olated from potato and sugar beet, produces an extracellular beta-fructofur anosidase inducible by sucrose, glucose, fructose, mannose, galactose, and raffinose. This enzyme, also known as invertase or sucrase (beta-D-fructofu ranoside fructohydrolase, EC 3.2.1.26), is found in the supernatant of C. m ichiganensis subsp. sepedonicus broth cultures. The maximal sucrase activit y in the culture medium was observed after 96 h of incubation. Very little of this enzyme was associated with the cells. The enzymatic activity in the supernatant was concentrated by ethanol precipitation and further purified by a procedure that included polyethylene glycol 1450 aqueous phase partit ion and preparative isoelectric focusing as the main steps. Enzyme preparat ions were separated into two fractions (I and II) by gel-filtration chromat ography. The preparative isoelectric focusing of the native enzyme showed t hat most activity was concentrated in the pi range of 4.0-5.0. The enzyme h ydrolyzed sucrose, raffinose, and stachyose but hydrolyzed very little inul in, levan, cellobiose, lactose, maltose, or melezitose. The enzyme demonstr ated glucosyltransferase activity toward [U-C-14]sucrose, cleaving the disa ccharide into glucose and fructose, with a small amount incorporated into a trisaccharide.