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

Abstract

Clavibacter michiganensis subsp. sepedonicus, a phytopathogenic bacteria isolated from potato and sugar beet, produces an extracellular β-fructofuranosidase inducible by sucrose, glucose, fructose, mannose, galactose, and raffinose. This enzyme, also known as invertase or sucrase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26), is found in the supernatant of C. michiganensis subsp. sepedonicus broth cultures. The maximal sucrase activity 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 partition and preparative isoelectric focusing as the main steps. Enzyme preparations were separated into two fractions (I and II) by gel-filtration chromatography. The preparative isoelectric focusing of the native enzyme showed that most activity was concentrated in the pI range of 4.0-5.0. The enzyme hydrolyzed sucrose, raffinose, and stachyose but hydrolyzed very little inulin, levan, cellobiose, lactose, maltose, or melezitose. The enzyme demonstrated glucosyltransferase activity toward [U-14C]sucrose, cleaving the disaccharide into glucose and fructose, with a small amount incorporated into a trisaccharide.Key words: levansucrase, glucosyltransferase, sucrase, dextransucrase, Solanum tuberosum