D. Baer et al., Partial characterization of an extracellular beta-fructofuranosidase from Clavibacter michiganensis subspecies sepedonicus, CAN J MICRO, 44(9), 1998, pp. 852-865
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.