1.6.4
PURIFICATION AND PROPERTIES OF AN EXTRACELLULAR BETA-FRUCTOFURANOSIDASE FROM CLAVIBACTER MICHIGANENSIS SUBSP. SEPEDONICUS

D BAER1, AR WHITE2 and NC GUDMESTAD1

1Department of Plant Pathology, North Dakota State University, Fargo, ND 58105, USA; 2Department of Botany, North Dakota State University, Fargo, ND 58105, USA

Background and objectives
Plant pathogenic corynebacteria have been traditionally considered host-specific. An exception to this was discovered when Clavibacter michiganensis subsp.sepedonicus was found to be capable of establishing an endophytic relationship in sugar beet [1]. To date, this is the only plant pathogenic coryneform found to have an association outside its host plant. It was hypothesized that the ability of C. m. sepedonicus to establish an association in sugar beet was due to its ability to utilize sucrose. This work was undertaken to determine the ability of C. m. sepedonicus strains to hydrolyse sucrose, the optimal culture conditions for extracellular sucrase production in culture, and some properties of the sucrase enzyme.

Materials and methods
Enzyme activity (U/ml) was determined by estimating mM reducing sugar released/min/ml from sucrose by the 3,5-dinitrosalicylic acid (DNS) method. Transferase activity was measured by determining the formation of new compounds using uniformly labelled, radioactive [U-14C]sucrose.

41 C. m. sepedonicus strains were tested for production of acid from sucrose. Localization of fructofuranosidase was determined by measuring activity in washed cells, sonicated cells, culture supernatant and cell washings. Enzyme was obtained from culture by ETOH precipitation and polyethylene glycol (PEG) phase partition. Optimum pH, substrate specificity, and inhibition effects of p-hydroxymercuribenzoate and DTT on the activity of enzyme from two strains, were examined. Determination of enzyme parameters Km and Vmax based on release of reducing sugar were calculated with a computer program based on the Lineweaver-Burke and Hanes-Wolf equations. Buffers used in preparative, non-denaturing electrophoresis were prepared to determine effect of buffer type and pH on enzyme activity. Proteins were assessed by PAGE under denaturing and non-denaturing conditions. The pI was determined by isoelectric focusing. Estimation of the molecular weight was done by gel filtration on a Sephacryl S-100 HR column. Enzyme transfer reactions were investigated with [U-14C]sucrose using size-exclusion chromatography. Fractions from size-exclusion chromatography containing radioactive saccharides produced during the enzyme incubation were separated by high-pH anion-exchange chromatography. Radioactive peaks were identified by comparing peaks and retention times on a Radiomatic Flo-One/Beta radiochromatography detector [2].

Results and conclusions
C. m. sepedonicus, isolated from potato and sugar beet, produces a fructofuranosidase (invertase, EC 3.2.1.26) in broth culture. The greatest enzyme activity was recovered in the early, stationary phase of the growth period in the culture supernatant. Very little activity was associated with the cells. Activity was influenced by the type of sugar used as a carbon source for culture. Fructofuranosidase activity was recovered in the supernatant of broth cultures containing sucrose, glucose, fructose, mannose, galactose or raffinose as a carbon source, but not trehalose, cellobiose and maltose. Isolation methods of the enzyme included ethanol precipitation, aqueous phase partition and isoelectric focusing. Enzyme preparations were separated into two fractions (I and II) by gel filtration chromatography. Active isoelectric focusing fractions containing a single protein band had a molecular weight of 77.3 kDa by SDS PAGE. The enzyme had an isoelectric point of 4.5, and was most active at pH 4.5-5.5. This enzyme hydrolysed sucrose, raffinose and stachyose, but hydrolysed very little inulin, levan, cellobiose, lactose, maltose or melezitose. Partially purified enzyme demonstrated transferase activity toward [U-14C]sucrose, cleaving the disaccharide into glucose and fructose, with a small amount incorporated into a group of larger sugars.

References
1. Bugbee WM, Gudmestad NC, 1988. Phytopathology 78, 205-208.
2. White AR, Xin Y, Pezeshk V, 1993. Biochemistry Journal 294, 231-238.