Fructosidic Linkages in Sucrose, Raffinose, and Related Glycosides (5-7,9,13)

J. Gen. Appl. Microbiol., 38, 533-539 (1992)

RAFFINOSE METABOLISM AND A NOVEL MODE OF SUCROSE SYNTHESIS IN COR YNEBACTERI UM MURISEPTICUM

MANGALA A. NADKARNI,* C. K. K. NAIR, V. N. PANDEY, AND D. S. PRADHAN

Radiation Biology and Biochemistry Division, Bhabha Atomic Research Centre, Trombay, Bombay 400 085, India

(Received March 11, 1992)

In C. murisepticum, a constitutive extracellular invertase hydrolyzes raffinose to melibiose and fructose. During growth on raffinose plus glucose, an unexpected appearance of sucrose was discovered in the growth medium. The enzyme preparation from raffinose-grown culture supernatant could synthesize in vitro, sucrose, and difructose from raffinose plus glucose or difructose from raffinose alone. The results thus bring to light a novel attribute of the extracellular invertase, of catalyzing the transfer of the fructose moiety of raffinose to glucose and fructose, thereby achieving the synthesis of sucrose, and difructose, respectively.

Several bacteria, yeasts and fungi have enzymes capable of hydrolyzing /-D- fructosidic linkages in sucrose, raffinose, and related glycosides (5-7,9,13). The hydrolysis of $-D-fructosidic linkage is usually catalyzed by invertase which also catalyzes transfructosylation in addition to its hydrolytic action (10). However, in bacteria, sucrose can also be metabolized by dextransucrase (a glucosyltransferase) and levansucrase (a fructosyltransferase) with subsequent formation of polymers- dextrans and levans- and/or disaccharides (2-4,8). None of the bacterial invertases is reported to have the ability to synthesize sucrose. There is only a solitary report of a fungal invertase capable of synthesizing sucrose (10). The present paper reports the first evidence for bacterial sucrose-synthesizing extracellular invertase from Corynebacterium murisepticum.

MATERIALS AND METHODS

Chemicals. Raffinose, sucrose, glucose, fructose- l -phosphate, fructose- 6-

* Address reprint requests to: Dr . Mangala A. Nadkarni, Radiation Biology and Biochemistry Division, Bhabha Atomic Research Centre, Trombay, Bombay 400 085, India.

533 534 NADKARNI, NAIR, PANDEY, and PRADHAN VOL. 38 phosphate, fructose-l,6-diphosphate, fructose, invertase (yeast), were purchased from Sigma Chemical Co. (St. Louis, M.O., U.S.A.). All other chemicals were of analytical grade. [U-14C]glucose (Sp. act. 210 mCi/mmol), [U-14C]fructose (Sp. act. 160 mCi/ mmol) were obtained from Isotope Division, BARC (Bombay, India). [G-3H]- raffinose (Sp. act. 7.6 Ci/mmol) was obtained from New England Nuclear (Boston, Massachusetts, U.S.A.). Bacterial strain. Corynebacterium murisepticum ATCC 21374 was obtained from American Type Culture Collection (U.S.A.). Media and culture conditions. For studying extracellular mode of raffinose or sucrose hydrolysis and sucrose synthesis, C. murisepticum cells were grown in M9 minimal medium (Na2HPO4 6 g, KH2PO4 3 g, NH4C1 1 g, CaCl2.2H2O 0.02 g, MgSO4.7H2O 0.2 g, distilled water 11, pH 7.0) supplemented with 0.4% raffinose; or 0.4% sucrose; or 0.4% each of raffinose plus glucose; or 0.4% each of raffinose and glucose along with [U-14C]glucose (0.5 ,uCi/ml, final conc. ); or 0.4% each of raffinose and glucose along with [U-14C]fructose (0.25 ,iCi/ml, final conc. ), or fructose- 1-phosphate, or fructose-6-phosphate, or fructose- l,6-diphosphate. For isolation of extracellular invertase-like activity, cells were grown in M9 minimal medium supplemented with 0.4% raffinose. The cultures were incubated with aeration at 30°C on orbital shaker set at 200 rpm. Isolation of extracellular invertase-like activity from C, murisepticum cells grown with rafnose as carbon source. After 24 h growth in M9 minimal medium supplemented with raffinose, cells were harvested at 3,000 X g for 10 min. From the extracellular culture supernatant, the enzyme was precipitated by the addition of ammonium sulfate to 90% saturation. The precipitated enzyme was recovered by centrifugation at 27,000 X g for 15 min, dissolved in 50 mM phosphate buffer pH 7.5 and dialyzed against 50 mM phosphate buffer pH 7.5 containing 50% glycerol and stored at -20°C. This enzyme preparation was used for in vitro studies. Chromatography. Thin-layer chromatography (TLC) was performed on silica gel plates using chloroform : acetic acid : water (60: 70: 10) as solvent system. The plate was sprayed with 1% p-aminophenol and 4% phosphoric acid in distilled alcohol to locate the position of sugar bands after baking at 100°C for 30 min. Paper chromatography was carried out using butanol : pyridine : 0.1 N HC1(50:30: 20) as the solvent-system. Corresponding sugar bands on the paper chromatogram were detected by cutting 0.5 cm strips along the spot up to the solvent front and the radioactivity was counted using toluene-based scintillator containing 0.5% PPO and 0.1% POPOP in liquid scintillation counter, in addition to autoradiography. Standard sugars, chromatographed along were detected by spraying the paper as described for TLC. 1992 Sucrose Synthesis in C murtsepticum 535

Fig. 1. Thin-layer chromatography of sugars in the culture supernatant of cells grown on sucrose. Ten microliters of cell-free supernatant of 3 h sucrose-grown culture (B) was applied on silica gel plate. Chromatographic separation and detection of sugars were performed as described in MATERIALSAND METHODS. (A) Spots for standard sugars (from bottom): raffinose, melibiose, sucrose, galactose, glucose and fructose.

RESULTS AND DISCUSSION

Extracellular hydrolysis of raffinose and sucrose during cell growth In our earlier work, the mode of raffinose utilization in C. murisepticum was followed by the analysis of metabolic products in medium of the raffinose-grown culture, by thin-layer chromatography (TLC). It revealed presence of melibiose and fructose when cells were grown with raffinose as carbon source (11). This suggested that raffinose, a trisaccharide [ a-D-galactopyranosyl(1~6)-a-D-glucopy- ranosyl- (l->2) -$-D-fructofuranoside] is not hydrolyzed extracellularly by a- galactosidase (for which the products should have been sucrose and galactose), but by an invertase. In the present study, it was found that sucrose was hydrolyzed similarly, presumably by the same extracellular invertase activity (Fig. 1). Howev- er, detection of invertase but not of a-galactosidase in the sucrose-grown cells indicated that the two enzymes are not co-regulated to be parts of the same operon, as observed in E. coli (1). In this organism, enzymes metabolizing raffinose or sucrose have been found on raf or suc operons, respectively, located on plasmids (12,14). 536 NADKARNI, NAIR, PANDEY, and PRADHAN VOL. 38

Fig. 2. Thin-layer chromatography of sugars in the culture supernatant of cells grown on raffinose plus glucose. Ten microliters of cell-free supernatant of 3 h raffinose plus glucose-grown culture (B) was applied on silica gel plate. Chromatographic separation and detection of sugars were performed as described in MATERIALSAND METHODS. (A) Spots for standard sugars (from bottom): raffinose, melibiose, sucrose, galactose, glucose and fructose.

Extracellular sucrose synthesis during cell growth During the growth of C. murisepticum in a medium containing raffinose (0.4%) and glucose (0.4%), de novo formation of sucrose was clearly evident from the thin-layer chromatography analysis of the supernatant of the 3 h growth culture (Fig. 2). The absence of extracellular a-galactosidase, as also catabolite repression of a galactosidase synthesis by glucose (data not shown), implied that formation of sucrose was not the consequence of the hydrolysis of raffinose by a-galactosidase. At this stage, thus, it was evident that presence of glucose in the medium, along with raffinose, has some definite role in extracellular sucrose synthesis. In support of this observation, further studies were performed using radiolabeled glucose. The cell-free supernatant of culture grown in the medium containing raffinose and [U-14C]glucose, was analyzed by paper chromatography. It indeed revealed synthesis of 14C-sucrose-this could be confirmed by hydrolysis of the labeled sucrose with yeast invertase (Fig. 3). It was found that 20% of the total radiolabel could be detected in sucrose moiety. In another experiment, it was seen that addition of [U-14C]fructose to the cells growing on raffinose plus glucose did not lead to incorporation of [U-14C]fructose into sucrose (results not shown). Likewise none of the fructose derivatives, viz, fructose-l-phosphate, fructose-6-phosphate, 1992 Sucrose Synthesis in C. murtsepticum 537

Fig. 3. Evidence for extracellular synthesis of 14Csucrose from 14Cglucose plus raffinose by C. murisepticum: paper chromatography, autoradiography and radioactivity determination. Lyophilized cell-free supernatant of 3 h raffinose plus-[U)4C] glucose-grown culture was suspended in 50 mM phosphate buffer, pH 7.5. An aliquot of 10 ~cl was spotted on Whatman No. 3 paper (B, -0-). A other aliquot of 10,ul was incubated with yeast invertase (3 IU) at 37°C for 3Omin an spotted on Whatman No. 3 paper (C, --•--). Chromatographic separation and detection of sugars were performed as described in MATERIALSAND METHODS. (a) Autoradiography was carried out using dried paper chromatogram. (b) The autoradiogram was graphically presented for the quantitative radioactivity determination by cutting the paper chromatogram along the loading and counting the same in liquid scintillation counter. (A) Spots for standard sugars (from bottom): raffinose, melibiose, sucrose, galactose, glucose and fructose. fructose-l,6-diphsphate, when added to the growth medium, along with glucose, led to the formation of sucrose (data not shown). These results suggested, therefore, that the cells growing on raffinose can effectively transfer the fructosyl moiety of raffinose to glucose (provided in the medium) to form sucrose but fail to achieve sucrose synthesis from glucose and fructose or from glucose and any of the fructose phosphates supplied in the medium.

Isolation of extracellular invertase-like activity from raffinose-grown cells The above observations were supported with help of the enzyme preparation isolated from the cell-free supernatant of raffinose-grown culture (3 h growth). It could exhibit not only invertase activity but the ability to synthesize sucrose in vitro, from raffinose plus glucose (Fig. 4). Interestingly the enzyme preparation 538 NADKARNI, NAIR, PANDEY, and PRADHAN VOL. 38

Fig. 4. Thin-layer chromatography showing in vitro raffinose hydrolysis and sucrose synthesis by the extracellular enzyme(s) isolated from raffinose-grown C. murisepticum Reaction mixture [0.4% each of raffinose (B) or raffinose plus glucose (C) in 50 mM phosphate buffer, pH 7.5, containing enzyme preparation (0.75 fig) from cell-free supernatant of raffinose-grown cells] was incubated at 30°C for 30min. Aliquot of 10 iil was spotted on silica gel plate. Chromatographic separation and detection of sugars were performed as described in MATERIALSAND METHODS. (A) Spots for standard sugars (from bottom): raffinose, melibiose, sucrose, galactose, glucose and fructose. could also synthesize difructose in vitro, from raffinose or raffinose plus glucose. This indicated that the fructose moiety for the in vitro synthesis of sucrose or difructose has to be essentially derived from enzymatic hydrolysis of raffinose. To provide a definitive proof for the ability of the enzyme preparation to synthesize sucrose in vitro from raffinose plus glucose, [G-3H] raffinose obtained from New England Nuclear (U.S.A.) was used. Surprisingly, no radioactivity could be detected in the paper chromatography spots corresponding to fructose and sucrose. The failure to detect label in sucrose was however due to the revelation that this compound did not contain any radioactive label in its fructose moiety, as found by us in our test using yeast invertase (data not shown). The use of uniformly labeled raffinose could have helped in the clear resolution of the problem whether the synthesis of sucrose by the raffinose-grown cells (or cell-free supernatant therefrom) can be achieved from the transfer of fructose group from raffinose to glucose (when present along with raffinose). 1992 Sucrose Synthesis in C. murisepticum 539

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