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required for complete reaction within 10 h is 1 U/mL IGT or 0.3 U/mL SGT (see Note ).

2. Reaction is carried out at 25°C under magnetic stirring.

3. End of the reaction: after 8-10 h, the reaction is stopped by heating the tubes at 90°C for 10 min (see Note —).

4. Elimination of dextran (see Note —): the GOS mixture is centrifuged for 5 min at 14 000g, room temperature, and then ultrafiltered using an Amicon hollow fiber system (membrane cutoff = 100,000).

5. Elimination of fructose: the GOS preparation obtained in 4 contains about 50 g/L fructose and 70 g/L of a mixture of maltose, a(1^2) and a(1^6) GOS. Fructose can be removed by chromatography on a strongly acidic column (Duolite C-204F) loaded with calcium.

6. Enrichment in a(1^2) GOS: the mixture can be enriched in a(1^2) GOS by incubation with endodextranase (20 U/mL) or glucoamylase (3 AGU/mL) (see Note 6).

• Reaction is carried out at 37°C, pH 5.4 (acetate buffer, 20 mM), with the GOS solution obtained in step 5 diluted by half.

• Reaction is nearly complete within 6 h, but it is allowed to continue for 15 h and stopped by heating the tubes at 90°C for 10 min.

7. All the GOS preparations (obtained in steps 3, 4, 5 and 6) can be stored a week at 4°C and several months at -18°C.

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HPLC Analysis

1. GOS are separated according to their size and structure by HPLC on a C18 column and eluted with 0.5 mL/min ultrapure water at room temperature (20-25°C). Products are detected by means of their refractive index with a refractometer. Typical chromatograms of the various preparations obtained are described in Note —.

2. The relative concentration of each molecule can be evaluated by calibration of the refractometer with maltose, sucrose, and fructose (0.5, 1, 2.5, and 5 g/L each) as references.

3. The apparent yield in oligosaccharides can be calculated by the following equation (see Notes 17 and

- 100 x Sum (final oligosaccharide concentrations) {maltose + U.474 sutrusc) initial concentrations

Notes

1. The various components are sterilized separately to avoid Maillard reactions.

2. The salt powders should not be mixed together before adding water. Otherwise, precipitation of most of the components will occur. In addition, care must be taken to mix the various products in the order indicated here.

3. A recent study of the medium composition was made to find the minimal concentration necessary for each component (17). Except for certain salts, all the elements in the medium were found to be essential for dextransucrase production. During the culture, sucrose is used by the bacteria as a carbon source for growth, but also as the dextransucrase inducer (18). Yeast extract provides the nitrogen components and vitamins necessary for growth. The phosphate buffer is used to slow down the pH fall owing to lactic acid and acetic acid production from sucrose metabolism by L. mesenteroides (19,20). A pH range between 6.5 to 6.9 is advised as the best compromise between enzyme stability and a cell physiology compatible with dextransucrase production (19,21).

4. After storage at low temperature (+4°C), standard medium must be heated to at least room temperature and at most 27°C before inoculation.

5. Do not use a higher polymerization degree than 1500, to avoid the precipitation of products other than dextran and dextransucrase.

6. Both enzymes convert the a(1^6) GOS into glucose residues. One unit of glucoamylase corresponds to the amount of enzyme that hydrolyzes 1 |mol of maltose/min at 25°C, pH 5.4 (acetate buffer 20 mM) in the presence of 100 g/L maltose. One unit of endodextranase corresponds to the amount of enzyme that produces 1 |mol of isomaltose/min from linear T70 dextran (Sigma, St. Louis, MO, 50 g/L) at 37°C and pH 6.0 (20 mM K2HPO4 buffer).

7. 27°C is the recommended temperature for the best compromise between rapid growth and enzyme stability; enzyme production is also possible at lower temperatures (20-25°C), but it is inadvisable to use a culture temperature higher than 27°C (21,22).

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8. Cell growth can be followed by measuring the absorbance at 650 nm. The optimum dilution range (distilled water) is between 5 and 20. After about 6 h, it reaches quite high values: about 20 absorbance units. These high values result from cell multiplication but also from dextran formation from sucrose owing to the extracellular dextransucrase activity. For additional details on enzyme production, see Dols et al. (17,20).

9. L. mesenteroides NRRL B-1299 dextransucrase is mainly insoluble: 90% of the enzyme is recovered in the pellet after centrifugation (23). Many unsuccessful attempts have been made to increase the enzyme solubility (9,24). Moreover, during batch or fed-batch cultures in stirred reactors, the proportion of the soluble form of dextransucrase remains unchanged even when it is possible to increase the level of activity produced significantly (17). Nevertheless, both soluble and insoluble dextransucrase produce a(^6) GOS (9), but the respective protocols necessary to concentrate each form of dextransucrase are different and thus they are generally used separately. The protocol described in Subheading 3.2. enables a standard activity recovery of 98% from the culture medium. The final SGT preparation contains soluble dextran associated with dextransucrase, but the insoluble preparation contains cells and insoluble dextran. Consequently, NaN3 is used to avoid cell metabolism in the presence of substrates, but it does not affect dextransucrase activity. No significant interfering activity (levansucrase, invertase, or sucrose-phosphorylase) can be detected in the two dextransucrase fractions produced by L. mesenteroides NRRL B-1299 (20).

10. Resuspension can be made easier by means of an Ultra Turrax (IKA ULTRA TURRAX ®T25, Janke & Kunkel, Labortechnik Staufen) mixer.

11. Standard activity measurements are carried out in order to measure the active enzyme content of either the culture medium or the final SGT and IGT preparations. They consist of measuring the velocity of fructose formation from sucrose during dextran synthesis by dextransucrase (6). Fructose is assayed as a reducing sugar by the method of Sumner and Howell (25). If, for dextransucrase production, the advised pH value is around 6.7 (see Note ), that for both enzyme purification and activity measurements is 5.4 (23).

12. In the presence of sucrose and maltose (acceptor molecule), dextransucrase catalyzes the transfer of glucosyl units to the nonreducing end of the acceptor (Fig. 2). The fructose produced accumulates in the medium or can act as an acceptor. The first product of the reaction on maltose is panose (6-O-a-D-glucosyl maltose). Panose is also an acceptor for dextransucrase resulting in an incease in molecule length and a higher mol-wt GOS (8). Beyond the degree of polymerization 4, both a(1^6) and a(1^2) bonds are formed. All the GOS bearing only a (1^6) linkages (except the a[1^4] bond coming from maltose) are easily digestible by glucoamylase ; they belong to the OD (oligodextran) family. On the other hand, the a(1^2) GOS withstand hydrolysis by such enzymes; they were called R, for resistant to hydrolysis (9). As shown in Fig. 2, dextran and leucrose are side-products of the reaction. Dextran polymer is directly synthesized from sucrose, wheras leucrose (5-O-a-D glucosyl D-fructopyranose) is the result of an acceptor reaction on fructose (26).

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Schematic representation of the various reactions catalyzed by L. mesenteroides NRRL B-1299 dextransucrase in the presence of maltose and sucrose.

Schematic representation of the various reactions catalyzed by L. mesenteroides NRRL B-1299 dextransucrase in the presence of maltose and sucrose.

13. Owing to its specific dextran content, SGT is three times more active than IGT in the presence of maltose, and it can be introduced in lower quantities into the reaction mixture (9,23).

14. If the reaction lasts more than 20 h, the composition of the oligosaccharide preparation obtained can be modified because of disproportionation reactions (27).

15. This protocol enables the elimination of the catalyst and both the insoluble and the soluble endogenous dextran, which would damage the HPLC columns.

16. The chromatogram of the oligosaccharide preparation (obtained after fructose elimination) is presented in Fig. 3A. The first peak eluted corresponds to fructose. The second one corresponds to maltose, which cannot be separated from leucrose, unless leucrose and maltose are present in equimolar quantities in the mixture. Just after this second peak, a third one could indicate the presence of remaining sucrose. Next, comes the peak of panose, followed by a series of GOS of degree of polymerization higher than 4.

Among these molecules, the a(1^2) GOS can be identified by comparison with the chromatogram of the glucoamylase treated GOS (Fig. 3B). These GOS are named Ri in Fig. 3, i being the degree of polymerization of the GOS, and the a(1^6) GOS are named ODi.

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HPLC analysis of the GOS synthesized with 100 g/L sucrose, 33 g/L

maltose, and 1 U/mL IGT. Products noted ODi represent the oligosaccharides of degree of polymerization i composed of a(l^6) linkages and a maltose residue at the reducing end. Products noted Ri represent oligosaccharides bearing an a(1^2) linkage. (A) Acceptor reaction products. (B) Acceptor reaction products after glucoamylase hydrolysis. (C) Acceptor reaction products after endo-dextranase hydrolysis. (D) Acceptor reaction products after glucoamylase and endo-dextranase hydrolysis.

HPLC analysis of the GOS synthesized with 100 g/L sucrose, 33 g/L

maltose, and 1 U/mL IGT. Products noted ODi represent the oligosaccharides of degree of polymerization i composed of a(l^6) linkages and a maltose residue at the reducing end. Products noted Ri represent oligosaccharides bearing an a(1^2) linkage. (A) Acceptor reaction products. (B) Acceptor reaction products after glucoamylase hydrolysis. (C) Acceptor reaction products after endo-dextranase hydrolysis. (D) Acceptor reaction products after glucoamylase and endo-dextranase hydrolysis.

As can be seen in Fig. 3C and D, the ODi GOS can also be eliminated by endodextranase treatment. This treatment enriches the medium mostly with R4 and R5. R7 can be eliminated by dextranase treatment, but a new a(1^2) GOS called R'4 appears.

17. The equation takes into account the weight of all the monosaccharides involved in the oligosaccharide structure: it comprises: (a) Numerator: all the oligosaccharide concentration (g/L); and (b)Denominator: the entire maltose concentration (g/L), but only the glucosyl moiety (MM = 180 g) of sucrose (MM = 342 g) after elimination of a water molecule (180-18 = 162 g); that is to say, that only 162/342 = 0.474 x the initial sucrose concentration (g/L) can take part in the oligosaccharide synthesis.

18. Under the test conditions, this yield is about 70%, the fraction of GOS bearing an a(1^2) linkage being approx 50%. These results can be modified by changing the reaction conditions, especially the sucrose/maltose (S/M) concentration ratio (Dols et al., in preparation).

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