TABLE 3.
Quantitation of U and X residue accumulation in cultures by 1H NMR
| Strain | U-1 and X-1 |
U-5 and X-5 |
% MeGXne | ||||
|---|---|---|---|---|---|---|---|
| Concn (mM) |
X-1/U-1 | Concn (mM) |
X-5/U-5 | ||||
| U-1a | X-1b | U-5c | X-5d | ||||
| 168 | 11.8 | 48.9 | 4.1 | 13.5 | 51.3 | 3.8 | 88 |
| MR42 | 11.1 | 55.6 | 5.0 | 12.4 | 53.2 | 4.3 | 83 |
| MR44 | 11.5 | 84.2 | 7.3 | 13.0 | 93.2 | 7.1 | 86 |
1H–U-1 determined as the integration ratio of 1H atom equivalents of MeG 1H–C-1 (5.28 to 5.32 ppm) to acetone [1H-(CH3)2; 2.23 ppm] set to 1.00 for 188 mM 1H atom equivalents.
1H–X-1 determined as the ratio of the sum of 1H integrations for α–X-1 (5.19 to 5.20 ppm), U–X-1 (4.60 to 4.68 ppm), β–X-1 (4.56 to 4.59 ppm), int–X-1 (4.47 to 4.5 ppm), and nr–X-1 (4.45 ppm) to 1.00 for acetone at 188 mM 1H atom equivalents.
1H–U-5 determined as the ratio of 1H integration (4.31 to 4.35 ppm) to 1.00 for acetone at 188 mM 1H atom equivalents.
1H–X-5 (axial only) determined as the ratio of 1H integration (4.08 to 4.12 ppm) to 1.00 for acetone at 188 mM 1H atom equivalents.
The percentage of the MeGXn substrate converted to accumulated aldouronate products by each culture was determined on the basis of the X/MeG ratio found in the products generated from 0.5% MeGXn substrate after complete digestion with pure XynC. XynC digestion generated exclusively U-XOS containing a single MeG with an X/MeG ratio of 6.9 for integration of X-1/U-1 by 1H NMR. The concentration of MeGXn in the uninoculated medium was 5 mg ml−1, and following the 3× concentration of 3.0 ml of culture medium during the process of D2O exchange prior to NMR analysis (see Materials and Methods), the accumulated products would have been derived from 15 mg ml−1 MeGXn. Using the molecular mass for the product MeGX6.9 (191 + 5.9 × 132 + 150 = 1,120 mg mmol−1), the concentration of MeGX6.9 equivalents, equal to the concentration of MeG equivalents in the uninoculated medium, was 15 mg ml−1/1,120 mg mmol−1, or 13.3 mM. This value, divided by the concentration of U-1, provides an estimate of the fraction (in percent) of the MeGXn accumulated as products of MeGXn digestion.