TABLE 1.
Catalytic activity of CtXyl5A and its variants
The enzymes were assayed at 60 °C in 50 mm sodium phosphate buffer, pH 7.0, containing substrate at a concentration of 1 mg ml−1. The reaction was monitored by the release of reducing sugar (14). The catalytic rate could be used to determine kcat/Km values as the substrate concentration was ≪ Km (the rate of reaction was directly proportion to substrate concentration up to 2 mg ml−1). Note that CtXyl5A is the full-length enzyme, and CtGH5-CBM6 and CtGH5 are derivatives of the enzymes containing the catalytic module appended to the CBM6 and the catalytic module, respectively.
| Proteins |
kcat/Km |
||
|---|---|---|---|
| Rye arabinoxylan | Wheat arabinoxylan | Oat spelt xylan | |
| min−1mg−1ml | |||
| CtXyl5A | 1322 ± 357 | 808 ± 76 | 15 ± 1.2 |
| CtXyl5A + 2 mm CaCl2 | 2271 ± 274 | 1658 ± 56 | NDa |
| CtXyl5A + 5 mm EDTA | 858 ± 102 | 343 ± 32 | ND |
| CtGH5-CBM6 | 1012 ± 83 | 652 ± 21 | 14 ± 2.2357 |
| W424A (CtGH5-CBM6) | 1656 ± 173 | 728 ± 127 | ND |
| F478A (CtGH5-CBM6) | 983 ± 52 | 713 ± 66 | ND |
| E279A (CtGH5-CBM6) | NAb | NA ± 357 | ND |
| E171A (CtGH5-CBM6) | NA | NA | ND |
| CtGH5 | 1.1 ± 0.45 | 0.6 ± 0.07 | NA |
a ND means not determined.
b NA means no activity detected.