TABLE 2.
Substrate specificity and kinetic parameters of CbXyn10Ca
| Substrate | Sp act (μmol/min/μmol of enzyme) | Km | kcat (s−1) | kcat/Km (ml s−1 mg−1) |
|---|---|---|---|---|
| Beech wood xylan | 39,000 ± 160 | 0.3 ± 0.1 mg ml−1 | 630 ± 16 | 2,100 |
| WAX | 51,000 ± 240 | 0.3 ± 0.0 mg ml−1 | 1,100 ± 15 | 3,700 |
| Barley β-glucan | 3,400 ± 15 | 2.7 ± 0.2 mg ml−1 | 85 ± 5.6 | 31 |
| CMC | 39 ± 3.3 | 4.3 ± 0.0 mg ml−1 | 1.8 ± 0.0 | 0.42 |
| pNPX | 19 ± 0.4 | 47 ± 0.1 μM | 0.1 ± 0.0 | 0.002 |
| pNPG | 0.5 ± 0.0 | 1.7 ± 0.0 μM | 0.02 ± 0.00 | 0.011 |
| Avicel | 3.4 ± 0.8 | ND | ND | ND |
| Filter paper | 5.4 ± 0.3 | ND | ND | ND |
| MCC | 2.3 ± 0.0 | ND | ND | ND |
| PASC | 1.2 ± 0.4 | ND | ND | ND |
| Lichenin | 180 ± 3.8 | ND | ND | ND |
The specific activities and kinetic parameters of CbXyn10C on konjac flour, arabic gum, and debranched arabinan were not determined in this study. No activity was detected on laminarin, pectin, carob bean gum, guar gum, locust bean gum, sugar beet arabinan, or xylogluan. The concentrations used for specific activity determination were 10 mg/ml for all polysaccharide substrates, while a series of concentrations (1 to 20 mg/ml) was used for estimation of the kinetic parameters of CbXyn10C on these substrates. The concentrations of pNPX and -pNPG used for specific activity measurement were 1 mM, while those used for kinetic studies were 0.1 to 4.0 mM. The released reducing sugars were determined by using the DNS method, and pNP was monitored spectrophotometrically at 405 nm. ND, not determined.