Table 1. Summary of enzymes used in this study and their action.
| Enzyme (source) | Reference | Activity [U/mg] | Shipping buffer | Buffer, pH and T (°C) used in this study for the hydrolysis experiments* | Linkage hydrolysis** |
|---|---|---|---|---|---|
| α-L-arabinofuranosidase Clostridium thermocellum | EC 3.2.1.55 | 125 | 35 mM NaHepes pH 7.5, 750 mM NaCl, 200 mM imidazol, 3.5 mM CaCl2, 0.02% sodium azide, 25% glycerol. | Phosphate buffer 50 mM, pH 7, 65 °C. | Terminal non-reducing α-L-arabinofuranoside. |
| β-glucuronidase Escherichia Coli | EC 3.2.1.31 | 37000 | 20 mM Tris-HCl pH 7.5, 50 mM NaCl, 0.1 mM EDTA plus 0.02% (w/v) sodium azide. | Phosphate buffer 100 mM, pH 6.8, 37 °C. | Terminal non-reducing β-D-glucuronic acid residues. |
| α-L-rhamnosidase Prokaryote | EC 3.2.1.40 | 190 | Ammonium sulphate suspension in 0.02% (w/v) sodium azide. | Phosphate buffer 20 mM, pH 6.5, 50 °C. | Terminal non-reducing α-L-rhamnose. |
| β-galactosidase Aspergillus niger | EC 3.2.1.23 | 175 | 3.2 M ammonium sulphate + 0.02% Na azide. | Acetate buffer 50 mM, pH 4.5, 60 °C. | Terminal non-reducing β-D-galactose. |
| exo-β-1,3-galactanase Clostridium thermocellum | EC 3.2.1.145 | 20 | 35 mM NaHepes pH 7.5, 750 mM NaCl, 200 mM imidazol, 3.5 mM CaCl2, 0.02% sodium azide, 25% glycerol | Phosphate buffer 50 mM, pH 6, 50 °C. | Terminal non-reducing β-D-galactose residues in β-1,3-galactan. |
| α-galactosidase Cellvibrio mixtus | EC. 3.2.1.22 | 150 | 35 mM NaHepes pH 7.5, 750 mM NaCl, 200 mM imidazol, 3.5 mM CaCl2, 0.02% sodium azide, 25% glycerol. | Tris-HCl buffer 25 mM, pH 8.5, 37 °C. | Terminal, non-reducing α-D-galactose. |
| endo-1,4-β-mannanase Cellvibrio japonicus | EC 3.2.1.78 | 2200 | 35 mM NaHepes pH 7.5, 750 mM NaCl, 200 mM imidazol, 3.5 mM CaCl2, 0.02% sodium azide, 25% glycerol. | Phosphate buffer 50 mM, pH 7.5, 45 °C. | (1–4)-β-D-Mannosidic linkages. |
| Enzyme cocktail: exo-β-1,3-galactanase Clostridium thermocellum/endo-1,4-β-mannanase Cellvibrio japonicus (v/v) | EC 3.2.1.145/EC 3.2.1.78 | 20/2200 | 35 mM NaHepes pH 7.5, 750 mM NaCl, 200 mM imidazol, 3.5 mM CaCl2, 0.02% sodium azide, 25% glycerol. | Phosphate buffer 50 mM, pH 7, 45 °C. |
*The enzymes’ activities were evaluated via experiments involving 25, 100, 500 and 1000 mU (see experimental part), the best results were obtained with 100 mU of exo-β-1,3-galactanase and 100 mU of endo-β-1,4-mannanase. The best incubation duration obtained in this study is 5 h.
**Information obtained by Megazyme (Wicklow, Ireland) and NZYtech (Lisboa, Portugal) suppliers, and by the on-line “The Comprehensive Enzyme Information System”, Braunschweig, Germany (http://www.brenda-enzymes.org/index.php4).