Abstract:
Cellulase purification from the broth of the mutant strain Trichoderma koningii SG0026 allowed for the investigation of the enzymatic properties of its cellulase system. Three electrophoretically pure components of the cellulase system (endo-β-1,4-glucanase, exo-β-1,4-glucanase, and β-glucosidase) were isolated and purified from the broth of the mutant strain Trichoderma koningii SG0026 by a combination of ammonium sulfate fractionation, Sephadex G-100 gel filtration, DEAE-Sepharose FF anion exchange column chromatography, and CM-Sepharose FF cation exchange chromatography. The specific enzyme activities of electrophoretically pure endo-β-1,4-glucanase, exo-β-1,4-glucanase, and β-glucosidase were measured to be 4.67 ± 0.06, 5.16 ± 0.08, and 12.52 ± 0.12 IU/mg, respectively. The relative molecular masses of three cellulases were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and were 78.1, 91.2, and 83.1 kDa, respectively. The Lineweaver-Burk method was used to calculate the kinetic parameters of the three cellulases; the Km values for endo-β-1,4-glucanase, exo-β-1,4-glucanase, and β-glucosidase were 3.84, 6.62, and 6.21 mg/mL, respectively and the Vmax values were 2.29, 1.74, and 2.19 mg/(min?mL), respectively. Based on the results, the reaction temperature and pH of these three cellulases were determined. The optimal temperatures for endo-β-1,4-glucanase, exo-β-1,4-glucanase, and β-glucosidase were 50, 50, and 55℃, respectively, and their optimum pH values were 5.0.
