Puification And Characterization Of An Engo-β-Glucanase From Trichoderma Koningii

Cellulose utilization depends largely on efficient cellulase, and therefore to carry out the separation, purification and characterization of cellulase enzyme, for the promotion of resource utilization of cellulose is important.An endo-β-glucanase was purified by methods of ammonium sulfate precipitation, Sephacryl S-200HR gel filtration chromatography, DEAE Sepharose FF ion exchange chromatography and Octrl Separose CL-4B hydrophobic interaction chromatography from the culture filtrate of Trichoderma koningii GIMP3.444. The molecular weight of the enzyme, which was identified as a single band by SDS-PAGE, is61.8kDa determined by gel imaging system。The final purified fold, specific activity and the yield of the purified enzyme were7.55,24.91IU·mg-1and3.72%, respectively.Characterization analysis showed that the optimum reaction temperature of the endo-β-glucanase was55℃, and the optimum pH was4.4. Besides, the endo-β-glucanase had relatively good stability under60℃,and the good pH stability range was3.2-6.2, while4.4-5.0was the best pH stability range. The values of Km and Vmax of the enzyme for CMC-Na obtained from Lineweaver-Burk were4.81mg/mL and2.48mg/(min·mL), respectively. And the activity of the endo-β-glucanase was inhibed in different degrees by different metal ions, wherein the inhibition of Mn2+was most obvious.Considering both the influence of pH on the substrate dissociation and the inhibition of product glucose on endo-p-glucanase, the values of Km’and Vmax’were measured at different pH to verify the kinetic model of influence of pH on endoglucanase activity. The result was that the optimum pH value gained by the kinetic model matched that got from experiment.