A method of purification, identification and characterization of beta-glucosidase from Trichoderma koningii AS3.2774.

摘要

In this study, we used native gradient-polyacrylamide gel electrophoresis and electroelution (NGGEE) to purify enzymatic proteins from Trichoderma koningii AS3.2774. With this method, we purified eight enzymatic proteins and classified them to the cellulase system by comparing secretions of T. koningii in inductive medium and in repressive medium. It resulted in 24-fold beta-glucosidase (BG) purification with a recovery rate of 5.5%, and a specific activity of 994.6 IUmg-1 protein. The final yield of BG reached 8 mug under purifying procedure of NGGEE. We also identified BG using the enzyme assay with thin-layer chromatography and MALDI-TOFMS. This BG had one subunit with a molecular mass of 69.1 kDa as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The hydrolytic activity of the BG had an optimal pH of 5.0, an optimal temperature of 50 degrees C, an isoelectric point of 5.68 and a Km for p-nitrophenyl- beta-D-glucopyranoside of 2.67mM. Taken together, we show that NGGEE is a reliable method through which mug grade of active proteins can be purified.