A method of purification, identification and characterization of β-glucosidase from Trichoderma koningii AS3.2774.
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 β-glucosidase (BG) purification with a recovery rate of 5.5%, and a specific activity of 994.6IUmg(-1) protein. The final yield of BG reached 8μg 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 69.1kDa 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°C, an isoelectric point of 5.68 and a K(m) for p-nitrophenyl-β-d-glucopyranoside of 2.67mM. Taken together, we show that NGGEE is a reliable method through which μg grade of active proteins can be purified.
Lin
Yuanshan
Y
College of Life Science & Technology, Guangxi University, Nanning 530004, China; College of Bioscience & Biotechnology, Hunan Agricultural University, Changsha 410128, China. wallslin@126.com
Chen
Guiguang
G
Ling
Min
M
Liang
Zhiqun
Z
D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D007700:Kinetics; D008970:Molecular Weight; D014242:Trichoderma / Q000737:chemistry / Q000201:enzymology*; D001617:beta-Glucosidase / Q000737:chemistry* / Q000302:isolation & purification*
D005656:Fungal Proteins; D001617:beta-Glucosidase