Purification and characterization of cellulasesfrom Trichoderma harzianum strain ETS 323

• Main Authors: Hua-Jing Jhan, 詹華景
• Other Authors: Kou-Cheng Peng
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/91352264158374179782

碩士 === 國立東華大學 === 生物技術研究所 === 93 === Trichoderma harzianum is the soil-borne filamentous fungus that is notable for biological control. The major reason is T. harzianum could secrete many cell-wall-degrading enzymes. In this study, two kinds of cellulases were purified from T. harzianum strain ETS 323. We also characterized their biochemical and biophysical properties. The two cellulases also exhibited β-glucosidase activity by TLC analysis. The molecular mass of cellulase A and cellulase B determined by SDS-PAGE was 23.5kDa and 61kDa, respectively. According to the ion exchange chromatography, the pI value of cellulase A is 8.3~8.5 and of cellulase B is 4.0~5.0. The specific activity of cellulase A is 7.4 U/mg, purification fold is 7.3, and the yield is 5 %. The specific activity of cellulase B is 24.6 U/mg, purification fold is 33.7, and the yield is 0.15 %. The pH range for the activity of cellulase A and B are pH 3.0~pH 6.5 and pH 3.0~pH 5.5, respectively, pH optimum for both cellases is pH 4.5. The pH stability of cellulase A and cellulase B was pH 4.0~pH 5.5 and pH 3.0~pH 5.5, respectively. The temperature range for the activity of cellulase A and B are 20~60 oC and 40~60 oC, and temperature optimum for both cellulases is 50oC. The temperature stabilities of cellulase A and cellulase B are 20~40 oC and 40~60 oC, respectivity. The Km and Vmax of cellulase A estimated for CMC was 23 mg/mL and 294 µM/min, respectively. Whereas the Km and Vmax of cellulase B estimated for CMC was 84.8 mg/mL and 384.6 µM/min, respectively. MnCl2 could stimulate the enzyme activities of both cellulases and raised the activities more than the 90% than original. As to specificity, Cellulase A has higher specifity, and cellulase B has could use xylan as substrate 60% enzyme activity in xylan. Both cellulases are stable at 38 oC, which is the temperature for Simultaneous Saccharification and Fermentation (SSF) in the industrial settings. In conclusion, it is the first time that purification and characterization of cellulases from T. harzianum strain ETS 323 had been reported. In the future both cellulases could be applied to the industry and increase the economical value.