里氏木霉纤维素酶的分离纯化与酶学性质研究-生物化工专业论文.docxVIP

Pu尚cation Pu尚cation and Characterization of CeHulases from the Fungus Tdchoderma reesei Yu Zhao-hai Supervised by Professor Jiang Hua (Conege of Chemical Engineering，Nanjing Forestry University,210037) June 2006 Abstract Using ultrafiltration，cationic(CM-Sepharose FD and anionic(DEAE-Sepharosc FF) chromatography and size exclusion(Sephadex G-100)，six cdlulolytic enzymes were purified from the culture broth of Trichoderma reesei：three endoglucanases(EG I,EG II，EGffl)，two ceUobiohydrolases(CBH I，CBH]I)and a肛gulcosidase(GB)．The six ceHulases displayed one single band on SDSrespectively．The specific activity of EG I正G II and EGm with eathoxyinethyl ceHulose was estimated to be 176．35 IU·mg1,153．96 IU·mg-1,64．22 IU．mg1 respectively．For CBH I and CBH II．血c specific activity was 16．86 IU．mgq,4．82 IU．mg-1 respectively with mierocellulose．The specific activity of the 13-gulcosidase witll salicin was 31_00 IU·na91． Basic enzymatic characteristic of obtained cellulases were studied．The relative molecular weightofEGI，EGII。EGⅢ，CBHI，CBHⅡandGBwas50kD，46kD，25kD，65 kD，58．1 kD and 75kD，respectively．Kinetics method showed the Km of EG I，EG II，EGIII for carboxymethyl cellulose was 6．70 meCmL，8．46 mg／mL，13．22 mgCmL respectively．For CBH I and CBHII，the Km With microceUulose was 1．37 mg／mL’and 3．46 mg／mL respectively．The Km ofB—gulcosidase with salicin was 2．20 mg／mL．For EG I，EG II，EGIII，CBH I，CBHⅡ and GB，the optimum temperture was 55C，50*(2，55C，60。C，45*(2 and 60。C respectively, while their optimum pH at 5．6，4．4，5．0，5．6，5．0 and 5．0． Besides，the eeUulases had the relatively good stability under 30。C，while in the temperture upon 80℃．they lost their activity quickly．The ceHulases were relatively stable within pH 5．ON 6．2．EG I和CBH I had the stronger stability,which had strong activity after being kept at pH 6．8 for5．0h． Key words：Trichoderma reesei；Cellulases；Purification；Charactedzation；Ultraffltration 致 致 谢 本论文是在导师江华副教授的悉心指导下完成的，从论文的选题到研究 内容的确立，从论文的撰写到最终的定稿，每个环节都倾注着导师大量的心 血。三年来，江老师以他严谨