里氏木霉纤维素酶的分离纯化及酶学性质

通过(NH4)2SO4分级沉淀、HiPrep 26/10 Desalting凝胶色谱脱盐、Source 15 Q阴离子交换色谱技术,里氏木霉(Rut C-30)纤维素酶主要组分得以初步分开,再经过Source 15 S阳离子交换色谱、HiPrep Sephacryl S-100 HR凝胶过滤色谱、Superdex 75 PrepGrade凝胶过滤色谱进一步分离纯化,得到2个纯化的内切葡聚糖酶组分EGⅡ、EGⅠ和一个外切葡聚糖酶组分CBHⅠ;经过SDS-PAGE电泳鉴定为电泳纯,测得相对分子质量分别为5.22×104,5.62×104和6.90×104。EGⅡ的最适反应pH是5.6,最适反应温度为65℃;EGⅠ的最适反应pH是4.4,最适反应温度为55℃;以羧甲基纤维素(CMC)为底物时,EGⅠ、EGⅡ的米氏常数(Km)分别为2.20 mg/mL、3.38 mg/mL。CBHⅠ的最适反应pH是5.8,最适反应温度为60℃,以对硝基苯基-β-D-纤维二糖苷(PNPC)为底物时,米氏常数(Km)为0.12 mg/mL。

Purification and characterization of cellulase from Trichoderma reesei

The cellulase of Trichoderma reesei Rut C-30 was separated by ammonium sulfate precipitation,HiPrep 26/10 Desalting gel layer chromatography and Source 15Q anion exchange.Two endoglucanases（EG II,EG I） and one cellobiohydrolase CBH I were purified to homogenety by using HiPrep Sephacryl S-100 HR,Superdex 75 PrepGrade gel layer chromatography and Source 15 S cation exchanger.The enzyme single subunit molecular weights of EG II,EG I and CBH I were about 5.22×104,5.62×104 and 6.90×104,which were identified by SDS-PAGE.The optimal reaction pH of EG II,EG I and CBH I was 5.6,4.4 and 5.8.The optimal reaction temperature of EG II、EG I and CBH I were 65,55 and 60 ℃.The Km values of EG II,EG I were 2.20 and 3.38 mg/mL by using CMC as the substrate,and that of CBH I was 0.12 mg/mL by using PNPC as the substrate.