灰绿曲霉(Aspergillus glaucus)发酵液通过硫酸铵盐析、Sephadex G-100分子筛、DEAE Sepharose Fast Flow离子交换柱和Phenyl Sepharose Fast Flow疏水层析,分离纯化一种外切葡聚糖酶(CBH)和一种内切葡聚糖酶(EG).通过SDS-PAGE和凝胶柱层析法测定分子质量表明:CBH全酶分子质量为71 ku,由两个分子质量为35 ku的同型亚基组成;EG为单体蛋白,全酶分子质量为32 ku.酶学性质研究表明:CBH催化pNPC的最适pH为6.0,最适温度为55℃,酶活在pH 5.0~8.0区间和温度低于55℃时稳定;EG催化CMC-Na的最适pH为4.0,最适温度为50℃,酶活在pH3.5~7.5区间和温度低于65℃时稳定.Na+、K+、Ba2+、Mg2+以及NO3-和SO42-对CBH和EG酶活均无影响;Ca2+和Mn2+对CBH有激活作用,Fe2+和Mn2+对EG有激活作用,而Zn2+、Cd2+和Cu2+对CBH和EG均有不同程度的抑制效应.酶动力学分析表明:CBH催化pNPC水解的米氏常数Km值为1.4 mmol/L(pH 6.0,55℃),EG催化CMC-Na水解的米氏常数Km值为5.0 mg/mL(pH 4.0,50℃).
A cellobiohydrolase(CBH) and an endoglucanase(EG) were purified from fermentation liquor of Aspergillus glaucus by following procedures: ammonium sulfate precipitation,gel filtration on Sephadex G-100,ion-exchange chromatography on DEAE Sepharos Fast Flow and hydrophobic chromatography on Phenyl Sepharose Fast Flow.By the methods of gel column chromatography and SDS-PAGE,the molecular weight of CBH was determined to be 71 ku,consisted of two 35 ku subunits,and the EG was determined to be a 32 ku monomer.Enzymatic properties showed that the optimum pH and temperature of CBH for the hydrolysis of pNPC was at pH 6.0 and 55 ℃,respectively;CBH was stable at pH 5.0~8.0 and below 55 ℃;the optimum pH and temperature of EG for the hydrolysis of CMC-Na was at pH 4.0 and 50 ℃,respectively;EG was stable at pH 3.5~7.5 and below 65 ℃.Na+,K+,Ba2+,Mg2+,NO-3 and SO2-4 had no effects on CBH and EG activities.Ca2+ and Mn2+ activated CBH activity,Fe2+ and Mn2+ activated EG activity,while Zn2+,Cd2+ and Cu2+ inhibited CBH and EG activities.Enzymatic kinetics indicated that Michaelis-Menten contant(Km) of the hydrolysis for pNPC by CBH was 1.4 mmol/L(pH 6.0,55 ℃) and Km of the hydrolysis for CMC-Na by EG was 5.0 mg/mL(pH 4.0,50 ℃).