灰绿曲霉产纤维素酶的研究  

Cellulase Production from Aspergillus glaucus

作　　者：陶毅明[1] 马素娟[1] 邬小兵[1] 龙敏南[1,2] 黄建忠[3] 陈清西[1,3] 

机构地区：[1]厦门大学生命科学学院细胞生物学与肿瘤细胞工程教育部重点实验室 [2]厦门大学能源研究院,福建厦门361005 [3]福建师范大学工业微生物教育部工程研究中心,福建福州351008

出　　处：《厦门大学学报（自然科学版）》2010年第3期391-395,共5页Journal of Xiamen University：Natural Science

基　　金：国家重点基础研究计划(2010CB732201);福建省自然科学基金(2009J01196);福建师范大学工业微生物教育部工程研究中心开放课题(IM200802)

摘　　要：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 ℃）.

关 键 词：灰绿曲霉 纤维素酶 外切葡聚糖酶 内切葡聚糖酶 分离纯化 酶学性质 

分 类 号：Q556.2[生物学—生物化学]