摘要
耐热纤维素酶具有广泛的应用前景,本研究室在利.用大肠杆菌表达来自Pyrococcus horikoshii的极端耐热β-1,4-内切纤维素酶基础上,建立了包括热处理、DEAE Sepharose Fast Flow阴离子层析的纯化工艺,分离纯化获得单一组分的重组极端耐热β-1,4-内切纤维素酶(EGPh),SDS-PAGE测定该酶纯度大于90%,以CMC-Na为底物,DNS法测定比活22.3 U/mg,活性收率为73.3%,纯化倍数为45倍.酶学性质研究表明,该酶的分子量为43 KD,等电点(PI)为5.3,催化CMC-Na最适反应温度为95℃,最适反应pH为6.0,Km值为3.61±0.26 mg/ml.酶学稳定性研究表明EGPh在pH4.0～9.0、温度85℃下稳定.纯化的EGPh表现了极好的热稳定性,95℃时半衰期为8 h.
Hyperthermophilicβ - 1,4 endo cellulose （EGPh） is a promising cellulase in industrial application. On the foundation of successful construction and expression the enzyme from Pyrococcus horikoshii in Escherichia coli, we established a high efficiency purification procedure, including heat treatment and an ion-exchange chromatography on DEAE Sepharose Fast Flow. Using the procedure, the purity of EGPh on sodium dodecylsulfate-polyacrylamide gel electrophoresis （SDS - PAGE） was over 95% with activity recovery of 73.3 % by 3, 5-dinitrosalicylic acid （DNS） method and purification multiple of 45 times. The specific activity of purified EGPh was 22.3 U/mg using CMC-Na as substrate. The study on the enzymatic properties showed, the enzyme has molecular mass of 43 KD and isoelectric point （PI） of 5.3. The optimum pH and temperature of EGPh for the hydrolysis of CMC-Na was pH6.0 and 95 ℃ with Km of 3.61 ±0.26 mg/ml. Study on enzymatic sta- bility showed that EGPh was stable at pH4.0 - 9.0 and below 85 ℃. The purified EGPh showed a remarkable thermostability with a half - life of 8 h at 95 ℃.
出处
《天津理工大学学报》
2013年第4期51-56,共6页
Journal of Tianjin University of Technology
基金
天津市滨海新区科技小巨人成长计划-科技型企业创新发展(科技创业)项目(2010-BK130070)
科技型中小企业技术创新基金项目(11C26211203970)
天津市滨海新区重大科技项目(2011-BK120014)
天津市科技计划项目(12ZCZDSY01800)
关键词
耐热纤维素酶
离子层析
纯化工艺
酶学性质
hyperthermophilic cellulose
ion- exchange chromatography
purification
enzymatic properties
