摘要
交联酶聚体(CLEA)是一种新型的无载体固定化技术.以胰蛋白酶为模型体系,系统地研究了CLEA技术的制备工艺、应用条件、稳定性及结构形貌.(1)在制备工艺中考察了各步骤对CLEA酶活保留的影响,重点分析了沉淀剂浓度、类型的影响,结果表明100%乙醇是较为理想的沉淀剂;(2)在应用条件确定中测定CLEA的最适催化温度为70℃,最适催化pH为9.0,并解释了温度-活性、pH-活性曲线的漂移现象;(3)稳定性研究结果表明CLEA技术大幅提高了胰蛋白酶的热稳定性、溶剂稳定性,且无酶泄漏;(4)在形貌表征中分别利用扫描电子显微镜、光学显微镜和激光粒度分析对CLEA的微观结构进行了多尺度研究,着重讨论了其独特结构与优良特性间的关系.本文所得结论为胰蛋白酶CLEA的应用提供基础数据,并为CLEA技术应用于其它酶种提供参考。
Cross-linked enzyme aggregation （CLEA） is a novel cartier-free immobilization technique. In this study, trypsin-CLEA has been prepared and its preparation routine, application condition, stability and structure have been studied systematically. （1） Every step for CLEA preparation was investigated to evaluate the effect on the enzyme-activity retention with special aims at the precipitant concentration and type. The experimental results indicated that pure alcohol was an ideal precipitant. （2） The best temperature and pH for CLEA catalysis were determined respectively as 70 ℃ and 9.0, and a detailed explanation was given to clarify the phenomena about shift of the temperature-activity and pH-activity curves. （3） The enzyme stability versus temperature and solvent was tested and the enzyme leakage was examined. This investigation result confirms that the CLEA technique can fundamentally improve the stability of trypsin. （4） The structure of CLEA was characterized by scan electronic microscopy, optical microscopy and laser particle size analysis, respectively. The relationship between the structure and property of CLEA was also discussed. The obtained result of the present research may benefit the further application of CLEA technique, and the observed rules can be applied to other enzyme-type CLEA.
出处
《化学学报》
SCIE
CAS
CSCD
北大核心
2008年第16期1929-1934,共6页
Acta Chimica Sinica
基金
教育部科学技术研究重点项目(No.108031)
国家高技术研究发展计划(863计划)专题课题(No.2008AA10Z318)资助项目
关键词
胰蛋白酶
交联酶聚体
固定化
酶的稳定性
trypsin
cross-linked enzyme aggregation （CLEA）
immobilization
enzyme stability
