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Analysis and improvement of hyperthermostable endo-glucanase from Archaea

古细菌超热稳定内切葡聚糖酶的分析与改进

基本信息

批准号：
17613009
负责人：
ISHIKAWA Kazuhiko
金额：
$ 2.3万
依托单位：
National Institute of Advanced Industrial Science and Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

In this study, I tried to analyze and improve the function of the hyperthermostable cellulose from hyperthermophilic archaea Pyrococcus horikoshii. I examined the function and role of the unique hyperthermostable cellulase that containing the membrane binding site with protein engineering method. As a result, I succeeded in mass expression and solubilization of the enzyme by improving the C terminus residue of the enzyme. Furthermore, I succeeded in structure and a functional analysis of the catalytic site of the enzyme and succeeded in identification of the unknown function protein in the cellulose gene with protein engineering method. The detailed role of the protein is in progress. Furthermore, I succeeded in preparation of the crystals of the enzyme by improving N or C terminus of the enzyme. By changing the crystallization condition, high quality crystals of the enzyme were obtained. Using molecular replacement method, I solved the crystal structure of the enzyme. The refinement of the structural analysis is in progress. From the present structural data, it was elucidated that the enzyme exhibits the TIM barrel structure and catalytic residues of the enzyme are similar to those of the well known cellulase, but there is a big difference in the substrate recognition loop structures among the cellulases. This loop structure seems to be related to substrate specificity. I feel that the activity toward the crystalline cellulose can be improved by analyzing the detailed part. Using this structural data, I succeeded in preparation of new chimera enzyme between this cellulose and hyperthermophilic chitinase from Pyrococcus fuiosus. It was elucidated this chimera enzyme exhibited the activity of two times as high as that of the cellulose.

本研究试图对超高温古菌Pyrococcus horikoshii的超热稳定纤维素的功能进行分析和改进。利用蛋白质工程的方法研究了具有膜结合位点的超耐热纤维素酶的功能和作用。结果，通过改进酶的C末端残基，我成功地实现了酶的大量表达和增溶。此外，还成功地对该酶的催化位点进行了结构和功能分析，并利用蛋白质工程方法成功地鉴定了纤维素基因中的未知功能蛋白。蛋白质的详细作用正在进行中。此外，我成功地制备了晶体的酶的N或C端的改进。通过改变结晶条件，获得了高质量的酶晶体。用分子置换法解出了该酶的晶体结构。结构分析的完善工作正在进行中。从目前的结构数据，它被阐明，该酶具有TIM桶结构和催化残基的酶是众所周知的纤维素酶的类似，但有一个很大的差异，在底物识别环结构的纤维素酶。这种环结构似乎与底物特异性有关。我觉得通过分析细节部分可以提高对结晶纤维素的活性。利用这些结构数据，我成功地制备了这种纤维素和来自Pyrococcus fuiosus的超嗜热几丁质酶之间的新嵌合酶。结果表明，该嵌合体酶的活性是纤维素酶的2倍。