Analysis and improvement of hyperthermostable endo-glucanase from Archaea

在本研究中，我试图分析和改进从嗜热古生菌中提取的耐热纤维素的功能。利用蛋白质工程方法对含有膜结合部位的独特的耐热纤维素酶的功能和作用进行了研究。因此，我通过改进酶的C末端残基，成功地实现了酶的大量表达和增溶。此外，我成功地对该酶的催化部位进行了结构和功能分析，并成功地用蛋白质工程方法鉴定了纤维素基因中的未知功能蛋白。该蛋白质的详细作用正在进行中。此外，通过改进酶的N端或C端，成功地制备了酶的晶体。通过改变结晶条件，获得了高质量的酶晶体。采用分子置换的方法，解决了该酶的晶体结构问题。结构分析的改进工作正在进行中。从目前的结构数据可以看出，该酶具有TIM桶状结构，酶的催化残基与众所周知的纤维素酶相似，但底物识别环结构在不同纤维素酶之间存在较大差异。这种环结构似乎与底物专一性有关。我觉得对结晶纤维素的活性可以通过分析详细的部分来提高。利用这些结构数据，我成功地制备了该纤维素与高温几丁质酶之间的新型嵌合酶。结果表明，该嵌合体酶的活性是纤维素酶的两倍。

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.