Immunogold localisation of catalytic domain of endocellulase in the cellulosic substrates
Yoon Soo Kim, Seung-Gon Wi, Kyu Ho Myung, K Ohmiya, S Karita
The reaction of catalytic domain of endocellulase on the cellulosic substrates was visualised by using immunogold labelling procedures to obtain a better understanding of the mechanism of enzymatic cellulose hydrolysis. Polyclonal antibody was produced against the catalytic domain of endo-l, 4-ß-glucanase (EGI) from a ruminal anaerobic bacterium Ruminococcus albus F-40. Immuno-TEM works showed the positive labelling of polyclonal antibody of catalytic domain endocellulase on the cellulosic structures. Gold particles occurred mostly in the disrupted terminal areas of cellulose microfibrils. In contrast, gold labelling on the central part of microfibril was not so much distributed as that in the terminal part. The catalytic domain of endocellulase was presented mainly in the outer membrane of bacterial cells, suggesting that this enzyme is a cell surface protein serving to degrade the cellulosic substrates. The labelling density between the whole endocellulase and the catalytic domain of endocellulase was different. Micromorphological characteristics of cellulose degradation by this bacterium were also examined by SEM and TEM.