Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria.
J Biol Chem. 2012 Jan 20;287(4):2759-65. doi: 10.1074/jbc.M111.257717. Epub 2011 Nov 29.
Enzymatic hydrolysis of cellulose is key for the production of second generation biofuels, which represent a long-standing leading area in the field of sustainable energy. Despite the wealth of knowledge about cellulase structure and function, the elusive mechanism by which these enzymes disintegrate the complex structure of their insoluble substrate, which is the gist of cellulose saccharification, is still unclear. We herein present a time-resolved structural characterization of the action of cellulases on a nano-flat cellulose preparation, which enabled us to overcome previous limitations, using atomic force microscopy (AFM). As a first step in substrate disintegration, elongated fissures emerge which develop into coniform cracks as disintegration continues. Detailed data analysis allowed tracing the surface evolution back to the dynamics of crack morphology. This, in turn, reflects the interplay between surface degradation inside and outside of the crack. We observed how small cracks evolved and initially increased in size. At a certain point, the crack diameter stagnated and then started decreasing again. Stagnation corresponds with a decrease in the total amount of surface which is fissured and thus leads to the conclusion that the surface hydrolysis "around" the cracks is proceeding more rapidly than inside the cracks. The mesoscopic view presented here is in good agreement with various mechanistic proposals from the past and allows a novel insight into the structural dynamics occurring on the cellulosic substrate through cellulase action.
纤维素的酶解是生产第二代生物燃料的关键，这是可持续能源领域长期以来的领先领域。尽管人们对纤维素酶的结构和功能有了丰富的了解，但这些酶分解其不溶性底物复杂结构的难以捉摸的机制仍然不清楚，而这正是纤维素糖化的关键。在此，我们通过原子力显微镜（AFM）对纤维素酶在纳米级平整纤维素制剂上的作用进行了时分辨析。作为底物分解的第一步，出现了细长的裂缝，随着分解的继续，这些裂缝发展成锥形裂缝。详细的数据分析允许我们将表面演化追溯到裂纹形态的动力学。这反过来又反映了裂纹内外表面降解之间的相互作用。我们观察了小裂缝是如何演变并最初增大的。在某个时刻，裂缝直径停滞，然后再次开始减小。停滞对应于发生裂缝的表面发生水解的总量减少，因此得出结论，“围绕”裂缝的表面水解比裂缝内部更快。这里呈现的介观观点与过去的各种机制建议非常吻合，并且允许通过纤维素酶作用对发生在纤维素底物上的结构动力学有新的认识。
