AVT-Aachener Verfahrenstechnik, Biochemical Engineering, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany.
Biotechnol Biofuels. 2010 Aug 18;3:18. doi: 10.1186/1754-6834-3-18.
It is important to generate biofuels and society must be weaned from its dependency on fossil fuels. In order to produce biofuels, lignocellulose is pretreated and the resulting cellulose is hydrolyzed by cellulases such as cellobiohydrolases (CBH) and endoglucanases (EG). Until now, the biofuel industry has usually applied impractical celluloses to screen for cellulases capable of degrading naturally occurring, insoluble cellulose. This study investigates how these cellulases adsorb and hydrolyze insoluble α-cellulose - considered to be a more practical substrate which mimics the alkaline-pretreated biomass used in biorefineries. Moreover, this study investigates how hydrodynamics affects cellulase adsorption and activity onto α-cellulose.
First, the cellulases CBH I, CBH II, EG I and EG II were purified from Trichoderma reesei and CBH I and EG I were utilized in order to study and model the adsorption isotherms (Langmuir) and kinetics (pseudo-first-order). Second, the adsorption kinetics and cellulase activities were studied under different hydrodynamic conditions, including liquid mixing and particle suspension. Third, in order to compare α-cellulose with three typically used celluloses, the exact cellulase activities towards all four substrates were measured.It was found that, using α-cellulose, the adsorption models fitted to the experimental data and yielded parameters comparable to those for filter paper. Moreover, it was determined that higher shaking frequencies clearly improved the adsorption of cellulases onto α-cellulose and thus bolstered their activity. Complete suspension of α-cellulose particles was the optimal operating condition in order to ensure efficient cellulase adsorption and activity. Finally, all four purified cellulases displayed comparable activities only on insoluble α-cellulose.
α-Cellulose is an excellent substrate to screen for CBHs and EGs. This current investigation shows in detail, for the first time, the adsorption of purified cellulases onto α-cellulose, the effect of hydrodynamics on cellulase adsorption and the correlation between the adsorption and the activity of cellulases at different hydrodynamic conditions. Complete suspension of the substrate has to be ensured in order to optimize the cellulase attack. In the future, screenings should be conducted with α-cellulose so that proper cellulases are selected to best hydrolyze the real alkaline-pretreated biomass used in biorefineries.
生产生物燃料很重要，社会必须摆脱对化石燃料的依赖。为了生产生物燃料，木质纤维素需要经过预处理，然后纤维素酶（如纤维二糖水解酶 (CBH) 和内切葡聚糖酶 (EG)）将其水解。到目前为止，生物燃料行业通常应用不切实际的纤维素来筛选能够降解天然存在的不溶性纤维素的纤维素酶。本研究调查了这些纤维素酶如何吸附和水解不溶性 α-纤维素——被认为是一种更实用的底物，可模拟生物精炼厂中使用的碱性预处理生物质。此外，本研究还调查了水动力如何影响纤维素酶对 α-纤维素的吸附和活性。
首先，从里氏木霉中纯化了 CBH I、CBH II、EG I 和 EG II 这 4 种纤维素酶，并利用 CBH I 和 EG I 来研究和模拟吸附等温线（朗缪尔）和动力学（拟一级）。其次，在不同的水动力条件下，包括液体混合和颗粒悬浮，研究了吸附动力学和纤维素酶活性。第三，为了将 α-纤维素与三种常用的纤维素进行比较，对所有四种底物的准确纤维素酶活性进行了测量。结果发现，使用 α-纤维素时，吸附模型拟合实验数据并得出的参数与滤纸的参数相当。此外，确定较高的摇床频率可明显提高纤维素酶对 α-纤维素的吸附，从而提高其活性。为了确保纤维素酶的有效吸附和活性，完全悬浮 α-纤维素颗粒是最佳操作条件。最后，只有在不溶性 α-纤维素上，四种纯化的纤维素酶才显示出相当的活性。
α-纤维素是筛选 CBH 和 EG 的绝佳底物。本研究首次详细展示了纯化纤维素酶在 α-纤维素上的吸附、水动力对纤维素酶吸附的影响以及不同水动力条件下吸附与纤维素酶活性之间的相关性。为了优化纤维素酶的攻击，必须确保底物完全悬浮。在未来，应该使用 α-纤维素进行筛选，以便选择最合适的纤维素酶来水解生物精炼厂中实际使用的碱性预处理生物质。
