Percival Zhang Y-H, Himmel Michael E, Mielenz Jonathan R
Biological Systems Engineering Department, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA.
Biotechnol Adv. 2006 Sep-Oct;24(5):452-81. doi: 10.1016/j.biotechadv.2006.03.003. Epub 2006 Mar 27.
Cellulose is the most abundant renewable natural biological resource, and the production of biobased products and bioenergy from less costly renewable lignocellulosic materials is important for the sustainable development of human beings. A reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. Here, we review quantitative cellulase activity assays using soluble and insoluble substrates, and focus on their advantages and limitations. Because there are no clear relationships between cellulase activities on soluble substrates and those on insoluble substrates, soluble substrates should not be used to screen or select improved cellulases for processing relevant solid substrates, such as plant cell walls. Cellulase improvement strategies based on directed evolution using screening on soluble substrates have been only moderately successful, and have primarily targeted improvement in thermal tolerance. Heterogeneity of insoluble cellulose, unclear dynamic interactions between insoluble substrate and cellulase components, and the complex competitive and/or synergic relationship among cellulase components limit rational design and/or strategies, depending on activity screening approaches. Herein, we hypothesize that continuous culture using insoluble cellulosic substrates could be a powerful selection tool for enriching beneficial cellulase mutants from the large library displayed on the cell surface.
纤维素是最丰富的可再生天然生物资源，利用成本较低的可再生木质纤维素材料生产生物基产品和生物能源对人类的可持续发展至关重要。降低纤维素酶生产成本、提高纤维素酶性能以及提高糖产量对于降低生物精炼厂的加工成本都至关重要。对于非复合纤维素酶混合物，可以通过基于合理设计或针对每种纤维素酶组分酶的定向进化以及纤维素酶组分的重组来进行纤维素酶工程，从而提高其比活性。在此，我们综述了使用可溶性和不溶性底物的定量纤维素酶活性测定方法，并重点介绍了它们的优点和局限性。由于纤维素酶在可溶性底物上的活性与在不溶性底物上的活性之间没有明确的关系，因此不应使用可溶性底物来筛选或选择用于处理相关固体底物（如植物细胞壁）的改良纤维素酶。基于在可溶性底物上进行筛选的定向进化的纤维素酶改良策略仅取得了一定程度的成功，并且主要针对耐热性的提高。不溶性纤维素的异质性、不溶性底物与纤维素酶组分之间不明确的动态相互作用以及纤维素酶组分之间复杂的竞争和/或协同关系限制了基于活性筛选方法的合理设计和/或策略。在此，我们假设使用不溶性纤维素底物进行连续培养可能是从细胞表面展示的大型文库中富集有益纤维素酶突变体的有力选择工具。
