Faculty of Biology, Microbiology Group, National and Kapodistrian University of Athens, Zografou Campus, 15784, Zografou, Attica, Greece.
Department of Biomolecular Sciences, The Weizmann Institute of Science, 76100, Rehovot, Israel.
Appl Microbiol Biotechnol. 2016 Oct;100(20):8731-43. doi: 10.1007/s00253-016-7594-5. Epub 2016 May 21.
Enzymatic breakdown of lignocellulose is a major limiting step in second generation biorefineries. Assembly of the necessary activities into designer cellulosomes increases the productivity of this step by enhancing enzyme synergy through the proximity effect. However, most cellulosomal components are obtained from mesophilic microorganisms, limiting the applications to temperatures up to 50 °C. We hypothesized that a scaffoldin, comprising modular components of mainly mesophilic origin, can function at higher temperatures when combined with thermophilic enzymes, and the resulting designer cellulosomes could be employed in higher temperature reactions. For this purpose, we used a tetravalent scaffoldin constituted of three cohesins of mesophilic origin as well as a cohesin and cellulose-binding module derived from the thermophilic bacterium Clostridium thermocellum. The scaffoldin was combined with four thermophilic enzymes from Geobacillus and Caldicellulosiruptor species, each fused with a dockerin whose specificity matched one of the cohesins. We initially verified that the biochemical properties and thermal stability of the resulting chimeric enzymes were not affected by the presence of the mesophilic dockerins. Then we examined the stability of the individual single-enzyme-scaffoldin complexes and the full tetravalent cellulosome showing that all complexes are stable and functional for at least 6 h at 60 °C. Finally, within this time frame and conditions, the full complex appeared over 50 % more efficient in the hydrolysis of corn stover compared to the free enzymes. Overall, the results support the utilization of scaffoldin components of mesophilic origin at relatively high temperatures and provide a framework for the production of designer cellulosomes suitable for high temperature biorefinery applications.
木质纤维素的酶解是第二代生物精炼厂的主要限制步骤。通过邻近效应增强酶协同作用，将必要的活性组装到设计的纤维小体中，可以提高这一步骤的生产力。然而，大多数纤维小体成分是从嗜温微生物中获得的，这将应用限制在 50°C 以下。我们假设，一个由主要嗜温来源的模块化组件组成的支架蛋白，当与嗜热酶结合时，可以在更高的温度下发挥作用，并且由此产生的设计纤维小体可以在更高温度的反应中使用。为此，我们使用了一个由三个来自嗜温微生物的黏合蛋白以及一个来自嗜热细菌 Clostridium thermocellum 的黏合蛋白和纤维素结合模块组成的四价支架蛋白。该支架蛋白与来自 Geobacillus 和 Caldicellulosiruptor 物种的四种嗜热酶结合，每个酶都融合了一个 dockerin，其特异性与其中一个黏合蛋白匹配。我们最初验证了所得嵌合酶的生化特性和热稳定性不受嗜温 dockerin 的存在影响。然后，我们检查了单个单酶-支架蛋白复合物和完整四价纤维小体的稳定性，结果表明所有复合物在 60°C 下至少稳定和功能 6 小时。最后，在这个时间范围内和条件下，与游离酶相比，完整复合物在玉米秸秆水解中的效率提高了 50%以上。总体而言，这些结果支持在相对较高的温度下使用嗜温来源的支架蛋白组件，并为生产适用于高温生物精炼应用的设计纤维小体提供了框架。
