Tanghe Magali, Danneels Barbara, Last Matthias, Beerens Koen, Stals Ingeborg, Desmet Tom
Centre for Synthetic Biology (CSB), Department of Biochemical and Microbial Technology, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium.
Industrial Catalysis and Adsorption Technology (INCAT), Faculty of Engineering and Architecture, Ghent University, Valentin Vaerwyckweg 1, B-9000 Ghent, Belgium.
Protein Eng Des Sel. 2017 May 1;30(5):401-408. doi: 10.1093/protein/gzx014.
Lytic polysaccharide monooxygenases (LPMOs) are crucial components of cellulase mixtures but their stability has not yet been studied in detail, let alone been engineered for industrial applications. In this work, we have evaluated the importance of disulfide bridges for the thermodynamic stability of Streptomyces coelicolor LPMO10C. Interestingly, this enzyme was found to retain 34% of its activity after 2-h incubation at 80°C while its apparent melting temperature (Tm) is only 51°C. When its three disulfide bridges were broken, however, irreversible unfolding occurred and no residual activity could be detected after a similar heat treatment. Based on these findings, additional disulfide bridges were introduced, as predicted by computational tools (MOdelling of DIsulfide bridges in Proteins (MODiP) and Disulfide by Design (DbD)) and using the most flexible positions in the structure as target sites. Four out of 16 variants displayed an improvement in Tm, ranging from 2 to 9°C. Combining the positive mutations yielded additional improvements (up to 19°C) but aberrant unfolding patterns became apparent in some cases, resulting in a diminished capacity for heat resistance. Nonetheless, the best variant, a combination of A143C-P183C and S73C-A115C, displayed a 12°C increase in Tm and was able to retain and was able to retain no less than 60% of its activity after heat treatment.
裂解多糖单加氧酶（LPMOs）是纤维素酶混合物的关键成分，但尚未对其稳定性进行详细研究，更不用说对其进行工业应用改造了。在这项工作中，我们评估了二硫键对天蓝色链霉菌LPMO10C热力学稳定性的重要性。有趣的是，该酶在80°C孵育2小时后仍保留34%的活性，而其表观解链温度（Tm）仅为51°C。然而，当其三个二硫键断裂时，会发生不可逆的解折叠，经过类似的热处理后未检测到残留活性。基于这些发现，通过计算工具（蛋白质中二硫键建模（MODiP）和设计二硫键（DbD））预测，并以结构中最灵活的位置为靶点，引入了额外的二硫键。16个变体中有4个的Tm有所提高，范围为2至9°C。将正向突变组合起来可进一步提高（高达19°C），但在某些情况下会出现异常的解折叠模式，导致耐热能力下降。尽管如此，最佳变体，即A143C-P183C和S73C-A115C的组合，Tm提高了12°C，热处理后能够保留不少于60%的活性。
