Improving an enzyme's (thermo-)stability or tolerance against solvents and detergents is highly relevant in protein engineering and biotechnology. Recent developments have tended toward data-driven approaches, where available knowledge about the protein is used to identify substitution sites with high potential to yield protein variants with improved stability, and subsequently, substitutions are engineered by site-directed or site-saturation (SSM) mutagenesis. However, the development and validation of algorithms for data-driven approaches have been hampered by the lack of availability of large-scale data measured in a uniform way and being unbiased with respect to substitution types and locations. Here, we extend our knowledge on guidelines for protein engineering following a data-driven approach by scrutinizing the impact of substitution sites on thermostability or/and detergent tolerance for Bacillus subtilis lipase A (BsLipA) at very large scale. We systematically analyze a complete experimental SSM library of BsLipA containing all 3439 possible single variants, which was evaluated as to thermostability and tolerances against four detergents under respectively uniform conditions. Our results provide systematic and unbiased reference data at unprecedented scale for a biotechnologically important protein, identify consistently defined hot spot types for evaluating the performance of data-driven protein-engineering approaches, and show that the rigidity theory and ensemble-based approach Constraint Network Analysis yields hot spot predictions with an up to ninefold gain in precision over random classification.

中文翻译：

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系统地研究取代位点对枯草芽孢杆菌脂肪酶A的热稳定性和洗涤剂耐受性的影响。

在蛋白质工程和生物技术中，提高酶对溶剂和去污剂的（热）稳定性或耐受性非常重要。最近的发展趋向于数据驱动的方法，其中有关蛋白质的可用知识用于鉴定具有高潜力以产生具有改进的稳定性的蛋白质变体的取代位点，随后，通过位点定向或位点饱和（SSM）设计取代诱变。但是，数据驱动方法算法的开发和验证因缺乏以统一方式测量的大规模数据的可用性而受到阻碍，并且在替代类型和位置方面没有偏见。这里，我们通过仔细研究取代位点对枯草芽孢杆菌脂肪酶A（BsLipA）的热稳定性或/和去污剂耐受性的影响，以数据驱动的方式扩展了我们对蛋白质工程指导原则的认识。我们系统地分析了BsLipA的完整实验SSM库，其中包含所有3439种可能的单个变体，并分别在相同的条件下评估了其对四种洗涤剂的热稳定性和耐受性。我们的研究结果为生物技术上重要的蛋白质提供了前所未有的规模化，系统化的参考数据，确定了一致定义的热点类型，以评估数据驱动的蛋白质工程方法的性能，