Systematically Scrutinizing the Impact of Substitution Sites on Thermostability and Detergent Tolerance for Bacillus subtilis Lipase A

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.

提升酶的热稳定性或对其溶剂和洗涤剂的耐受性在蛋白质工程与生物技术领域具有极高的相关性。近年来，研究发展趋向于数据驱动的方法，其中利用关于蛋白质的可获得知识来识别具有高潜在性产生改进稳定性蛋白质变体的替换位点，随后通过位点定向或位点饱和（SSM）突变工程化地实现这些替换。然而，由于缺乏大规模数据，这些数据以统一的方式测量并且对替换类型和位置无偏见，数据驱动方法算法的开发与验证受到了阻碍。在此，我们通过审视对芽孢杆菌子囊孢子脂酶A（BsLipA）的热稳定性或/及洗涤剂耐受性影响的替换位点，在极大规模上扩展了我们关于数据驱动蛋白质工程指南的知识。我们系统地分析了包含所有3439种可能单变体的BsLipA的完整实验SSM文库，该文库在分别统一的条件下对热稳定性和对四种洗涤剂的耐受性进行了评估。我们的结果为一种生物技术重要蛋白质提供了前所未有的系统性、无偏见的参考数据，并确定了用于评估数据驱动蛋白质工程方法性能的一致性定义的热点类型，同时表明，刚体理论和基于集合的约束网络分析方法在热点预测方面，与随机分类相比，精确度提高了九倍。