Xiang Xia, Wan Sidi, Zhang Songjing, Zhu Enheng, Lin Xuejun, Han Nanyu
School of Life Sciences, Yunnan Normal University, Kunming, China.
School of Biological Sciences, Nanyang Technological University, Singapore.
Enzyme Microb Technol. 2025 Oct;190:110702. doi: 10.1016/j.enzmictec.2025.110702. Epub 2025 Jun 23.
Lipases serve as indispensable biocatalysts in many industrial applications due to their versatile catalytic abilities. To ensure their thermal resilience of the harsh biological treatment in industry, it is crucial to identify key residues which might impact thermostability. Here, computational design was adopted to decode the stability-determining residues in Thermomyces lanuginosus lipase (TLL). Systematic Gibbs free energy profiling of potent TLL single-point mutational candidates predicted proline 256 (P256) as a thermal liability hotspot. Saturation mutagenesis at P256 discovered that among nineteen P256 variants: (1) five P256 variants exhibited increased melting temperature (ΔT up to 2°C); (2) six variants displayed an optimum temperature with 5-10°C elevation; (3) five P256 variants retained up to 21 % higher residual activity after incubation at 80°C. Furthermore, both P256E and P256I demonstrated synergistic improvements in biodiesel conversion rates, P256I specifically exhibited long-term and recycling stability. Molecular dynamics simulations revealed that the substitutions in P256A/E/I/K with compensatory main-chain rotational freedom, facilitating hydrogen bonding network with both upstream and downstream residues, thereby preserving local structural stability. This study pioneers the identification of P256 as a critical residue governing TLL thermostability. Furthermore, our Gibbs-guided engineering strategy generates multi-property-enhanced lipase variants, directly addressing industrial demands.
脂肪酶因其多样的催化能力，在许多工业应用中是不可或缺的生物催化剂。为确保它们在工业苛刻生物处理条件下的热稳定性，识别可能影响热稳定性的关键残基至关重要。在此，采用计算设计来解析嗜热栖热放线菌脂肪酶（TLL）中决定稳定性的残基。对有效的TLL单点突变候选物进行系统的吉布斯自由能分析预测脯氨酸256（P256）是一个热不稳定热点。在P256处进行饱和诱变发现，在19个P256变体中：（1）5个P256变体的解链温度升高（ΔT高达2°C）；（2）6个变体的最适温度升高了5 - 10°C；（3）5个P256变体在80°C孵育后保留的残余活性高达21%。此外，P256E和P256I在生物柴油转化率方面都表现出协同提高，P256I尤其表现出长期和循环稳定性。分子动力学模拟表明，P256A/E/I/K中的取代具有补偿性主链旋转自由度，促进了与上游和下游残基的氢键网络，从而保持了局部结构稳定性。本研究首次鉴定出P256是控制TLL热稳定性的关键残基。此外，我们基于吉布斯自由能指导的工程策略产生了多性能增强的脂肪酶变体，直接满足了工业需求。
