Bi Jiahua, Chen Shuhui, Zhao Xianghan, Nie Yao, Xu Yan
School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China.
Suqian Industrial Technology Research Institute of Jiangnan University, Suqian, 223814, China.
Appl Microbiol Biotechnol. 2020 Sep;104(17):7551-7562. doi: 10.1007/s00253-020-10764-z. Epub 2020 Jul 7.
Pullulanases are widely used in food, medicine, and other industries because they specifically hydrolyze α-1,6-glycosidic linkages in starch and oligosaccharides. In addition, high-temperature thermostable pullulanase has multiple advantages, including decreasing saccharification solution viscosity accompanied with enhanced mass transfer and reducing microbial contamination in starch hydrolysis. However, thermophilic pullulanase availability remains limited. Additionally, most do not meet starch-manufacturing requirements due to weak thermostability. Here, we developed a computation-aided strategy to engineer the thermophilic pullulanase from Bacillus thermoleovorans. First, three computational design predictors (FoldX, I-Mutant 3.0, and dDFIRE) were combined to predict stability changes introduced by mutations. After excluding conserved and catalytic sites, 17 mutants were identified. After further experimental verification, we confirmed six positive mutants. Among them, the G692M mutant had the highest thermostability improvement, with 3.8 °C increased T and 2.1-fold longer half-life than the wild type at 70 °C. We then characterized the mechanism underlying increased thermostability, such as rigidity enhancement, closer conformation, and strengthened motion correlation using root mean square fluctuation (RMSF), principal component analysis (PCA), dynamic cross-correlation map (DCCM), and free energy landscape (FEL) analysis. KEY POINTS: • A computation-aided strategy was developed to engineer pullulanase thermostability. • Seventeen mutants were identified by combining three computational design predictors. • The G692M mutant was obtained with increased Tand half-life at 70 °C.
支链淀粉酶因其能特异性水解淀粉和寡糖中的α-1,6-糖苷键而被广泛应用于食品、医药和其他行业。此外，高温热稳定支链淀粉酶具有多种优势，包括降低糖化溶液粘度并增强传质，以及减少淀粉水解过程中的微生物污染。然而，嗜热支链淀粉酶的可用性仍然有限。此外，由于热稳定性较弱，大多数支链淀粉酶不符合淀粉制造的要求。在此，我们开发了一种计算辅助策略来改造嗜热栖热芽孢杆菌的嗜热支链淀粉酶。首先，结合三种计算设计预测工具（FoldX、I-Mutant 3.0和dDFIRE）来预测突变引入的稳定性变化。在排除保守位点和催化位点后，鉴定出17个突变体。经过进一步的实验验证，我们确认了6个正向突变体。其中，G692M突变体的热稳定性提高最为显著，在70℃时，其熔点增加了3.8℃，半衰期比野生型延长了2.1倍。然后，我们使用均方根波动（RMSF）、主成分分析（PCA）、动态互相关图（DCCM）和自由能景观（FEL）分析等方法，对热稳定性提高的潜在机制进行了表征，如刚性增强、构象更紧密以及运动相关性增强。要点：• 开发了一种计算辅助策略来改造支链淀粉酶的热稳定性。• 通过结合三种计算设计预测工具鉴定出17个突变体。• 获得了在70℃时熔点和半衰期增加的G692M突变体。
