Computation-aided engineering of starch-debranching pullulanase fromBacillus thermoleovoransfor enhanced thermostability
Computation-aided engineering of starch-debranching pullulanase fromBacillus thermoleovoransfor enhanced thermostability
复制标题
用于增强热稳定性的嗜热芽孢杆菌淀粉脱支支链淀粉酶的计算辅助工程
DOI：
10.1007/s00253-020-10764-z
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发表时间：
2020-07-07
影响因子：
5
通讯作者：
Xu, Yan
中科院分区：
文献类型：
--
作者：
Bi, Jiahua;Chen, Shuhui;Xu, Yan
普鲁兰酶广泛用于食品、医药和其他工业，因为它们特异性地水解淀粉和寡糖中的α-1，6-糖苷键。此外，高温热稳定普鲁兰酶具有多种优点，包括降低糖化溶液粘度伴随增强的传质和减少淀粉水解中的微生物污染。然而，嗜热普鲁兰酶的可用性仍然有限。此外，由于热稳定性差，大多数不符合淀粉制造要求。在这里，我们开发了一种计算机辅助的策略，工程嗜热普鲁兰酶从嗜热食芽孢杆菌。首先，将三种计算设计预测因子（FoldX、I-Mutant 3.0和dDFIRE）组合以预测突变引起的稳定性变化。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个阳性突变体。其中，G692 M突变体的热稳定性改善最高，在70 ℃时，T（m）增加了3.8 ℃，半衰期比野生型延长了2.1倍。然后，我们的特点增加的热稳定性，如刚性增强，更紧密的构象，并加强运动相关性，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）分析的机制。
Pullulanases are widely used in food, medicine, and other industries because they specifically hydrolyze alpha-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 fromBacillus 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 degrees C increasedT(m)and 2.1-fold longer half-life than the wild type at 70 degrees 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.