Abstract

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_m 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 T_mand half-life at 70 °C.

中文翻译：

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来自热芽孢杆菌的淀粉脱支支链淀粉酶的计算机辅助工程设计，可提高热稳定性。

摘要

支链淀粉酶被广泛地用于食品，医药和其他行业，因为它们特异性地水解淀粉和低聚糖中的α-1,6-糖苷键。此外，高温热稳定的支链淀粉酶具有多种优势，包括降低糖化溶液的粘度，同时增强传质能力，并减少淀粉水解过程中的微生物污染。但是，嗜热支链淀粉酶的可用性仍然有限。另外，由于弱的热稳定性，大多数不满足淀粉制造要求。在这里，我们开发了一种计算辅助策略来工程化嗜热芽孢杆菌的嗜热支链淀粉酶。首先，将三个计算设计预测变量（FoldX，I-Mutant 3.0和dDFIRE）组合在一起，以预测由突变引起的稳定性变化。排除保守和催化位点后，鉴定出17个突变体。经过进一步的实验验证，我们确认了六个阳性突变体。其中，G692M突变体的热稳定性提高最高，在70°C时，T _m升高3.8°C ，半衰期比野生型长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）和自由能态图来表征提高热稳定性的机制，例如刚性增强，更紧密的构型和增强的运动相关性。 （FEL）分析。

关键点

•开发了一种计算辅助策略来设计支链淀粉酶的热稳定性。

•通过组合三个计算设计预测因子，鉴定出17个突变体。

•获得的G692M突变体在70°C下具有更高的T _m和半衰期。