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 Tm 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 Tmand half-life at 70 °C.
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We thank Editage (www.editage.cn) for the English language editing.
Funding
This work was supported by the National Natural Science Foundation of China (31872891, 21676120), the Program of Introducing Talents of Discipline to Universities (111-2-06), the High-End Foreign Experts Recruitment Program (G20190010083), the Program for Advanced Talents within Six Industries of Jiangsu Province (2015-NY-007), the National Program for Support of Top-notch Young Professionals, the Fundamental Research Funds for the Central Universities (JUSRP51504), the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, Top-notch Academic Programs Project of Jiangsu Higher Education Institutions, the Program for the Key Laboratory of Enzymes of Suqian (M201803), and the National First-Class Discipline Program of Light Industry Technology and Engineering (LITE2018-09).
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J.B., Y.N., and Y.X. conceived and designed the experiments. J.B., S.C., and X.Z. performed the experiments. J.B. and Y.N. analyzed experimental data. J.B. and Y.N. wrote the main manuscript. All authors reviewed the manuscript.
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Bi, J., Chen, S., Zhao, X. et al. Computation-aided engineering of starch-debranching pullulanase from Bacillus thermoleovorans for enhanced thermostability. Appl Microbiol Biotechnol 104, 7551–7562 (2020). https://doi.org/10.1007/s00253-020-10764-z
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DOI: https://doi.org/10.1007/s00253-020-10764-z