Protein engineering of Bacillus acidopullulyticus pullulanase for enhanced thermostability using in silico data driven rational design methods |
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| Institution: | 1. National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China;2. The Key Laboratory of Industrial Biotechnology and The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China;3. School of Biochemical Engineering, Anhui Polytechnic University, Wuhu 241000, China;4. University of Strathclyde, Glasgow G1 1XQ, UK;1. Key Laboratory of Renewable Energy, Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, Guangzhou 510640, PR China;2. University of Chinese Academy of Sciences, Beijing 100049, PR China;3. Guangdong Key Laboratory of New and Renewable Energy Research and Development, Chinese Academy of Sciences, Guangzhou 510640, PR China;4. Collaborative Innovation Centre of Biomass Energy, Zhengzhou 450002, Henan Province, PR China;5. South China Agricultural University, Guangzhou Guangdong 510642, PR China;1. The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, PR China;2. Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, PR China;3. Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH 43210, USA;1. School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China;2. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China;3. The 2011 Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, China |
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| Abstract: | Thermostability has been considered as a requirement in the starch processing industry to maintain high catalytic activity of pullulanase under high temperatures. Four data driven rational design methods (B-FITTER, proline theory, PoPMuSiC-2.1, and sequence consensus approach) were adopted to identify the key residue potential links with thermostability, and 39 residues of Bacillus acidopullulyticus pullulanase were chosen as mutagenesis targets. Single mutagenesis followed by combined mutagenesis resulted in the best mutant E518I-S662R-Q706P, which exhibited an 11-fold half-life improvement at 60 °C and a 9.5 °C increase in Tm. The optimum temperature of the mutant increased from 60 to 65 °C. Fluorescence spectroscopy results demonstrated that the tertiary structure of the mutant enzyme was more compact than that of the wild-type (WT) enzyme. Structural change analysis revealed that the increase in thermostability was most probably caused by a combination of lower stability free-energy and higher hydrophobicity of E518I, more hydrogen bonds of S662R, and higher rigidity of Q706P compared with the WT. The findings demonstrated the effectiveness of combined data-driven rational design approaches in engineering an industrial enzyme to improve thermostability. |
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| Keywords: | Pullulanase Thermostability Rational design Protein engineering |
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