Protein engineering of Bacillus acidopullulyticus pullulanase for enhanced thermostability using in silico data driven rational design methods
| Autor: | Ana Chen, Nie Jianqi, Brian McNeil, Laura Jeffrey, Zhonghu Bai, Yankun Yang, Yamei Li |
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| Rok vydání: | 2015 |
| Předmět: |
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Molecular Glycoside Hydrolases Protein Conformation Molecular Sequence Data Mutant Mutagenesis (molecular biology technique) Bacillus Bioengineering Biology Protein Engineering Applied Microbiology and Biotechnology Biochemistry Protein structure Bacterial Proteins Enzyme Stability Computer Simulation Amino Acid Sequence Thermostability Binding Sites Sequence Homology Amino Acid Pullulanase Temperature Rational design Protein engineering Hydrogen-Ion Concentration Protein tertiary structure Kinetics Amino Acid Substitution Mutagenesis Site-Directed Biotechnology |
| Zdroj: | Enzyme and Microbial Technology. 78:74-83 |
| ISSN: | 0141-0229 |
| DOI: | 10.1016/j.enzmictec.2015.06.013 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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