title: Protein engineering of Bacillus acidopullulyticus pullulanase for enhanced thermostability using in silico data driven rational design methods.
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.
authors.affiliation: National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China; 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; School of Biochemical Engineering, Anhui Polytechnic University, Wuhu 241000, China.
authors.forename: Ana
authors.initials: A
authors.lastname: Chen
authors.affiliation: National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China; 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.
authors.forename: Yamei
authors.initials: Y
authors.lastname: Li
authors.affiliation: National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China; 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.
authors.forename: Jianqi
authors.initials: J
authors.lastname: Nie
authors.affiliation: University of Strathclyde, Glasgow G1 1XQ, UK.
authors.forename: Brian
authors.initials: B
authors.lastname: McNeil
authors.affiliation: University of Strathclyde, Glasgow G1 1XQ, UK.
authors.forename: Laura
authors.initials: L
authors.lastname: Jeffrey
authors.affiliation: National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China; 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. Electronic address: yangyankun@jiangnan.edu.cn.
authors.forename: Yankun
authors.initials: Y
authors.lastname: Yang
authors.affiliation: National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China; 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. Electronic address: baizhonghu@jiangnan.edu.cn.
authors.forename: Zhonghu
authors.initials: Z
authors.lastname: Bai
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D003198:Computer Simulation; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D006026:Glycoside Hydrolases; C100181:pullulanase
keywords: Protein engineering; Pullulanase; Rational design; Thermostability