title: Structure and sequence analysis-based engineering of pullulanase from Anoxybacillus sp. LM18-11 for improved thermostability.
abstract: Pullulanase (EC 3.2.1.41) is a well-known starch-debranching enzyme. Enhancing the thermostability of Pullulanase is required for industrial application. In this study, we used two methods to improve the thermostability of the pullulanase from Anoxybacillus sp. LM18-11; these methods were the modified amino acid consensus method combined with the analyses of the residue water-exposed surface (ACC) and the deletion of flexible domains. Four mutants (Y477A, Y175C, L215C and R473E) were obtained via the modified consensus method exhibited varying degrees of improvements in terms of thermostability. One deletion mutant termed D3 (residues(686-688)) was obtained and exhibited enhanced thermostability due to deletion of the flexible region at the C-terminus. The combination of the two strategies yielded the mutant M18 (Y477A/D3/Y175C/L215P/R473E). It retained 66% of its initial activity after incubation at 60 °C for 72 hrs, whereas that of the wild-type enzyme was only 35%. After incubation at 65 °C for 4 h, M18 retained 50.6% of its initial activity, whereas that of the wild-type was only 16.8%, respectively. Additionally, kinetic studies revealed that the Km of M17 (Y477A/D3/Y175C/L215P) was decreased by 33.9% and that the Kcat/Km value of M17 increased by 50%, while M18 exhibited Km and Kcat/Km values that were similar to those of the wild-type enzyme. The attractive improved thermostability and the high catalytic efficiency made M17 and M18 more suitable for industrial application.
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences,32 XiQiDao, Tianjin Airport Economic Park, Tianjin 300308, China.
authors.forename: Shu-fang
authors.initials: SF
authors.lastname: Li
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences,32 XiQiDao, Tianjin Airport Economic Park, Tianjin 300308, China.
authors.forename: Jian-yong
authors.initials: JY
authors.lastname: Xu
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences,32 XiQiDao, Tianjin Airport Economic Park, Tianjin 300308, China.
authors.forename: Yun-juan
authors.initials: YJ
authors.lastname: Bao
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences,32 XiQiDao, Tianjin Airport Economic Park, Tianjin 300308, China.
authors.forename: Hong-chen
authors.initials: HC
authors.lastname: Zheng
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences,32 XiQiDao, Tianjin Airport Economic Park, Tianjin 300308, China. Electronic address: song_h@tib.cas.cn.
authors.forename: Hui
authors.initials: H
authors.lastname: Song
mesh_terms: D058677:Anoxybacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D017434:Protein Structure, Tertiary; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D006026:Glycoside Hydrolases; C100181:pullulanase
keywords: Anoxybacillus sp. LM18-11; Consensus method; Flexible region; Pullulanase; Thermostability