title: Improving the Thermostability of Acidic Pullulanase from Bacillus naganoensis by Rational Design.
abstract: Pullulanase (EC 3.2.1.41) plays an important role in the specific hydrolysis of branch points in amylopectin. Enhancing its thermostability is required for its industrial application. In this study, rational protein design was used to improve the thermostability of PulB from Bacillus naganoensis (AB231790.1), which has strong enzymatic properties. Three positive single-site mutants (PulB-D328H, PulB-N387D, and PulB-A414P) were selected from six mutants. After incubation at 65°C for 5 min, the residual activities of PulB-D328H, PulB-N387D, and PulB-A414P were 4.5-, 1.7-, and 1.47-fold higher than PulB-WT, and their Tm values (the temperature at which half protein molecule denature) were 1.8°C, 0.4°C, and 0.9°C higher than PulB-WT, respectively. Then the final combined mutant PulB-328/387/414 was constructed. The t1/2 of it was 12.9-fold longer than that of PulB-WT at 65°C and the total increase in Tm of it (5.0°C) was almost 60% greater than the sum of individual increases (3.1°C). In addition, kinetic studies revealed that the kcat and the kcat/Km of PulB-328/387/414 increased by 38.8% and 12.9%. The remarkable improvement in thermostability and the high catalytic efficiency of PulB-328/387/414 make it suitable for industrial applications.
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China.
authors.forename: Meihui
authors.initials: M
authors.lastname: Chang
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Chu
authors.affiliation: Institute of Food Science and Engineering, Jiangxi Agricultural University, Nanchang, 330045, China.
authors.forename: Jinzhi
authors.initials: J
authors.lastname: Lv
authors.affiliation: College of Biological Sciences, China Agricultural University, Beijing, 100094, China.
authors.forename: Qingbin
authors.initials: Q
authors.lastname: Li
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Tian
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China.
authors.forename: Ningfeng
authors.initials: N
authors.lastname: Wu
mesh_terms: D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D019295:Computational Biology / Q000379:methods*; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation; D040681:Structural Homology, Protein; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D006026:Glycoside Hydrolases; C100181:pullulanase