Open AccessImproving the Thermostability of Acidic Pullulanase from Bacillus naganoensis by Rational Design
Author(s) -
Meihui Chang,
Xiaoyu Chu,
Lv JinZhi,
Qingbin Li,
Jian Tian,
Ningfeng Wu
Publication year - 2016
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0165006
Subject(s) - thermostability , pullulanase , enzyme kinetics , hydrolysis , chemistry , rational design , mutant , kinetics , amylopectin , enzyme , biochemistry , starch , biology , active site , physics , quantum mechanics , gene , amylose , genetics
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 T m 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 t 1/2 of it was 12.9-fold longer than that of PulB-WT at 65°C and the total increase in T m 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 k cat and the k cat / K m 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.