Improving the Thermostability of Acidic Pullulanase from Bacillus naganoensis by Rational Design.
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 (AB...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2016-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC5072709?pdf=render |
| _version_ | 1819086867035848704 |
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| author | Meihui Chang Xiaoyu Chu Jinzhi Lv Qingbin Li Jian Tian Ningfeng Wu |
| author_facet | Meihui Chang Xiaoyu Chu Jinzhi Lv Qingbin Li Jian Tian Ningfeng Wu |
| author_sort | Meihui Chang |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-21T21:27:04Z |
| format | Article |
| id | doaj.art-c5aae2486320498a98878ca353e96e6f |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-21T21:27:04Z |
| publishDate | 2016-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-c5aae2486320498a98878ca353e96e6f2022-12-21T18:49:44ZengPublic Library of Science (PLoS)PLoS ONE1932-62032016-01-011110e016500610.1371/journal.pone.0165006Improving the Thermostability of Acidic Pullulanase from Bacillus naganoensis by Rational Design.Meihui ChangXiaoyu ChuJinzhi LvQingbin LiJian TianNingfeng WuPullulanase (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.http://europepmc.org/articles/PMC5072709?pdf=render |
| spellingShingle | Meihui Chang Xiaoyu Chu Jinzhi Lv Qingbin Li Jian Tian Ningfeng Wu Improving the Thermostability of Acidic Pullulanase from Bacillus naganoensis by Rational Design. PLoS ONE |
| title | Improving the Thermostability of Acidic Pullulanase from Bacillus naganoensis by Rational Design. |
| title_full | Improving the Thermostability of Acidic Pullulanase from Bacillus naganoensis by Rational Design. |
| title_fullStr | Improving the Thermostability of Acidic Pullulanase from Bacillus naganoensis by Rational Design. |
| title_full_unstemmed | Improving the Thermostability of Acidic Pullulanase from Bacillus naganoensis by Rational Design. |
| title_short | Improving the Thermostability of Acidic Pullulanase from Bacillus naganoensis by Rational Design. |
| title_sort | improving the thermostability of acidic pullulanase from bacillus naganoensis by rational design |
| url | http://europepmc.org/articles/PMC5072709?pdf=render |
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