Directed Modification of a GHF11 Thermostable Xylanase AusM for Enhancing Inhibitory Resistance towards SyXIP-I and Application of AusM<sup>PKK</sup> in Bread Making
To reduce the inhibition sensitivity of a thermoresistant xylanase AusM to xylanase inhibitor protein (XIP)-type in wheat flour, the site-directed mutagenesis was conducted based on the computer-aided redesign. First, fourteen single-site variants and one three-amino acid replacement variant in the...
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MDPI AG
2023-09-01
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| author | Dong Zhang Jing Huang Youyi Liu Xingyi Chen Tiecheng Gao Ning Li Weining Huang Minchen Wu |
| author_facet | Dong Zhang Jing Huang Youyi Liu Xingyi Chen Tiecheng Gao Ning Li Weining Huang Minchen Wu |
| author_sort | Dong Zhang |
| collection | DOAJ |
| description | To reduce the inhibition sensitivity of a thermoresistant xylanase AusM to xylanase inhibitor protein (XIP)-type in wheat flour, the site-directed mutagenesis was conducted based on the computer-aided redesign. First, fourteen single-site variants and one three-amino acid replacement variant in the thumb region of an AusM-encoding gene (<i>AusM</i>) were constructed and expressed in <i>E. coli</i> BL21(DE3), respectively, as predicted theoretically. At a molar ratio of 100:1 between SyXIP-I/xylanase, the majority of mutants were nearly completely inactivated by the inhibitor SyXIP-I, whereas AusM<sup>N127A</sup> retained 62.7% of its initial activity and AusM<sup>PKK</sup> retained 100% of its initial activity. The optimal temperature of the best mutant AusM<sup>PKK</sup> was 60 °C, as opposed to 60–65 °C for AusM, while it exhibited improved thermostability, retaining approximately 60% of its residual activity after heating at 80 °C for 60 min. Furthermore, AusM<sup>PKK</sup> at a dosage of 1000 U/kg was more effective than AusM at 4000 U/kg in increasing specific bread loaf volume and reducing hardness during bread production and storage. Directed evolution of AusM significantly reduces inhibition sensitivity, and the mutant enzyme AusM<sup>PKK</sup> is conducive to improving bread quality and extending its shelf life. |
| first_indexed | 2024-03-10T21:44:51Z |
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| institution | Directory Open Access Journal |
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| language | English |
| last_indexed | 2024-03-10T21:44:51Z |
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| series | Foods |
| spelling | doaj.art-95dd211289a742c9830c175e36193c0f2023-11-19T14:22:48ZengMDPI AGFoods2304-81582023-09-011219357410.3390/foods12193574Directed Modification of a GHF11 Thermostable Xylanase AusM for Enhancing Inhibitory Resistance towards SyXIP-I and Application of AusM<sup>PKK</sup> in Bread MakingDong Zhang0Jing Huang1Youyi Liu2Xingyi Chen3Tiecheng Gao4Ning Li5Weining Huang6Minchen Wu7Wuxi School of Medicine, Jiangnan University, Wuxi 214122, ChinaState Key Laboratory of Food Science and Technology, and the Laboratory of Baking and Fermentation Science, Cereals/Sourdough and Nutritional Functionality Research, School of Food Science and Technology, Jiangnan University, Wuxi 214122, ChinaWuxi School of Medicine, Jiangnan University, Wuxi 214122, ChinaWuxi School of Medicine, Jiangnan University, Wuxi 214122, ChinaGuangzhou Puratos Food Co., Ltd., Guangzhou 511400, ChinaGuangzhou Puratos Food Co., Ltd., Guangzhou 511400, ChinaState Key Laboratory of Food Science and Technology, and the Laboratory of Baking and Fermentation Science, Cereals/Sourdough and Nutritional Functionality Research, School of Food Science and Technology, Jiangnan University, Wuxi 214122, ChinaWuxi School of Medicine, Jiangnan University, Wuxi 214122, ChinaTo reduce the inhibition sensitivity of a thermoresistant xylanase AusM to xylanase inhibitor protein (XIP)-type in wheat flour, the site-directed mutagenesis was conducted based on the computer-aided redesign. First, fourteen single-site variants and one three-amino acid replacement variant in the thumb region of an AusM-encoding gene (<i>AusM</i>) were constructed and expressed in <i>E. coli</i> BL21(DE3), respectively, as predicted theoretically. At a molar ratio of 100:1 between SyXIP-I/xylanase, the majority of mutants were nearly completely inactivated by the inhibitor SyXIP-I, whereas AusM<sup>N127A</sup> retained 62.7% of its initial activity and AusM<sup>PKK</sup> retained 100% of its initial activity. The optimal temperature of the best mutant AusM<sup>PKK</sup> was 60 °C, as opposed to 60–65 °C for AusM, while it exhibited improved thermostability, retaining approximately 60% of its residual activity after heating at 80 °C for 60 min. Furthermore, AusM<sup>PKK</sup> at a dosage of 1000 U/kg was more effective than AusM at 4000 U/kg in increasing specific bread loaf volume and reducing hardness during bread production and storage. Directed evolution of AusM significantly reduces inhibition sensitivity, and the mutant enzyme AusM<sup>PKK</sup> is conducive to improving bread quality and extending its shelf life.https://www.mdpi.com/2304-8158/12/19/3574arabinoxylanxylanaseinhibition sensitivitycomputer-aided re-designbread baking |
| spellingShingle | Dong Zhang Jing Huang Youyi Liu Xingyi Chen Tiecheng Gao Ning Li Weining Huang Minchen Wu Directed Modification of a GHF11 Thermostable Xylanase AusM for Enhancing Inhibitory Resistance towards SyXIP-I and Application of AusM<sup>PKK</sup> in Bread Making Foods arabinoxylan xylanase inhibition sensitivity computer-aided re-design bread baking |
| title | Directed Modification of a GHF11 Thermostable Xylanase AusM for Enhancing Inhibitory Resistance towards SyXIP-I and Application of AusM<sup>PKK</sup> in Bread Making |
| title_full | Directed Modification of a GHF11 Thermostable Xylanase AusM for Enhancing Inhibitory Resistance towards SyXIP-I and Application of AusM<sup>PKK</sup> in Bread Making |
| title_fullStr | Directed Modification of a GHF11 Thermostable Xylanase AusM for Enhancing Inhibitory Resistance towards SyXIP-I and Application of AusM<sup>PKK</sup> in Bread Making |
| title_full_unstemmed | Directed Modification of a GHF11 Thermostable Xylanase AusM for Enhancing Inhibitory Resistance towards SyXIP-I and Application of AusM<sup>PKK</sup> in Bread Making |
| title_short | Directed Modification of a GHF11 Thermostable Xylanase AusM for Enhancing Inhibitory Resistance towards SyXIP-I and Application of AusM<sup>PKK</sup> in Bread Making |
| title_sort | directed modification of a ghf11 thermostable xylanase ausm for enhancing inhibitory resistance towards syxip i and application of ausm sup pkk sup in bread making |
| topic | arabinoxylan xylanase inhibition sensitivity computer-aided re-design bread baking |
| url | https://www.mdpi.com/2304-8158/12/19/3574 |
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