Study of key amino acid residues of GH66 dextranase for producing high-degree polymerized isomaltooligosaccharides and improving of thermostability
Obtaining high-degree polymerized isomaltose is more difficult while achieving better prebiotic effects. We investigated the mutation specificity and enzymatic properties of SP5-Badex, a dextranase from the GH66 family of Bacillus aquimaris SP5, and determined its mutation sites through molecular do...
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Frontiers Media S.A.
2022-08-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2022.961776/full |
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| author | Qianru Lin Qianru Lin Huanyu Wang Huanyu Wang Yingying Xu Yingying Xu Dongxue Dong Dongxue Dong Qingzhen Miao Qingzhen Miao Jing Lu Jing Lu Mingsheng Lyu Mingsheng Lyu Shujun Wang Shujun Wang |
| author_facet | Qianru Lin Qianru Lin Huanyu Wang Huanyu Wang Yingying Xu Yingying Xu Dongxue Dong Dongxue Dong Qingzhen Miao Qingzhen Miao Jing Lu Jing Lu Mingsheng Lyu Mingsheng Lyu Shujun Wang Shujun Wang |
| author_sort | Qianru Lin |
| collection | DOAJ |
| description | Obtaining high-degree polymerized isomaltose is more difficult while achieving better prebiotic effects. We investigated the mutation specificity and enzymatic properties of SP5-Badex, a dextranase from the GH66 family of Bacillus aquimaris SP5, and determined its mutation sites through molecular docking to obtain five mutants, namely E454K, E454G, Y539F, N369F, and Y153N. Among them, Y539F and Y153N exhibited no enzymatic activity, but their hydrolysates included isomaltotetraose (IMO4). The enzymatic activity of E454G was 1.96 U/ml, which was 3.08 times higher than that before mutation. Moreover, 70% of the enzymatic activity could be retained after holding at 45°C for 180 min, which was 40% higher than that of SP5-Badex. Furthermore, its IMO4 content was 5.62% higher than that of SP5-Badex after hydrolysis at 30°C for 180 min. To investigate the effect of different amino acids on the same mutation site, saturation mutation was induced at site Y153, and the results showed that the enzyme activity of Y153W could be increased by 2 times, and some of the enzyme activity could still be retained at 50°C. Moreover, the enzyme activity increased by 50% compared with that of SP5-Badex after holding at 45°C for 180 min, and the IMO4 content of Y153W was approximately 64.97% after hydrolysis at 30°C for 180 min, which increased by approximately 12.47% compared with that of SP5-Badex. This site is hypothesized to rigidly bind to nonpolar (hydrophobic) amino acids to improve the stability of the protein structure, which in turn improves the thermal stability and simultaneously increases the IMO4 yield. |
| first_indexed | 2024-04-11T21:31:46Z |
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| institution | Directory Open Access Journal |
| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-04-11T21:31:46Z |
| publishDate | 2022-08-01 |
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| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-ff3684f67bf84acab36b5fcad31520d42022-12-22T04:01:57ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852022-08-011010.3389/fbioe.2022.961776961776Study of key amino acid residues of GH66 dextranase for producing high-degree polymerized isomaltooligosaccharides and improving of thermostabilityQianru Lin0Qianru Lin1Huanyu Wang2Huanyu Wang3Yingying Xu4Yingying Xu5Dongxue Dong6Dongxue Dong7Qingzhen Miao8Qingzhen Miao9Jing Lu10Jing Lu11Mingsheng Lyu12Mingsheng Lyu13Shujun Wang14Shujun Wang15Jiangsu Key Laboratory of Marine Bioresources and Environment/Jiangsu Key Laboratory of Marine Biotechnology, Jiangsu Ocean University, Lianyungang, ChinaCo-Innovation Center of Jiangsu Marine Bio-industry Technology, Jiangsu Ocean University, Lianyungang, ChinaJiangsu Key Laboratory of Marine Bioresources and Environment/Jiangsu Key Laboratory of Marine Biotechnology, Jiangsu Ocean University, Lianyungang, ChinaCo-Innovation Center of Jiangsu Marine Bio-industry Technology, Jiangsu Ocean University, Lianyungang, ChinaJiangsu Key Laboratory of Marine Bioresources and Environment/Jiangsu Key Laboratory of Marine Biotechnology, Jiangsu Ocean University, Lianyungang, ChinaCo-Innovation Center of Jiangsu Marine Bio-industry Technology, Jiangsu Ocean University, Lianyungang, ChinaJiangsu Key Laboratory of Marine Bioresources and Environment/Jiangsu Key Laboratory of Marine Biotechnology, Jiangsu Ocean University, Lianyungang, ChinaCo-Innovation Center of Jiangsu Marine Bio-industry Technology, Jiangsu Ocean University, Lianyungang, ChinaJiangsu Key Laboratory of Marine Bioresources and Environment/Jiangsu Key Laboratory of Marine Biotechnology, Jiangsu Ocean University, Lianyungang, ChinaCo-Innovation Center of Jiangsu Marine Bio-industry Technology, Jiangsu Ocean University, Lianyungang, ChinaJiangsu Key Laboratory of Marine Bioresources and Environment/Jiangsu Key Laboratory of Marine Biotechnology, Jiangsu Ocean University, Lianyungang, ChinaCo-Innovation Center of Jiangsu Marine Bio-industry Technology, Jiangsu Ocean University, Lianyungang, ChinaJiangsu Key Laboratory of Marine Bioresources and Environment/Jiangsu Key Laboratory of Marine Biotechnology, Jiangsu Ocean University, Lianyungang, ChinaCo-Innovation Center of Jiangsu Marine Bio-industry Technology, Jiangsu Ocean University, Lianyungang, ChinaJiangsu Key Laboratory of Marine Bioresources and Environment/Jiangsu Key Laboratory of Marine Biotechnology, Jiangsu Ocean University, Lianyungang, ChinaCo-Innovation Center of Jiangsu Marine Bio-industry Technology, Jiangsu Ocean University, Lianyungang, ChinaObtaining high-degree polymerized isomaltose is more difficult while achieving better prebiotic effects. We investigated the mutation specificity and enzymatic properties of SP5-Badex, a dextranase from the GH66 family of Bacillus aquimaris SP5, and determined its mutation sites through molecular docking to obtain five mutants, namely E454K, E454G, Y539F, N369F, and Y153N. Among them, Y539F and Y153N exhibited no enzymatic activity, but their hydrolysates included isomaltotetraose (IMO4). The enzymatic activity of E454G was 1.96 U/ml, which was 3.08 times higher than that before mutation. Moreover, 70% of the enzymatic activity could be retained after holding at 45°C for 180 min, which was 40% higher than that of SP5-Badex. Furthermore, its IMO4 content was 5.62% higher than that of SP5-Badex after hydrolysis at 30°C for 180 min. To investigate the effect of different amino acids on the same mutation site, saturation mutation was induced at site Y153, and the results showed that the enzyme activity of Y153W could be increased by 2 times, and some of the enzyme activity could still be retained at 50°C. Moreover, the enzyme activity increased by 50% compared with that of SP5-Badex after holding at 45°C for 180 min, and the IMO4 content of Y153W was approximately 64.97% after hydrolysis at 30°C for 180 min, which increased by approximately 12.47% compared with that of SP5-Badex. This site is hypothesized to rigidly bind to nonpolar (hydrophobic) amino acids to improve the stability of the protein structure, which in turn improves the thermal stability and simultaneously increases the IMO4 yield.https://www.frontiersin.org/articles/10.3389/fbioe.2022.961776/fulldextranasesite-directed mutationhigh-degree polymerizationisomaltooligosaccharidemolecular docking |
| spellingShingle | Qianru Lin Qianru Lin Huanyu Wang Huanyu Wang Yingying Xu Yingying Xu Dongxue Dong Dongxue Dong Qingzhen Miao Qingzhen Miao Jing Lu Jing Lu Mingsheng Lyu Mingsheng Lyu Shujun Wang Shujun Wang Study of key amino acid residues of GH66 dextranase for producing high-degree polymerized isomaltooligosaccharides and improving of thermostability Frontiers in Bioengineering and Biotechnology dextranase site-directed mutation high-degree polymerization isomaltooligosaccharide molecular docking |
| title | Study of key amino acid residues of GH66 dextranase for producing high-degree polymerized isomaltooligosaccharides and improving of thermostability |
| title_full | Study of key amino acid residues of GH66 dextranase for producing high-degree polymerized isomaltooligosaccharides and improving of thermostability |
| title_fullStr | Study of key amino acid residues of GH66 dextranase for producing high-degree polymerized isomaltooligosaccharides and improving of thermostability |
| title_full_unstemmed | Study of key amino acid residues of GH66 dextranase for producing high-degree polymerized isomaltooligosaccharides and improving of thermostability |
| title_short | Study of key amino acid residues of GH66 dextranase for producing high-degree polymerized isomaltooligosaccharides and improving of thermostability |
| title_sort | study of key amino acid residues of gh66 dextranase for producing high degree polymerized isomaltooligosaccharides and improving of thermostability |
| topic | dextranase site-directed mutation high-degree polymerization isomaltooligosaccharide molecular docking |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2022.961776/full |
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