Degradation of Natural <i>Undaria pinnatifida</i> into Unsaturated Guluronic Acid Oligosaccharides by a Single Alginate Lyase
Here, we report on a bifunctional alginate lyase (Vnalg7) expressed in <i>Pichia pastoris</i>, which can degrade natural <i>Undaria pinnatifida</i> into unsaturated guluronic acid di- and trisaccharide without pretreatment. The enzyme activity of Vnalg7 (3620.00 U/mL-culture)...
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MDPI AG
2024-10-01
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| Series: | Marine Drugs |
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| author | Hui Wang Jiaqi Wen Nuraliya Ablimit Kun Deng Wenzhuo Wang Wei Jiang |
| author_facet | Hui Wang Jiaqi Wen Nuraliya Ablimit Kun Deng Wenzhuo Wang Wei Jiang |
| author_sort | Hui Wang |
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| description | Here, we report on a bifunctional alginate lyase (Vnalg7) expressed in <i>Pichia pastoris</i>, which can degrade natural <i>Undaria pinnatifida</i> into unsaturated guluronic acid di- and trisaccharide without pretreatment. The enzyme activity of Vnalg7 (3620.00 U/mL-culture) was 15.81-fold higher than that of the original <i>alg</i> (228.90 U/mL-culture), following engineering modification. The degradation rate reached 52.75%, and reducing sugar reached 30.30 mg/mL after combining Vnalg7 (200.00 U/mL-culture) and 14% (<i>w</i>/<i>v</i>) <i>U. pinnatifida</i> for 6 h. Analysis of the action mode indicated that Vnalg7 could degrade many substrates to produce a variety of unsaturated alginate oligosaccharides (AOSs), and the minimal substrate was tetrasaccharide. Site-directed mutagenesis showed that Glu<sup>238</sup>, Glu<sup>241</sup>, Glu<sup>312</sup>, Arg<sup>236</sup>, His<sup>307</sup>, Lys<sup>414</sup>, and Tyr<sup>418</sup> are essential catalytic sites, while Glu<sup>334</sup>, Glu<sup>344</sup>, and Asp<sup>311</sup> play auxiliary roles. Mechanism analysis revealed the enzymatic degradation pattern of Vnalg7, which mainly recognizes and attacks the third glycosidic linkage from the reducing end of oligosaccharide substrate. Our findings provide a novel alginate lyase tool and a sustainable and commercial production strategy for value-added biomolecules using seaweeds. |
| format | Article |
| id | doaj-art-9bbef1d1a6b64da9b1ee0539b46c0881 |
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| issn | 1660-3397 |
| language | English |
| publishDate | 2024-10-01 |
| publisher | MDPI AG |
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| series | Marine Drugs |
| spelling | doaj-art-9bbef1d1a6b64da9b1ee0539b46c08812025-08-20T02:10:56ZengMDPI AGMarine Drugs1660-33972024-10-01221045310.3390/md22100453Degradation of Natural <i>Undaria pinnatifida</i> into Unsaturated Guluronic Acid Oligosaccharides by a Single Alginate LyaseHui Wang0Jiaqi Wen1Nuraliya Ablimit2Kun Deng3Wenzhuo Wang4Wei Jiang5State Key Laboratory of Animal Biotech Breeding, College of Biological Sciences, China Agricultural University, Yuan Ming Yuan West Road No. 2, Haidian District, Beijing 100193, ChinaState Key Laboratory of Animal Biotech Breeding, College of Biological Sciences, China Agricultural University, Yuan Ming Yuan West Road No. 2, Haidian District, Beijing 100193, ChinaState Key Laboratory of Animal Biotech Breeding, College of Biological Sciences, China Agricultural University, Yuan Ming Yuan West Road No. 2, Haidian District, Beijing 100193, ChinaState Key Laboratory of Animal Biotech Breeding, College of Biological Sciences, China Agricultural University, Yuan Ming Yuan West Road No. 2, Haidian District, Beijing 100193, ChinaState Key Laboratory of Animal Biotech Breeding, College of Biological Sciences, China Agricultural University, Yuan Ming Yuan West Road No. 2, Haidian District, Beijing 100193, ChinaState Key Laboratory of Animal Biotech Breeding, College of Biological Sciences, China Agricultural University, Yuan Ming Yuan West Road No. 2, Haidian District, Beijing 100193, ChinaHere, we report on a bifunctional alginate lyase (Vnalg7) expressed in <i>Pichia pastoris</i>, which can degrade natural <i>Undaria pinnatifida</i> into unsaturated guluronic acid di- and trisaccharide without pretreatment. The enzyme activity of Vnalg7 (3620.00 U/mL-culture) was 15.81-fold higher than that of the original <i>alg</i> (228.90 U/mL-culture), following engineering modification. The degradation rate reached 52.75%, and reducing sugar reached 30.30 mg/mL after combining Vnalg7 (200.00 U/mL-culture) and 14% (<i>w</i>/<i>v</i>) <i>U. pinnatifida</i> for 6 h. Analysis of the action mode indicated that Vnalg7 could degrade many substrates to produce a variety of unsaturated alginate oligosaccharides (AOSs), and the minimal substrate was tetrasaccharide. Site-directed mutagenesis showed that Glu<sup>238</sup>, Glu<sup>241</sup>, Glu<sup>312</sup>, Arg<sup>236</sup>, His<sup>307</sup>, Lys<sup>414</sup>, and Tyr<sup>418</sup> are essential catalytic sites, while Glu<sup>334</sup>, Glu<sup>344</sup>, and Asp<sup>311</sup> play auxiliary roles. Mechanism analysis revealed the enzymatic degradation pattern of Vnalg7, which mainly recognizes and attacks the third glycosidic linkage from the reducing end of oligosaccharide substrate. Our findings provide a novel alginate lyase tool and a sustainable and commercial production strategy for value-added biomolecules using seaweeds.https://www.mdpi.com/1660-3397/22/10/453alginate lyaseaction modealginate oligosaccharidecatalytic mechanism<i>Undaria pinnatifida</i> |
| spellingShingle | Hui Wang Jiaqi Wen Nuraliya Ablimit Kun Deng Wenzhuo Wang Wei Jiang Degradation of Natural <i>Undaria pinnatifida</i> into Unsaturated Guluronic Acid Oligosaccharides by a Single Alginate Lyase Marine Drugs alginate lyase action mode alginate oligosaccharide catalytic mechanism <i>Undaria pinnatifida</i> |
| title | Degradation of Natural <i>Undaria pinnatifida</i> into Unsaturated Guluronic Acid Oligosaccharides by a Single Alginate Lyase |
| title_full | Degradation of Natural <i>Undaria pinnatifida</i> into Unsaturated Guluronic Acid Oligosaccharides by a Single Alginate Lyase |
| title_fullStr | Degradation of Natural <i>Undaria pinnatifida</i> into Unsaturated Guluronic Acid Oligosaccharides by a Single Alginate Lyase |
| title_full_unstemmed | Degradation of Natural <i>Undaria pinnatifida</i> into Unsaturated Guluronic Acid Oligosaccharides by a Single Alginate Lyase |
| title_short | Degradation of Natural <i>Undaria pinnatifida</i> into Unsaturated Guluronic Acid Oligosaccharides by a Single Alginate Lyase |
| title_sort | degradation of natural i undaria pinnatifida i into unsaturated guluronic acid oligosaccharides by a single alginate lyase |
| topic | alginate lyase action mode alginate oligosaccharide catalytic mechanism <i>Undaria pinnatifida</i> |
| url | https://www.mdpi.com/1660-3397/22/10/453 |
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