Molecular Modification and Enzymatic Properties of the Novel α-Glucosidase Aga432
This study aimed to clone the novel α-glucosidase gene Aga432 from Paenibacillus sp. and enhance its catalytic activity through site-directed mutagenesis. A gene fragment encoding α-glucosidase was successfully amplified from the genomic DNA of Paenibacillus sp., comprehensive sequence analysis was...
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The editorial department of Science and Technology of Food Industry
2025-06-01
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| Series: | Shipin gongye ke-ji |
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| Online Access: | http://www.spgykj.com/cn/article/doi/10.13386/j.issn1002-0306.2024100117 |
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| author | Han LU Ting FANG Xiaoxu NIU Xiaomin WENG Fen YAN |
| author_facet | Han LU Ting FANG Xiaoxu NIU Xiaomin WENG Fen YAN |
| author_sort | Han LU |
| collection | DOAJ |
| description | This study aimed to clone the novel α-glucosidase gene Aga432 from Paenibacillus sp. and enhance its catalytic activity through site-directed mutagenesis. A gene fragment encoding α-glucosidase was successfully amplified from the genomic DNA of Paenibacillus sp., comprehensive sequence analysis was performed, and homology modeling and molecular docking were employed to construct gene-engineered strains. Eight positive mutant strains were identified, among which the enzymatic properties of recombinant Aga432 and the highest relative activity mutant AT-2 were characterized. Additionally, the dispersing effects of recombinant α-glucosidases Aga432 and AT-2 on biofilms were explored, and their toxicity to mouse embryo fibroblasts was evaluated. The results revealed that the specific activity of Aga432 was 45.05 U/mg, while the mutant AT-2 exhibited a significantly enhanced specific activity of 84.09 U/mg. Although the optimal reaction temperature and pH for AT-2 were essentially unaltered relative to Aga432, its thermal stability was significantly enhanced, and it exhibited heightened stability under acidic conditions. The Km of mutant AT-2 was 2.18 times that of Aga432, the Vmax was 3.19 times, the Kcat was 2.33 times, and the Kcat/Km was 1.07 times that of Aga432. In vitro cellular assays indicated that Aga432 and AT-2 at concentrations of 15.0~30.0 μg/mL were non-toxic and exhibited good cell compatibility. Biofilm dispersal assays demonstrated that both recombinant α-glucosidases at concentrations ranging from 10.0 to 50.0 μg/mL significantly dispersed bacterial biofilms (P<0.0001). The thermostability of α-glucosidase Aga432 was successfully enhanced through molecular modification in this study, laying a foundation for the development of novel α-glucosidases and providing a reference for future targeted modification research. |
| format | Article |
| id | doaj-art-12cf95c5dd8a4157a9b79264beb8a39a |
| institution | Kabale University |
| issn | 1002-0306 |
| language | zho |
| publishDate | 2025-06-01 |
| publisher | The editorial department of Science and Technology of Food Industry |
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| series | Shipin gongye ke-ji |
| spelling | doaj-art-12cf95c5dd8a4157a9b79264beb8a39a2025-08-20T03:47:33ZzhoThe editorial department of Science and Technology of Food IndustryShipin gongye ke-ji1002-03062025-06-01461118519310.13386/j.issn1002-0306.20241001172024100117-11Molecular Modification and Enzymatic Properties of the Novel α-Glucosidase Aga432Han LU0Ting FANG1Xiaoxu NIU2Xiaomin WENG3Fen YAN4College of Biological Science and Engineering, Fuzhou University, Fuzhou 350108, ChinaCollege of Biological Science and Engineering, Fuzhou University, Fuzhou 350108, ChinaCollege of Biological Science and Engineering, Fuzhou University, Fuzhou 350108, ChinaCollege of Biological Science and Engineering, Fuzhou University, Fuzhou 350108, ChinaCollege of Biological Science and Engineering, Fuzhou University, Fuzhou 350108, ChinaThis study aimed to clone the novel α-glucosidase gene Aga432 from Paenibacillus sp. and enhance its catalytic activity through site-directed mutagenesis. A gene fragment encoding α-glucosidase was successfully amplified from the genomic DNA of Paenibacillus sp., comprehensive sequence analysis was performed, and homology modeling and molecular docking were employed to construct gene-engineered strains. Eight positive mutant strains were identified, among which the enzymatic properties of recombinant Aga432 and the highest relative activity mutant AT-2 were characterized. Additionally, the dispersing effects of recombinant α-glucosidases Aga432 and AT-2 on biofilms were explored, and their toxicity to mouse embryo fibroblasts was evaluated. The results revealed that the specific activity of Aga432 was 45.05 U/mg, while the mutant AT-2 exhibited a significantly enhanced specific activity of 84.09 U/mg. Although the optimal reaction temperature and pH for AT-2 were essentially unaltered relative to Aga432, its thermal stability was significantly enhanced, and it exhibited heightened stability under acidic conditions. The Km of mutant AT-2 was 2.18 times that of Aga432, the Vmax was 3.19 times, the Kcat was 2.33 times, and the Kcat/Km was 1.07 times that of Aga432. In vitro cellular assays indicated that Aga432 and AT-2 at concentrations of 15.0~30.0 μg/mL were non-toxic and exhibited good cell compatibility. Biofilm dispersal assays demonstrated that both recombinant α-glucosidases at concentrations ranging from 10.0 to 50.0 μg/mL significantly dispersed bacterial biofilms (P<0.0001). The thermostability of α-glucosidase Aga432 was successfully enhanced through molecular modification in this study, laying a foundation for the development of novel α-glucosidases and providing a reference for future targeted modification research.http://www.spgykj.com/cn/article/doi/10.13386/j.issn1002-0306.2024100117α-glucosidasehomology modelingmolecular dockingenzymatic propertiesbiofilm dispersing effect |
| spellingShingle | Han LU Ting FANG Xiaoxu NIU Xiaomin WENG Fen YAN Molecular Modification and Enzymatic Properties of the Novel α-Glucosidase Aga432 Shipin gongye ke-ji α-glucosidase homology modeling molecular docking enzymatic properties biofilm dispersing effect |
| title | Molecular Modification and Enzymatic Properties of the Novel α-Glucosidase Aga432 |
| title_full | Molecular Modification and Enzymatic Properties of the Novel α-Glucosidase Aga432 |
| title_fullStr | Molecular Modification and Enzymatic Properties of the Novel α-Glucosidase Aga432 |
| title_full_unstemmed | Molecular Modification and Enzymatic Properties of the Novel α-Glucosidase Aga432 |
| title_short | Molecular Modification and Enzymatic Properties of the Novel α-Glucosidase Aga432 |
| title_sort | molecular modification and enzymatic properties of the novel α glucosidase aga432 |
| topic | α-glucosidase homology modeling molecular docking enzymatic properties biofilm dispersing effect |
| url | http://www.spgykj.com/cn/article/doi/10.13386/j.issn1002-0306.2024100117 |
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