Characterization of an α-Amylase from the Honeybee Chalk Brood Pathogen <i>Ascosphaera apis</i>
The insect pathogenic fungus, <i>Ascosphaera apis</i>, is the causative agent of honeybee chalk brood disease. Amylases are secreted by many plant pathogenic fungi to access host nutrients through the metabolism of starch, and the identification of new amylases can have important biotech...
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MDPI AG
2023-11-01
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| Series: | Journal of Fungi |
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| Online Access: | https://www.mdpi.com/2309-608X/9/11/1082 |
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| author | Jincheng Li Sen Liu Chenjie Yang Nemat O. Keyhani Huili Pu Longbin Lin Xiaoxia Li Peisong Jia Dongmei Wu Jieming Pan Philip C. Stevenson G Mandela Fernández-Grandon Liaoyuan Zhang Yuxi Chen Xiayu Guan Junzhi Qiu |
| author_facet | Jincheng Li Sen Liu Chenjie Yang Nemat O. Keyhani Huili Pu Longbin Lin Xiaoxia Li Peisong Jia Dongmei Wu Jieming Pan Philip C. Stevenson G Mandela Fernández-Grandon Liaoyuan Zhang Yuxi Chen Xiayu Guan Junzhi Qiu |
| author_sort | Jincheng Li |
| collection | DOAJ |
| description | The insect pathogenic fungus, <i>Ascosphaera apis</i>, is the causative agent of honeybee chalk brood disease. Amylases are secreted by many plant pathogenic fungi to access host nutrients through the metabolism of starch, and the identification of new amylases can have important biotechnological applications. Production of amylase by <i>A. apis</i> in submerged culture was optimized using the response surface method (RSM). Media composition was modeled using Box–Behnken design (BBD) at three levels of three variables, and the model was experimentally validated to predict amylase activity (<i>R</i><sup>2</sup> = 0.9528). Amylase activity was highest (45.28 ± 1.16 U/mL, mean ± SE) in media composed of 46 g/L maltose and1.51 g/L CaCl<sub>2</sub> at a pH of 6.6, where total activity was ~11-fold greater as compared to standard basal media. The enzyme was purified to homogeneity with a 2.5% yield and 14-fold purification. The purified enzyme had a molecular weight of 75 kDa and was thermostable and active in a broad pH range (> 80% activity at a pH range of 7–10), with optimal activity at 55 °C and pH = 7.5. Kinetic analyses revealed a <i>K</i><sub>m</sub> of 6.22 mmol/L and a <i>V</i><sub>max</sub> of 4.21 μmol/mL·min using soluble starch as the substrate. Activity was significantly stimulated by Fe<sup>2+</sup> and completely inhibited by Cu<sup>2+</sup>, Mn<sup>2+</sup>, and Ba<sup>2+</sup> (10 mM). Ethanol and chloroform (10% <i>v</i>/<i>v</i>) also caused significant levels of inhibition. The purified amylase essentially exhibited activity only on hydrolyzed soluble starch, producing mainly glucose and maltose, indicating that it is an endo-amylase (α-amylase). Amylase activity peaked at 99.38 U/mL fermented in a 3.7 L-bioreactor (2.15-fold greater than what was observed in flask cultures). These data provide a strategy for optimizing the production of enzymes from fungi and provide insight into the α-amylase of <i>A. apis</i>. |
| first_indexed | 2024-03-09T16:41:58Z |
| format | Article |
| id | doaj.art-549ec6207eb84338892dab847790eca4 |
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| language | English |
| last_indexed | 2024-03-09T16:41:58Z |
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| spelling | doaj.art-549ec6207eb84338892dab847790eca42023-11-24T14:51:04ZengMDPI AGJournal of Fungi2309-608X2023-11-01911108210.3390/jof9111082Characterization of an α-Amylase from the Honeybee Chalk Brood Pathogen <i>Ascosphaera apis</i>Jincheng Li0Sen Liu1Chenjie Yang2Nemat O. Keyhani3Huili Pu4Longbin Lin5Xiaoxia Li6Peisong Jia7Dongmei Wu8Jieming Pan9Philip C. Stevenson10G Mandela Fernández-Grandon11Liaoyuan Zhang12Yuxi Chen13Xiayu Guan14Junzhi Qiu15State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, ChinaState Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, ChinaState Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, ChinaDepartment of Biological Sciences, University of Illinois, Chicago, IL 60607, USAState Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, ChinaState Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, ChinaState Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, ChinaInstitute of Plant Protection, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, ChinaBiotechnology Research Institute, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi 832061, ChinaCollege of Biology & Pharmacy, Yulin Normal University, Yulin 537000, ChinaNatural Resources Institute, University of Greenwich, Chatham Maritime ME4 4TB, UKNatural Resources Institute, University of Greenwich, Chatham Maritime ME4 4TB, UKState Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, ChinaState Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, ChinaCollege of Horticulture, Fujian Agriculture and Forestry University, Fuzhou 350002, ChinaState Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, ChinaThe insect pathogenic fungus, <i>Ascosphaera apis</i>, is the causative agent of honeybee chalk brood disease. Amylases are secreted by many plant pathogenic fungi to access host nutrients through the metabolism of starch, and the identification of new amylases can have important biotechnological applications. Production of amylase by <i>A. apis</i> in submerged culture was optimized using the response surface method (RSM). Media composition was modeled using Box–Behnken design (BBD) at three levels of three variables, and the model was experimentally validated to predict amylase activity (<i>R</i><sup>2</sup> = 0.9528). Amylase activity was highest (45.28 ± 1.16 U/mL, mean ± SE) in media composed of 46 g/L maltose and1.51 g/L CaCl<sub>2</sub> at a pH of 6.6, where total activity was ~11-fold greater as compared to standard basal media. The enzyme was purified to homogeneity with a 2.5% yield and 14-fold purification. The purified enzyme had a molecular weight of 75 kDa and was thermostable and active in a broad pH range (> 80% activity at a pH range of 7–10), with optimal activity at 55 °C and pH = 7.5. Kinetic analyses revealed a <i>K</i><sub>m</sub> of 6.22 mmol/L and a <i>V</i><sub>max</sub> of 4.21 μmol/mL·min using soluble starch as the substrate. Activity was significantly stimulated by Fe<sup>2+</sup> and completely inhibited by Cu<sup>2+</sup>, Mn<sup>2+</sup>, and Ba<sup>2+</sup> (10 mM). Ethanol and chloroform (10% <i>v</i>/<i>v</i>) also caused significant levels of inhibition. The purified amylase essentially exhibited activity only on hydrolyzed soluble starch, producing mainly glucose and maltose, indicating that it is an endo-amylase (α-amylase). Amylase activity peaked at 99.38 U/mL fermented in a 3.7 L-bioreactor (2.15-fold greater than what was observed in flask cultures). These data provide a strategy for optimizing the production of enzymes from fungi and provide insight into the α-amylase of <i>A. apis</i>.https://www.mdpi.com/2309-608X/9/11/1082fungal pathogen<i>Ascosphaera apis</i>α-amylasemedium factor optimizationpurificationlarge-scale production |
| spellingShingle | Jincheng Li Sen Liu Chenjie Yang Nemat O. Keyhani Huili Pu Longbin Lin Xiaoxia Li Peisong Jia Dongmei Wu Jieming Pan Philip C. Stevenson G Mandela Fernández-Grandon Liaoyuan Zhang Yuxi Chen Xiayu Guan Junzhi Qiu Characterization of an α-Amylase from the Honeybee Chalk Brood Pathogen <i>Ascosphaera apis</i> Journal of Fungi fungal pathogen <i>Ascosphaera apis</i> α-amylase medium factor optimization purification large-scale production |
| title | Characterization of an α-Amylase from the Honeybee Chalk Brood Pathogen <i>Ascosphaera apis</i> |
| title_full | Characterization of an α-Amylase from the Honeybee Chalk Brood Pathogen <i>Ascosphaera apis</i> |
| title_fullStr | Characterization of an α-Amylase from the Honeybee Chalk Brood Pathogen <i>Ascosphaera apis</i> |
| title_full_unstemmed | Characterization of an α-Amylase from the Honeybee Chalk Brood Pathogen <i>Ascosphaera apis</i> |
| title_short | Characterization of an α-Amylase from the Honeybee Chalk Brood Pathogen <i>Ascosphaera apis</i> |
| title_sort | characterization of an α amylase from the honeybee chalk brood pathogen i ascosphaera apis i |
| topic | fungal pathogen <i>Ascosphaera apis</i> α-amylase medium factor optimization purification large-scale production |
| url | https://www.mdpi.com/2309-608X/9/11/1082 |
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