Characterization of an α-Amylase from the Honeybee Chalk Brood Pathogen Ascosphaera apis.
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jof-09-01082-v2__1_.pdf
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Resource type
Journal article
Date published
November 5, 2023
Abstract
The insect pathogenic fungus, Ascosphaera apis, 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 A. apis 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 (R2 = 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 CaCl2 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 Km of 6.22 mmol/L and a Vmax of 4.21 μmol/mL·min using soluble starch as the substrate. Activity was significantly stimulated by Fe2+ and completely inhibited by Cu2+, Mn2+, and Ba2+ (10 mM). Ethanol and chloroform (10% v/v) 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 A. apis.
Project(s)
Priority 2: Trait Diversity and Function
Funder
| Funder name | Awards |
National Kew Research and Development Program of China, China | 2017YFE0122000 |
National Natural Science Foundation of China, China | No. 32270029 - No. U1803232 - No. 31670026 |
Key Project from Fujian Provincial Department of Science and Technology, China | No. 2020N5005 |
Fujian Provincial Major Science and Technology Project, China | 2022NZ029017 |
Young and Middle-aged Teacher Education Research Project of Fujian Province, China | JAT210075 |
Fujian Agriculture and Forestry University Social Service Team Support Program Project | No, 11899170165 |
Journal title
Journal of Fungi
Volume
9
Issue
11
Article number
1082
Publisher
MDPI AG
Place of publication
Basel, Switzerland
eISSN
2309-608X
Date accepted
November 2, 2023
Official URL
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In Copyright
Additional information
IF = 5.724 (2022-2023)