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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\u2013Behnken 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 \u00b1 1.16 U\/mL, mean \u00b1 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 (&gt; 80% activity at a pH range of 7\u201310), with optimal activity at 55 \u00b0C and pH = 7.5. Kinetic analyses revealed a Km of 6.22 mmol\/L and a Vmax of 4.21 \u03bcmol\/mL\u00b7min 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 (\u03b1-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 \u03b1-amylase of A. apis.<\/jats:p>","DOI":"10.3390\/jof9111082","type":"journal-article","created":{"date-parts":[[2023,11,5]],"date-time":"2023-11-05T07:35:06Z","timestamp":1699169706000},"page":"1082","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Characterization of an \u03b1-Amylase from the Honeybee Chalk Brood Pathogen Ascosphaera apis"],"prefix":"10.3390","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1472-2448","authenticated-orcid":false,"given":"Jincheng","family":"Li","sequence":"first","affiliation":[{"name":"State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sen","family":"Liu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chenjie","family":"Yang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nemat O.","family":"Keyhani","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, University of Illinois, Chicago, IL 60607, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Huili","family":"Pu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of 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