Phytases are widely used as feed additives because they improve phosphorus utilization from plant-derived feed ingredients and reduce phosphorus excretion into the environment. Most recombinant AppA phytases produced in Komagataella phaffii have been obtained using methanol-inducible expression systems, whereas information on constitutive methanol-free production remains limited. In this study, Escherichia coli AppA phytase was expressed in K. phaffii under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. Cultivation temperature and initial glucose concentration were evaluated in shake-flask cultures, while the selected conditions were assessed in a 2 L stirred-tank bioreactor. Extracellular phytase activity reached 114.17 ± 2.71 U/mL in shake flasks at 27 °C with 3% initial glucose. The K. phaffii-produced enzyme was N-glycosylated, with an apparent molecular weight of approximately 54 kDa compared with approximately 45 kDa after deglycosylation. Compared with purified rAppA-E, the rAppA-P preparation showed a lower temperature optimum, a narrower pH–activity profile, and lower apparent thermostability. In a single bioreactor run, extracellular phytase activity reached 186.81 ± 0.25 U/mL, approximately 1.6-fold higher than the maximum mean shake-flask value. These findings demonstrate constitutive, methanol-free extracellular production of AppA in K. phaffii and support further development of controlled cultivation strategies.

В настоящем исследовании успешно клонирована, экспрессирована в клетках Escherichia coli и очищена α-амилаза из казахстанского штамма Bacillus paralicheniformis, обладающего высокой амилолитической активностью. Ген α-амилазы был амплифицирован методом полимеразной цепной реакции с использованием специфических праймеров, подобранных на основе данных полногеномного секвенирования штамма B. paralicheniformis T7 (GenBank accession # CP124861). Ген был клонирован в векторе pET-28c(+) и с помощью плазмидной экспрессии была получена рекомбинантная α-амилаза. С использованием металлоаффинной хроматографии был очищен рекомбинантый белок, выход которого составил 1,26 мг/л. Рекомбинантная α-амилаза обладает максимальной активностью при 85°С и устойчива к часовой инкубации при 70°С и 80°С. Оптимальное значение рН для рекомбинантного фермента составляет 6,0. Удельная активность рекомбинантной α-амилазы в оптимальных условиях составляет 2512.6 ± 54.7 Ед/мг. Ионы Ca2+, Cu2+ Co2+ в 1,5-2 раза увеличивают, а ионы Fe3+ на 90% уменьшают активность рекомбинантной α-амилазы. Основными продуктами гидролиза картофельного крахмала при использовании рекомбинантной α-амилазы выступают: глюкоза, мальтоза, мальтотреоза, мальтотетраоза и мальтопентаоза. В целом, результаты свидетельствуют о высоком потенциале использования α-амилазы из B. paralicheniformis в переработке крахмала и крахмалсодержащего сырья.

Phytases are industrial enzymes that enhance the availability of phytate-bound phosphorus and can reduce the environmental burden associated with undigested phytate. Bacillus paralicheniformis T7 is a spore-forming strain exhibiting multiple hydrolytic activities, including phytase activity. The phyL gene encoding β-propeller phytase was cloned from B. paralicheniformis T7 and expressed in Escherichia coli, followed by purification and biochemical characterization of the recombinant enzyme—rPhyL. The molecular mass of rPhyL was 41.0 kDa, and maximal activity was observed at pH 8.0 and 60 °C. The enzyme displayed alkaline stability, retaining 90% and 93% of its initial activity after 5 h at pH 8.0 and 10.0, respectively. Following 5 h of incubation at 50 °C and 55 °C, residual activities were 83% and 46%, respectively. rPhyL was strongly Ca²⁺-dependent, with maximal activity at 0.6–1.0 mM CaCl₂. Kinetic analysis using sodium phytate as the substrate yielded an apparent K_m of 1.21 mM, an apparent V_max of 328.6 μM·min⁻¹, and an apparent K_i of 1.34 mM under the specified assay conditions. Under the tested conditions, rPhyL exhibited detectable activity toward sodium phytate, whereas no detectable activity was observed toward ATP, sodium pyrophosphate, β-glycerophosphate, or p-nitrophenyl phosphate. These findings characterize rPhyL as a Ca²⁺-dependent bacillary β-propeller phytase with alkaline stability, expanding the enzymatic repertoire of the spore-forming B. paralicheniformis T7 strain.