Methanol-Free Production of Recombinant AppA Phytase in Komagataella phaffii: Evaluation of Cultivation Conditions, Bioreactor Cultivation, and Biochemical Characterization

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.