Cephalosporin C acylase (CCA), a proton-producing enzyme, was covalently bound on an epoxy-activated porous support. The microenvironmental pH change in immobilized CCA during the reaction was detected using pH-sensitive fluorescein labeling. The high catalytic velocity of the initial stage of conversion resulted in a sharp intraparticle pH gradient, which was likely the key factor relating to low operational stability. Accordingly, a novel strategy for a two-stage catalytic process was developed to reduce the reaction rate of stage I at a low temperature to preserve enzymatic activity and to shorten the duration of catalysis at a high reaction temperature in stage II. The reaction using the two-stage catalytic process (10-37 degrees C shift at 30 min) showed significantly improved stability compared with that of the single-temperature reaction at 37 degrees C (29 batches versus five batches, respectively) and a shorter catalytic period than the reaction at 10 degrees C (40 min versus 70 min, respectively). (C) 2016 Elsevier Ltd. All rights reserved.