title: Microenvironmental pH changes in immobilized cephalosporin C acylase during a proton-producing reaction and regulation by a two-stage catalytic process.
abstract: 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°C shift at 30min) showed significantly improved stability compared with that of the single-temperature reaction at 37°C (29 batches versus five batches, respectively) and a shorter catalytic period than the reaction at 10°C (40min versus 70min, respectively).
authors: Department of Biological Science and Engineering, University of Science and Technology Beijing, Beijing 100083, China. Electronic address: luohui@ustb.edu.cn.
authors: Hui
authors: H
authors: Luo
authors: Department of Environmental Engineering, University of Science and Technology Beijing, Beijing 100083, China; Beijing Key Laboratory of Resource-oriented Treatment of Industrial Pollutants, Beijing 100083, China.
authors: Linlin
authors: L
authors: Zhu
authors: Department of Environmental Engineering, University of Science and Technology Beijing, Beijing 100083, China; Beijing Key Laboratory of Resource-oriented Treatment of Industrial Pollutants, Beijing 100083, China.
authors: Yanhong
authors: Y
authors: Chang
authors: Department of Biological Science and Engineering, University of Science and Technology Beijing, Beijing 100083, China; Department of Environmental Engineering, University of Science and Technology Beijing, Beijing 100083, China.
authors: Xiuhong
authors: X
authors: Liu
authors: Department of Biological Science and Engineering, University of Science and Technology Beijing, Beijing 100083, China; Department of Environmental Engineering, University of Science and Technology Beijing, Beijing 100083, China.
authors: Zijia
authors: Z
authors: Liu
authors: Department of Biological Science and Engineering, University of Science and Technology Beijing, Beijing 100083, China.
authors: Hongxu
authors: H
authors: Sun
authors: Department of Biological Science and Engineering, University of Science and Technology Beijing, Beijing 100083, China.
authors: Xi
authors: X
authors: Li
authors: Department of Chemical Engineering, Tsinghua University, Beijing 100084, China.
authors: Huimin
authors: H
authors: Yu
authors: Department of Chemical Engineering, Tsinghua University, Beijing 100084, China.
authors: Zhongyao
authors: Z
authors: Shen
mesh_terms: D000581:Amidohydrolases; D002384:Catalysis; D002511:Cephalosporins / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000378:metabolism; D019793:Fluorescein; D006863:Hydrogen-Ion Concentration; D011522:Protons; D013696:Temperature
chemical_list: D002511:Cephalosporins; D004800:Enzymes, Immobilized; D011522:Protons; C025163:cephalosporin C; D000581:Amidohydrolases; C019665:amidase; D019793:Fluorescein
keywords: Cephalosporin C acylase; Immobilized enzyme; Intraparticle pH; Operational stability; Two-stage catalytic process