Media improvement for 10 L bioreactor production of rPOXA 1B laccase by P. pastoris

Leidy D. Ardila-Leal, Diego A. Albarracín-Pardo, Claudia M. Rivera-Hoyos, Edwin D. Morales-Álvarez, Raúl A. Poutou-Piñales, Angela M. Cardozo-Bernal, Balkys E. Quevedo-Hidalgo, Aura M. Pedroza-Rodríguez, Dennis J. Díaz-Rincón, Alexander Rodríguez-López, Carlos J. Alméciga-Díaz, Claudia L. Cuervo-Patiño

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12 Scopus citations

Abstract

In this work, we statistically improved culture media for rPOXA 1B laccase production, expressed in Pichia pastoris containing pGAPZαA-LaccPost-Stop construct and assayed at 10 L bioreactor production scale (6 L effective work volume). The concentrated enzyme was evaluated for temperature and pH stability and kinetic parameter, characterized by monitoring oxidation of different ABTS [2, 20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] substrate concentrations. Plackett–Burman experimental design (PBED) implementation improved previous work results by 3.05-fold, obtaining a laccase activity of 1373.72 ± 0.37 U L−1 at 168 h of culture in a 500 mL shake flask. In contrast, one factor experimental design (OFED) applied after PBED improved by threefold the previous study, additionally increasing the C/N ratio. Employing OFED media at 10 L bioreactor scale was capable of producing 3159.93 ± 498.90 U L−1 at 192 h, representing a 2.4-fold increase. rPOXA 1B concentrate remained stable between 10 and 50 °C and retained over 70% residual enzymatic activity at 60 °C and 50% at 70 °C. Concerning pH stability, the enzyme was stable at pH 4.0 ± 0.2 with a residual activity greater than 90%. The lowest residual activity (60%) was obtained at pH 10.0 ± 0.2. Furthermore, the apparent kinetic parameters were Vmax of 3.163 × 10−2 mM min−1 and Km of 1.716 mM. Collectively, regarding enzyme stability our data provide possibilities for applications involving a wide range of pH and temperatures.

Original languageEnglish
Article number447
Journal3 Biotech
Volume9
Issue number12
DOIs
StatePublished - 01 Dec 2019

Keywords

  • Enzyme kinetics
  • Enzyme stability
  • One-factor experimental design
  • Pichia pastoris
  • Plackett–Burman experimental design
  • Recombinant laccase

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