3 Biotech

, 9:447 | Cite as

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-PiñalesEmail author
  • 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
Original Article


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.


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



Multicopper oxidases


2, 20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)


Pulping black liquor


Low density polyethylene


Alcohol oxidase promoter


Glyceraldehyde-3-phosphate dehydrogenase promoter


Laccase from P. ostreatus


Recombinant laccase from P. ostreatus


Maximum reaction rate


Michaelis constant


Yeast extract, peptone and glucose culture media


Master cell bank


Yeast extract, peptone, glucose-zeocin culture media


Effective work volume


Plackett–Burman experimental design


One-factor experimental design


Air volume per media volume


Specific growth rate


Duplication time


Revolutions per minute


Biomass/substrate yield


Enzyme/substrate yield


Biomass productivity


Enzyme productivity


Standard deviation


Green fluorescent protein


5′-AMP-activated protein kinase subunit gamma


Carbon catabolite-derepressing protein kinase


Enzyme glyceraldehyde-3-phosphate dehydrogenase



This research was funded by the following grants: Grant ID: 00005575 (Correlación entre la expresión constitutiva la concentración de proteína y la actividad biológica de las lacasas recombinantes POXA1 B de Pleurotus ostreatus y GLlac 1 de Ganoderma lucidum en Pichia pastoris); Grant ID: 00006337 (Optimización del medio de cultivo para producción de la lacasa recombinante POXA 1B de Pleurotus ostreatus en Pichia pastoris) from Pontificia Universidad Javeriana; Grant ID: 00006169 (Joven Investigador Colciencias 2014), Bogotá, D.C. Colombia; Grant ID: 00007885 (Estudio de la estabilidad a tiempo real del concentrado de la lacasa rPOXA 1B de Pleurotus ostreatus producida en Pichia pastoris) from Pontificia Universidad Javeriana. The financing entity had no role in the study design, data collection, or analysis, decision to publish, or preparation of the manuscript. The authors thank Fiona Raikes and María Lucía Gutiérrez, Ph.D., for English editing.

Compliance with ethical standards

Conflict of interest

The authors declare they have no competing interests.


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Copyright information

© King Abdulaziz City for Science and Technology 2019

Authors and Affiliations

  • Leidy D. Ardila-Leal
    • 1
  • Diego A. Albarracín-Pardo
    • 1
  • Claudia M. Rivera-Hoyos
    • 1
  • Edwin D. Morales-Álvarez
    • 2
  • Raúl A. Poutou-Piñales
    • 1
    Email author
  • Angela M. Cardozo-Bernal
    • 3
  • Balkys E. Quevedo-Hidalgo
    • 4
  • Aura M. Pedroza-Rodríguez
    • 5
  • Dennis J. Díaz-Rincón
    • 6
  • Alexander Rodríguez-López
    • 6
  • Carlos J. Alméciga-Díaz
    • 6
  • Claudia L. Cuervo-Patiño
    • 7
  1. 1.Laboratorio de Biotecnología Molecular, Grupo de Biotecnología Ambiental e Industrial (GBAI), Departamento de Microbiología, Facultad de CienciasPontificia Universidad Javeriana (PUJ)Bogotá D.C.Colombia
  2. 2.Departamento de Química, Facultad de Ciencias Exactas y NaturalesUniversidad de CaldasManizalesColombia
  3. 3.Joven Investigador COLCIENCIAS, Laboratorio de Biotecnología Molecular, Grupo de Biotecnología Ambiental e Industrial (GBAI), Departamento de Microbiología, Facultad de CienciasPontificia Universidad Javeriana (PUJ)Bogotá D.C.Colombia
  4. 4.Laboratorio de Biotecnología Aplicada, Grupo de Biotecnología Ambiental e Industrial (GBAI), Departamento de Microbiología, Facultad de CienciasPontificia Universidad Javeriana (PUJ)Bogotá D.C.Colombia
  5. 5.Laboratorio de Microbiología Ambiental y de Suelos, Grupo de Biotecnología Ambiental e Industrial (GBAI), Departamento de Microbiología. Facultad de Ciencias. PontificiaUniversidad Javeriana (PUJ)Bogotá D.C.Colombia
  6. 6.Laboratorio de Expresión de Proteínas, Instituto de Errores Innatos del Metabolismo (IEIM), Facultad de CienciasPontificia Universidad Javeriana (PUJ)Bogotá D.C.Colombia
  7. 7.Laboratorio de Parasitología Molecular, Grupo de Enfermedades Infecciosas, Facultad de CienciasPontificia Universidad Javeriana (PUJ)Bogotá D.C.Colombia

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