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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
  • 61 Downloads

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.

Keywords

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

Abbreviations

MCOs

Multicopper oxidases

ABTS

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

PBL

Pulping black liquor

LDPE

Low density polyethylene

pAOX1

Alcohol oxidase promoter

pGAP

Glyceraldehyde-3-phosphate dehydrogenase promoter

POXA 1B

Laccase from P. ostreatus

rPOXA 1B

Recombinant laccase from P. ostreatus

Vmax

Maximum reaction rate

KM

Michaelis constant

YPG

Yeast extract, peptone and glucose culture media

MCB

Master cell bank

YPG-Z

Yeast extract, peptone, glucose-zeocin culture media

EWV

Effective work volume

PBED

Plackett–Burman experimental design

OFED

One-factor experimental design

v.v.m.

Air volume per media volume

µx

Specific growth rate

Td

Duplication time

r.p.m.

Revolutions per minute

Y(x/s)

Biomass/substrate yield

Y(p/s)

Enzyme/substrate yield

P(x)

Biomass productivity

P(p)

Enzyme productivity

SD

Standard deviation

GFP

Green fluorescent protein

Snf4

5′-AMP-activated protein kinase subunit gamma

Snf1

Carbon catabolite-derepressing protein kinase

GAPDH

Enzyme glyceraldehyde-3-phosphate dehydrogenase

Notes

Acknowledgements

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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