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Enzyme Research
Volume 2017, Article ID 5947581, 10 pages
https://doi.org/10.1155/2017/5947581
Research Article

Plackett-Burman Design for rGILCC1 Laccase Activity Enhancement in Pichia pastoris: Concentrated Enzyme Kinetic Characterization

1Laboratorio de Microbiología Ambiental y de Suelos, Grupo de Biotecnología Ambiental e Industrial (GBAI), Departamento de Microbiología, Facultad de Ciencias, Pontificia Universidad Javeriana (PUJ), Bogotá, Colombia
2Departamento de Química, Facultad de Ciencias Exactas y Naturales, Universidad de Caldas, Manizales, Caldas, Colombia
3Laboratorio de Biotecnología Molecular, Grupo de Biotecnología Ambiental e Industrial (GBAI), Departamento de Microbiología, Facultad de Ciencias, Pontificia Universidad Javeriana (PUJ), Bogotá, Colombia
4Laboratorio de Expresión de Proteínas, Instituto de Errores Innatos del Metabolismo (IEIM), Facultad de Ciencias, Pontificia Universidad Javeriana (PUJ), Bogotá, Colombia
5Laboratorio de Parasitología Molecular, Grupo de Enfermedades Infecciosas, Facultad de Ciencias, Pontificia Universidad Javeriana (PUJ), Bogotá, Colombia

Correspondence should be addressed to Raúl A. Poutou-Piñales; oc.ude.anairevaj@uotuopr

Received 11 January 2017; Revised 27 February 2017; Accepted 9 March 2017; Published 21 March 2017

Academic Editor: Hartmut Kuhn

Copyright © 2017 Edwin D. Morales-Álvarez et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Laccases are multicopper oxidases that catalyze aromatic and nonaromatic compounds with concomitant reduction of molecular oxygen to water. They are of great interest due to their potential biotechnological applications. In this work we statistically improved culture media for recombinant GILCC1 (rGILCC1) laccase production at low scale from Ganoderma lucidum containing the construct pGAPZαA-GlucPost-Stop in Pichia pastoris. Temperature, pH stability, and kinetic parameter characterizations were determined by monitoring concentrate enzyme oxidation at different ABTS substrate concentrations. Plackett-Burman Design allowed improving enzyme activity from previous work 36.08-fold, with a laccase activity of 4.69 ± 0.39 UL−1 at 168 h of culture in a 500 mL shake-flask. Concentrated rGILCC1 remained stable between 10 and 50°C and retained a residual enzymatic activity greater than 70% at 60°C and 50% at 70°C. In regard to pH stability, concentrated enzyme was more stable at pH 4.0 ± 0.2 with a residual activity greater than 90%. The lowest residual activity greater than 55% was obtained at pH 10.0 ± 0.2. Furthermore, calculated apparent enzyme kinetic parameters were a of 6.87 × 10−5 mM s−1, with an apparent of 5.36 × 10−2 mM. Collectively, these important stability findings open possibilities for applications involving a wide pH and temperature ranges.