Artículos de investigación

 

Heterologous expression of Trichoderma atroviride endochitinase ech42 in Pichia pastoris at low and high dissolved oxygen tensions

 

Expresión heteróloga de la endoquitinasa ech42 de Trichoderma atroviride en Pichia pastoris a bajas y elevadas tensiones de oxígeno disuelto

 

A.S. Pérez-Martínez^1,2, A.P. Barba de la Rosa¹ and A. De León-Rodríguez¹*

 

¹ División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica A.C. Camino a la Presa San José 2055, Lomas 4a secc., C.P. 78216, San Luis Potosí, S.L.P. México. *Corresponding author. E-mail: aleonr@ipicyt.edu.mx.

² Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, Av. Manuel Nava # 6, Zona Universitaria, C.P. 78210, San Luis Potosí, S.L.P., México.

 

Received June 11, 2013.
Accepted January 23, 2014.

 

Abstract

The expression of recombinant Trichoderma atroviride endochitinase ech42 (rech42) in Pichia pastoris driven by the constitutive P_GAP promoter was tested at low (3%) and high (40%) dissolved oxygen tension (DOT) conditions. Maximum volumetric enzyme activities of 18,600±1053 and 12,330±1000 U1⁻¹ were attained at 3% and 40% DOT, respectively. Whereas maximum specific enzyme activities were 89.4±4.6 y 88.8±4.0 U mg⁻¹_protein. We found that conjunction of low DOT and the overexpression of rech42 affected the cell size of the P. pastoris but no the rech42 specific enzyme activity.

Keywords: chitin, cell size, recombinant endochitinase, oxygen-limited fermentation.

 

Resumen

La expresión de la Endoquitinasa de 42 kDa de Trichoderma atroviride recombinante (rech42) en Pichia pastoris bajo la dirección del promotor constitutivo, P_GAP, fue analizada a baja (3%) y alta (40%) tensión de oxígeno disuelto (TOD). Las actividades enzimáticas volumétricas máximas logradas en cada condición fueron 18,600±1053 y 12,330±1000 U 1⁻¹, a TOD de 3% y 40%, respectivamente. Mientras que las actividades específicas máximas fueron 89.4±4.6 y 88.8±4.0 U mg⁻¹_proteína. Se encontró que la conjunción de una baja TOD y la sobreexpresión de rech42 afectó el tamaño de las células de Pichia pastoris pero no afectó la actividad enzimática específica de rech42.

Palabras clave: quitina, tamaño celular, endoquitinasa recombinante, fermentación limitada por oxígeno.

 

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Acknowledgements

We thank María Magdalena Martínez and Ferdinando Tristán (Nanosciences and Nanotechnology Division from IPICyT, Mexico) for helping us with AFM techniques, and Leandro G. Ordoñez for his comments. We also thank J. Simpson Williamson PhD. (Genetic Engineering Department, CINVESTAV, Mexico) for allowing us to carry out fluorescence microscopy. We thank partial financial support from CONACyT Grant No 56787 (Laboratory for Nanoscience and Nanotechnology Research-LINAN).

 

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