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Evaluation of Strategies to Improve the Production of Alkaline Protease PrtA from Aspergillus nidulans

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Abstract

Aspergillus nidulans produces several proteases. The prtA gene encodes a major protease, and two approaches were explored to achieve the overproduction of this enzyme. Molecular cloning of the mature form of this enzyme in Pichia pastoris resulted in the production of an inactive form. In addition, the presence of this enzyme was toxic for the host and resulted in cell lysis. The modification of the culture medium constituents resulted in a 6.4-fold increase in enzyme production. The main effect was achieved through the use of organic nitrogen sources. Although it was previously shown that the PrtA protease shows promiscuous esterase activity, the production of this enzyme was not induced by lipidic sources.

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Abbreviations

dNTPs:

Deoxynucleotide triphosphates

PCR:

Polymerase chain reaction

DNA:

Deoxyribonucleic acid

OD:

Optical density

SDS:

Sodium dodecyl sulfate

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

PVDF:

Polyvinylidene fluoride

AP-conjugated anti-His (C-term):

Alkaline phosphatase (AP)-conjugated antibodies that recognizes a polyhistidine amino acid sequence at the carboxy-terminus of protein

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Acknowledgments

The authors would like to thank Bsc. Norma Ballesteros and Ana Alva for technical assistance. Financial support for this project was obtained from PAPIIT-DGAPA-UNAM IN2148092. Denise Castro received a scholarship from CONACyT. The authors would also like to thank the Proteomic Unit of the Institute of Biotechnology, UNAM, for the LC/MS/MS analysis.

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Authors and Affiliations

  1. Department of Food Science and Biotechnology, Faculty of Chemistry, Universidad Nacional Autónoma de México (UNAM), Ciudad Universitaria, México City, DF, 04510, Mexico

    Denise Castro-Ochoa, Carolina Peña-Montes & Amelia Farrés

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  1. Denise Castro-Ochoa
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  2. Carolina Peña-Montes
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  3. Amelia Farrés
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Correspondence to Amelia Farrés.

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Castro-Ochoa, D., Peña-Montes, C. & Farrés, A. Evaluation of Strategies to Improve the Production of Alkaline Protease PrtA from Aspergillus nidulans . Appl Biochem Biotechnol 169, 1672–1682 (2013). https://doi.org/10.1007/s12010-013-0091-8

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  • DOI: https://doi.org/10.1007/s12010-013-0091-8

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