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Expression and Biochemical Characterization of a Yersinia intermedia Phytase Expressed in Escherichia coli

[ Vol. 10 , Issue. 2 ]

Author(s):

Mariana S. Vieira, Vinícius V. Pereira, Alice da Cunha Morales Álvares, Lais M. Nogueira, William J.N. Lima, Paulo A. Granjeiro, Daniel B. Gonçalves, Mariana Campos-da-Paz, Sonia M. de Freitas and Alexsandro S. Galdino*   Pages 131 - 139 ( 9 )

Abstract:


Background: Phytases are enzymes capable of degrading phytic acid and used in animal feed supplementation in order to improve digestibility through the release of minerals such as phosphorus.

Objective: The main goal of this study was to express and characterize a Yersinia intermedia phytase expressed in Escherichia coli cells.

Methods: The Y. intermedia phytase gene was synthesized and overexpressed in Escherichia coli cells. The phytase recombinante (rPHY) was purified to homogeneity using a Ni-NTA column. The biochemical and biophysical properties of the rPHY were measured in order to fully characterize the recombinant enzyme. The following patents database were consulted: Espacenet, USPTO, LATIPAT, Patent Scope, WIPO and Google Patents.

Results: The results showed that the rPHY is active at 37-40ºC and presented an optimal pH and temperature of 8.0 and 40°C, respectively. The phytase rPHY was activated by Cu2+ ion and showed resistance to trypsin and pepsin, retaining 55% of the activity at the ratio of 0.02. Furthermore, the dissociation constant (Kd = 1.1150 ± 0.0087 mM), as estimated by a fluorescence binding assay, suggests a medium affinity of the enzyme with the substrate.

Conclusion: The results of this article can be considered as innovative and for this reason, they were protected by Intellectual Property Law in Brazil. Take together, the biochemical properties of the rPHY could be useful in future for its industrial application of this enzyme as an additive in the monogastric feed.

Keywords:

Phytase, Yersinia intermedia, emzyme kinetics, recombinant enzyme, protein structure, fluorescence quenching.

Affiliation:

Laboratorio de Biotecnologia de Microrganismos, Universidade Federal de Sao Joao Del-Rei, Divinópolis, MG, 35501-296, Laboratorio de Biotecnologia de Microrganismos, Universidade Federal de Sao Joao Del-Rei, Divinópolis, MG, 35501-296, Laboratorio de BiofIsica, Universidade de BrasIlia, BrasIlia, DF, 70910-900, Laboratorio de Biotecnologia de Microrganismos, Universidade Federal de Sao Joao Del-Rei, Divinópolis, MG, 35501-296, Laboratorio de Biotecnologia, Instituto de Ciencias Agrarias, Universidade Federal de Minas Gerais, Montes Claros, MG, 39404- 547, Laboratorio de Processos Biotecnologicos e Purificacao de Macromoleculas, Universidade Federal de Sao Joao Del-Rei, MG, 35501-296, Laboratorio de Processos Biotecnologicos e Purificacao de Macromoleculas, Universidade Federal de Sao Joao Del-Rei, MG, 35501-296, Laboratorio de Nanobiotecnologia, Universidade Federal de Sao Joao Del- Rei, MG, 35501-296, Laboratorio de BiofIsica, Universidade de BrasIlia, BrasIlia, DF, 70910-900, Laboratorio de Biotecnologia de Microrganismos, Universidade Federal de Sao Joao Del-Rei, Divinópolis, MG, 35501-296

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