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Int. J. Mol. Sci. 2014, 15(9), 15571-15591; doi:10.3390/ijms150915571

Expression of Aspergillus nidulans phy Gene in Nicotiana benthamiana Produces Active Phytase with Broad Specificities

1
Department of Biology & Medicinal Science, Pai Chai University, Daejeon 302-735, Korea
2
Research Institute for Subtropical Agriculture and Animal Biotechnology, Jeju National University, Jeju-si 690-756, Jeju Special Self-Governing Province, Korea
3
Faculty of Biotechnology, Jeju National University, Jeju-si 690-756, Jeju Special Self-Governing Province, Korea
4
Department of Biochemistry, Government Arts College (Autonomous), Kumbakonam-612 001, Tamilnadu, India
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed.
Received: 7 February 2014 / Revised: 9 July 2014 / Accepted: 22 August 2014 / Published: 3 September 2014
(This article belongs to the Special Issue Pharmaceuticals and Nutraceuticals by Molecular Farming)
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Abstract

A full-length phytase gene (phy) of Aspergillus nidulans was amplified from the cDNA library by polymerase chain reaction (PCR), and it was introduced into a bacterial expression vector, pET-28a. The recombinant protein (rPhy-E, 56 kDa) was overexpressed in the insoluble fraction of Escherichia coli culture, purified by Ni-NTA resin under denaturing conditions and injected into rats as an immunogen. To express A. nidulans phytase in a plant, the full-length of phy was cloned into a plant expression binary vector, pPZP212. The resultant construct was tested for its transient expression by Agrobacterium-infiltration into Nicotiana benthamiana leaves. Compared with a control, the agro-infiltrated leaf tissues showed the presence of phy mRNA and its high expression level in N. benthamiana. The recombinant phytase (rPhy-P, 62 kDa) was strongly reacted with the polyclonal antibody against the nonglycosylated rPhy-E. The rPhy-P showed glycosylation, two pH optima (pH 4.5 and pH 5.5), an optimum temperature at 45~55 °C, thermostability and broad substrate specificities. After deglycosylation by peptide-N-glycosidase F (PNGase-F), the rPhy-P significantly lost the phytase activity and retained 1/9 of the original activity after 10 min of incubation at 45 °C. Therefore, the deglycosylation caused a significant reduction in enzyme thermostability. In animal experiments, oral administration of the rPhy-P at 1500 U/kg body weight/day for seven days caused a significant reduction of phosphorus excretion by 16% in rat feces. Besides, the rPhy-P did not result in any toxicological changes and clinical signs. View Full-Text
Keywords: Aspergillus nidulans; phy gene; Escherichia coli; polyclonal antibody; transient expression; Agrobacterium infiltration; oral administration Aspergillus nidulans; phy gene; Escherichia coli; polyclonal antibody; transient expression; Agrobacterium infiltration; oral administration
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Oh, T.-K.; Oh, S.; Kim, S.; Park, J.S.; Vinod, N.; Jang, K.M.; Kim, S.C.; Choi, C.W.; Ko, S.-M.; Jeong, D.K.; Udayakumar, R. Expression of Aspergillus nidulans phy Gene in Nicotiana benthamiana Produces Active Phytase with Broad Specificities. Int. J. Mol. Sci. 2014, 15, 15571-15591.

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