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

International Journal of Molecular Sciences

Tae-Kyun Oh¹  Sung Oh¹  Seongdae Kim¹  Jae Sung Park¹  Nagarajan Vinod¹  Kyung Min Jang¹  Sei Chang Kim¹  Chang Won Choi¹  Suk-Min Ko²  Dong Kee Jeong³ 

[1]Department of Biology & Medicinal Science, Pai Chai University, Daejeon 302-735, Korea

[2] E-Mails:

[3]Research Institute for Subtropical Agriculture and Animal Biotechnology, Jeju National University, Jeju-si 690-756, Jeju Special Self-Governing Province, Korea

[4] E-Mail:

[5]Faculty of Biotechnology, Jeju National University, Jeju-si 690-756, Jeju Special Self-Governing Province, Korea

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关键词: Aspergillus nidulans;    phy gene;    Escherichia coli;    polyclonal antibody;    transient expression;    Agrobacterium infiltration;    oral administration;   

DOI  :  10.3390/ijms150915571

来源: mdpi

【 摘 要 】

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.

【 授权许可】

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© 2014 by the authors; licensee MDPI, Basel, Switzerland.