Expression of Aspergillus nidulans phy Gene in Nicotiana benthamiana Produces Active Phytase with Broad Specificities
AbstractA 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
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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.
Oh T-K, Oh S, Kim S, Park JS, Vinod N, Jang KM, Kim SC, Choi CW, Ko S-M, Jeong DK, Udayakumar R. Expression of Aspergillus nidulans phy Gene in Nicotiana benthamiana Produces Active Phytase with Broad Specificities. International Journal of Molecular Sciences. 2014; 15(9):15571-15591.Chicago/Turabian Style
Oh, Tae-Kyun; Oh, Sung; Kim, Seongdae; Park, Jae S.; Vinod, Nagarajan; Jang, Kyung M.; Kim, Sei C.; Choi, Chang W.; Ko, Suk-Min; Jeong, Dong K.; Udayakumar, Rajangam. 2014. "Expression of Aspergillus nidulans phy Gene in Nicotiana benthamiana Produces Active Phytase with Broad Specificities." Int. J. Mol. Sci. 15, no. 9: 15571-15591.