title: Expression of Aspergillus nidulans phy gene in Nicotiana benthamiana produces active phytase with broad specificities.
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.
authors.affiliation: Department of Biology & Medicinal Science, Pai Chai University, Daejeon 302-735, Korea. ohtk0@pcu.ac.kr.
authors.forename: Tae-Kyun
authors.initials: TK
authors.lastname: Oh
authors.affiliation: Department of Biology & Medicinal Science, Pai Chai University, Daejeon 302-735, Korea. 5star@pcu.ac.kr.
authors.forename: Sung
authors.initials: S
authors.lastname: Oh
authors.affiliation: Department of Biology & Medicinal Science, Pai Chai University, Daejeon 302-735, Korea. kimsc@pcu.ac.kr.
authors.forename: Seongdae
authors.initials: S
authors.lastname: Kim
authors.affiliation: Department of Biology & Medicinal Science, Pai Chai University, Daejeon 302-735, Korea. parkjs@kgicenter.com.
authors.forename: Jae Sung
authors.initials: JS
authors.lastname: Park
authors.affiliation: Department of Biology & Medicinal Science, Pai Chai University, Daejeon 302-735, Korea. biovinz@yahoo.com.
authors.forename: Nagarajan
authors.initials: N
authors.lastname: Vinod
authors.affiliation: Department of Biology & Medicinal Science, Pai Chai University, Daejeon 302-735, Korea. cardgame@hanafos.com.
authors.forename: Kyung Min
authors.initials: KM
authors.lastname: Jang
authors.affiliation: Department of Biology & Medicinal Science, Pai Chai University, Daejeon 302-735, Korea. kimsc@pcu.ac.kr.
authors.forename: Sei Chang
authors.initials: SC
authors.lastname: Kim
authors.affiliation: Department of Biology & Medicinal Science, Pai Chai University, Daejeon 302-735, Korea. choicw@pcu.ac.kr.
authors.forename: Chang Won
authors.initials: CW
authors.lastname: Choi
authors.affiliation: Research Institute for Subtropical Agriculture and Animal Biotechnology, Jeju National University, Jeju-si 690-756, Jeju Special Self-Governing Province, Korea. heyna@jejunu.ac.kr.
authors.forename: Suk-Min
authors.initials: SM
authors.lastname: Ko
authors.affiliation: Faculty of Biotechnology, Jeju National University, Jeju-si 690-756, Jeju Special Self-Governing Province, Korea. dkjeong@jejunu.ac.kr.
authors.forename: Dong Kee
authors.initials: DK
authors.lastname: Jeong
authors.affiliation: Department of Biochemistry, Government Arts College (Autonomous), Kumbakonam-612 001, Tamilnadu, India. udayabiochem@yahoo.co.in.
authors.forename: Rajangam
authors.initials: R
authors.lastname: Udayakumar
mesh_terms: D010832:6-Phytase / Q000235:genetics / Q000378:metabolism* / Q000493:pharmacokinetics; D000818:Animals; D001233:Aspergillus nidulans / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism* / Q000493:pharmacokinetics; D006031:Glycosylation; D066237:Intestinal Elimination; D008297:Male; D010758:Phosphorus / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000493:pharmacokinetics; D013379:Substrate Specificity; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D010758:Phosphorus; D010832:6-Phytase