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.

Fungal Phytase Production in Different Hosts: A Brief Review

“…The genus Nicotianahas been studied as an expression system by several researchers Ullah, et al (1999), George et al (2005) and Oh et al (2014) [10,30,31]. They verified the expression of phytase enzymes from A. ficuum, A. niger and A. nidulans.…”

Characterization of the Catalytic Structure of Plant Phytase, Protein Tyrosine Phosphatase-Like Phytase, and Histidine Acid Phytases and Their Biotechnological Applications

“…Many enzymes with phytase activity can be obtained or expressed in plants, animals, and microorganisms [ 18 , 36 , 37 ]. They are classified into the following four groups according to their catalytic mechanisms: histidine acid phosphatases (HAPs), PAPs, Cys or β -helix phosphatases, and protein tyrosine phosphatase-like enzymes (PTP) [ 38 ].…”

Engineering of Yersinia Phytases to Improve Pepsin and Trypsin Resistance and Thermostability and Application Potential in the Food and Feed Industry

“…show a broad substrate specificity and high enzyme activity for AMP, ATP, ADP, tripolyphosphate, pyrophosphate, acetyl phosphate, p-nitrophenyl phosphate, and sodium phytate. 5,6 In contrast to phytase, phosphatases, e.g., alkaline phosphatases nonspecifically hydrolyze almost all phosphate monoesters in various molecules including nucleotides, proteins, alkaloids, etc. 7 The enzymes are classified into three families, 3-, 5-, or 6phytases, based on the position of the first removed phosphate in the inositol ring.…”

“…Other fungal phytases from Aspergillus sp. show a broad substrate specificity and high enzyme activity for AMP, ATP, ADP, tripolyphosphate, pyrophosphate, acetyl phosphate, p -nitrophenyl phosphate, and sodium phytate. , In contrast to phytase, phosphatases, e.g., alkaline phosphatases nonspecifically hydrolyze almost all phosphate monoesters in various molecules including nucleotides, proteins, alkaloids, etc …”