Expression of Aspergillus nidulans phy gene in Nicotiana benthamiana produces active phytase with broad specificities.

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.

Comparison between ethanolic and aqueous extracts from Chinese juniper berries for hypoglycaemic and hypolipidemic effects in alloxan-induced diabetic rats.

AIM OF THE STUDY: Hypoglycaemic and hypolipidemic properties of the ethanolic and aqueous extracts, respectively, from Chinese juniper (Juniperus chinensis L.) berries were investigated in alloxan-induced diabetic rats. MATERIALS AND METHODS: After oral administration of each extract singly or repeatedly to alloxan-induced diabetic rats, the blood glucose, glutamate-pyruvate transferase (GPT), glutamate-oxaloacetate transaminase (GOT), total cholesterol (TC) and triglyceride (TG) levels were assayed. RESULTS: The blood glucose levels after a single oral administration of the ethanolic extract significantly reduced in a time-dependent manner, which is much faster and more than that of glibenclamide. The blood glucose levels of alloxan-induced diabetic rats treated with the ethanolic extract were reduced to 94, 81%, 66%, 45% and 40% at 1, 3, 5, 7 and 9h, respectively (p<0.05), while the aqueous extract had no effect at all. Repeated oral administration of the ethanolic extract also effectively reduced the GPT value to 58% of the diabetic rats, but slightly reduced the GOT value to 87% of the diabetic rats (p<0.05). On the other hand, the repeated oral administration of aqueous extract effectively reduced the GOT value to 43% of the diabetic rats, without affecting the GPT level. Effects of both extracts on the TC and TG levels were different. There was no significant difference in the TC and TG levels between diabetic control and diabetic groups when repeatedly administered orally with ethanolic extract. On the other hand, the aqueous extract brought down the TC value to 57% and the TG value to 37% of the diabetic control rats (p<0.05). CONCLUSIONS: The results suggested that the ethanolic extract of Chinese juniper berries possesses a potential hypoglycaemic effect while the aqueous extract has a potential hypolipidemic effect.