Fungal phyA gene expressed in potato leaves produces active and stable phytase
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- 作者：Ullah ; Abul H.J. ; Sethumadhavan ; Kandan ; Mullaney ; Edward J. ; Ziegelhoffer ; Thomas ; Austin-Phillips ; Sandra
- 刊名：Biochemical and Biophysical Research Communications
- 出版年：2003
- 出版时间：June 27, 2003
- 年：2003
- 卷：306
- 期：2
- 页码：603-609
- 全文大小：202 K
文摘
Fungal phyA gene from Aspergillus ficuum (niger) was cloned and expressed in potato leaves. The recombinant enzyme was stable and catalytically active. The expressed protein in the leaves of the dicotyledonous plant retained most physical and catalytic properties of the benchmark A. ficuum phytase. The expressed enzyme was, however, 15 % less glycosylated than the native phytase. The usual bi-hump pH optima profile, which is characteristic of the fungal phytase, was altered; however, the pH optimum at 5.0 was unchanged for phytate and at 4.0 for synthetic substrate p-nitrophenyl phosphate. The temperature was, however, unchanged. The expressed phytase was found to be as sensitive as the native enzyme to the inhibitory action of pseudo substrate, myo-inositol hexasulfate, while losing about 90 % of the activity at 20μM inhibitor concentration. Similar to the benchmark phytase, the expressed phytase in leaves was completely inactivated by Arg modifier phenylglyoxal at 60nM. In addition, the expressed phytase in the leaves was inhibited by antibody raised against a 20-mer internal peptide, which is present on the surface of the molecule as shown by the X-ray deduced 3D structure of fungal phytase. Taken together, the biochemical evidences indicate that fungal phytase when cloned and expressed in potato leaves produces a stable and active biocatalyst. ‘Biofarming,’ therefore, is an alternative way to produce functional hydrolytic enzymes as exemplified by the expression of A. ficuum (niger) phyA gene in potato leaf.