Efficient Expression of Escherichia coli AppA Phytase in Pichia pastoris
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Abstract:
In this study, the phytase gene, AppA, derived from Escherichia coli B48 was synthesized by whole gene, and cloned into PHKA vector for secretion expression in Pichia pastoris GS115. In order to further increase the expression of phytase in Pichia pastoris, this study adopted the optimization method based on the combination of signal peptide, promoter, gene dose and protein folding flux, which led to an ultimate increase of the phytase activity in Pichia pastoris from 329.41 U/mL to 1892.51 U/mL (5.75 times as high as that of the original strain). The basic enzymatic properties of phytase AppA were preliminarily determined, and the optimum temperature and pH were 60 ℃ and 4.5, respectively. After a 60-min treatment at 60 ℃, 40% of the phytase activity was retained, indicating poor thermal stability. After a 6-h treatment at pH 6.0~7.0, 87% of the enzyme activity was retained, indicating good stability in a weak acid environment. The effect of divalent metal ions on phytase activity was also studied. Fe2+ could activate phytase AppA, while ions such as Cu2+, Ni2+ and Mn2+ may inhibit the activity of phytase AppA through forming a complex with phytic acid. The high stability of the enzyme under acidic conditions is advantageous for its application in the field of food processing.