大肠杆菌植酸酶AppA在毕赤酵母中的高效表达

Efficient Expression of Escherichia coli AppA Phytase in Pichia pastoris

摘要:

本研究全基因合成大肠杆菌B48来源的植酸酶AppA基因，并将它克隆到PHKA载体上，在毕赤酵母GS115中进行分泌表达。为了进一步提高毕赤酵母中植酸酶的表达量，本研究结合信号肽、启动子、基因剂量和蛋白质折叠通量优化的方法，最终将毕赤酵母中植酸酶的活力从329.41 U/mL提高到了1892.51 U/mL，是初始菌株的5.75倍。初步测定了植酸酶AppA的基本酶学性质，其最适温度和pH分别为60 ℃和4.5；在60 ℃处理60 min后，植酸酶活力剩余40%，温度稳定性较差；在pH 6.0~7.0处理6 h后，酶活力剩余87%，该酶在弱酸环境中有较好的稳定性；同时研究了二价金属离子对植酸酶活力的影响，Fe2+能够激活植酸酶AppA的活力，而Cu2+、Ni2+、Mn2+等离子通过与植酸形成络合物而抑制植酸酶AppA的活力。该酶在酸性条件下的强稳定性有利于将其应用于食品加工领域。

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.