双功能域β-折叠桶碱性植酸酶蛋白序列分析与酶学特性

Sequencing and characterization of dual-domain β-propeller alkaline phytase

摘要:

[目的]表达、纯化来源于Bacillus sp. HJB17的双功能域β-折叠桶碱性植酸酶（phyHT），并对该酶进行蛋白序列以及酶学特征分析，为该酶的广泛应用奠定基础。[方法]应用生物信息学技术，对phyHT蛋白序列进行了生物信息学分析。表达、变复性、分离纯化得到具有酶活性的可溶性phyHT蛋白溶液，通过硫酸亚铁-钼蓝法对phyHT的酶学特性进行了研究。[结果]序列分析结果显示，phyHT由633个氨基酸组成，含有2个植酸酶结构域，属于典型的BPP植酸酶，相对分子量（M_W）为69321.68 Da，理论等电点为5.37，为亲水性蛋白。二级结构中，phyHT中α-螺旋（helix）、β-片层（sheet）占主要的比例，高级结构以β片层形成的桶状结构为主。酶学性质研究表明，该酶的最适反应温度为35，在45以下比较稳定。最适的pH为8.0，在pH为6.0-12.0条件之间比较稳定。低浓度的Ca²⁺以及Mg²⁺对酶活性有促进作用，Fe²⁺、Mn²⁺、Zn²⁺、Cu²⁺、Ni²⁺等金属离子对酶活性有抑制作用。[结论]本研究成功对phyHT进行了蛋白序列分析以及酶学性质研究，phyHT属于碱性植酸酶，酶活性高、稳定性好，在理论研究和工业生产方面均有很大应用价值。

关键词:β-折叠桶碱性植酸酶;生物信息学分析;酶学特性

Abstract:

[Objective] The aim of this study was to purify and characterize a dual-domain β-propeller alkaline phytase (phyHT) from Bacillus sp. HJB17 and to analyze the sequence features for application of the enzyme.[Methods] The amino acid sequence of phyHT was analyzed by bioinformatics methods. PhyHT protein was expressed, refolded, purified and the enzymatic properties were determined by ferrous sulfate and molybdenum blue method.[Results] Sequence analysis suggested that phyHT consisted of 633 amino acids and contained 2 phytase domain and belonged to typical BPP phytase. PhyHT was a hydrophilic protein and the molecular weight and theoretical isoelectric point were 69321.68 Da and 5.37, respectively. In the secondary structure, phyHT mainly consists of α-helix and β-sheet. Its three-dimensional structure mainly consists of β-sheet propeller. The optimum temperature of phyHT was 37℃, and stable at 45℃. PhyHT has an optimum pH of 8.0, and stable between pH 6.0 and 12.0. Low concentration of Ca²⁺ and Mg²⁺ promoted the enzymatic activity, whereas phytase activity was strongly inhibited by Fe²⁺, Mn²⁺, Zn²⁺, Cu²⁺ and Ni²⁺.[Conclusion] phyHT has important applications in theoretical research and in industry.

Key words:β-propeller alkaline phytases;bioinformatics analysis;enzyme properties