蜂房哈夫尼菌植酸酶基因appA的克隆、表达及酶学性质研究

Gene Cloning, Expression and Characterization of a Novel Phytase from Hafnia alvei

摘要:

从蜂房哈夫尼菌（Hafnia alvei）中克隆获得一个植酸酶编码基因appA, 该基因全长1335bp，编码444个氨基酸，其中前33个氨基酸为信号肽，成熟蛋白的理论分子量为45.2kD。将基因appA 克隆到大肠杆菌E. coli表达载体pET-22b(+)，并在大肠杆菌中表达, 表达产物具有植酸酶活性。对表达的酶蛋白进行纯化，并初步研究了该酶的酶学性质，结果表明：酶的作用最适pH值为4.5；在pH 2.0～10.0范围内, 酶活性保留80％以上；酶的作用最适温度为60℃；酶的比活性为356.7U/mg，酶动力学分析表明其K,/i>_m为0.49mmol/L，V_max为238U/mg；该酶对胰蛋白酶和胃蛋白酶有一定的抗性。该研究为哈夫尼菌属来源植酸酶的首次报道。

Abstract:

A gene appA encoding a novel phytase was firstly cloned from Hafnia alvei by PCR and sequenced. The gene was consisted of 1335 bp, encoding 444 amino acids. The calculated molecular weight of the mature APPA was about 45.2 kD. The gene appA was expressed in E. coli BL21(DE3). Recombinant APPA was purified and its enzymatic properties were determined. The optimum pH for the enzyme was 4.5 and the optimum temperature was 60℃.The pH stability of r-APPA is good, the relative phytase activity was above 80% after treated in buffers of pH2.0～10.0. The specific activity of r-APPA is 356.7 U/mg, and the K_m value was 0.49 mmol/L and V_max of 238 U/mg. The enzyme showed resistance to pepsin and trypsin treatment.