植酸酶基因在毕赤酵母中表达及表达酶性质研究

Expression of gene(phyA Ⅰ) encoding phytase in pichia pastoris and study of properties of phytase

PCR扩增无花果曲霉 As3 .3 2 4的植酸酶基因 phy A ,测序分析表明 ,phy A 全长1 5 1 5 bp,其中 1 1 1个核苷酸为内含子序列 ,共编码 467个氨基酸 ,N端的 1 9个氨基酸为信号肽 ,序列中存在 1 0个潜在的 N-糖基化位点 .构建 phy A 的酵母转化载体 p PIC9K/phy A ,电击法转化毕赤酵母 GS1 1 5 ,获得植酸酶基因重组酵母菌 GS1 1 5 /phy A .其发酵酶活性比 As3 .3 2 4提高了 3 3倍 .GS1 1 5 /phy A 植酸酶作用 p H有两个峰值 ,分别为 2 .5和4.5 ,最适作用温度为 60℃ .与 As3 .3 2 4相比 ,重组毕赤酵母 GS1 1 5 /phy A 植酸酶的热稳定性有显著提高 .

Gene phyAⅠ encoding phytase from Aspergillus ficuum As3.324 was amplified by PCR .Sequencing analysis shows that the gene phyA Ⅰis 1 515 bp in length and contains an intron of 111 nucleodides.The putative protein is composed of 467 amino acids and contains 10 potencial N\|glycosylation sites. There is a signal peptide from the 19 residues of the N\|terminus.The expression plasmid vector pPIC9K/ phyAⅡ for tranforming yeast was constructed.The pPIC9K/phyAⅡ was transformed into pichia pastoris GS115 by electroporation and the recombinant yeast GS115/ phyAⅡ was obtained. The phytase activity of the yeast recombinant GS115/ phyAⅡ is 33 times higher than that of A.ficuum As3.324 . The phytase of GS115/phyAⅡ has two activity peaks at pH 2.5 and 4.5, an optimum temperature of 60℃ and its thermostability is also improved significantly in comparison with that of the phytase produced by As3.324.