来源于Penicillium sp.C1的水产用中性植酸酶基因在毕赤酵母中的表达及性质研究

通过简并引物和TAIL-PCR技术从青霉Penicillium sp. C1菌株的基因组DNA中克隆得到一个新的植酸酶基因,命名为phyC1。其结构基因全长1 477 bp,5’端包含58 bp的内含子序列,该基因编码472个氨基酸和一个终止密码子,前23个氨基酸为信号肽。将PhyC1的成熟蛋白的编码基因在毕赤酵母菌GS115中高效分泌表达,重组蛋白经硫酸铵沉淀和阴离子交换柱和分子筛纯化后达到电泳纯。对其酶学性质分析表明:重组植酸酶PhyC1最适pH为6.5,在pH5.0-10.0的条件下具有较好的稳定性;重组植酸酶PhyC1最适温度为50℃。与现有商品植酸酶相比,PhyC1具有两个显著的特性,如低温条件下具有高活性,尤其是在低于20℃时;在中性pH 7.0条件下仍具有90%以上的相对酶活性,因此,植酸酶PhyC1的优良特性为其在水产行业的应用奠定基础。

Expression and Characterization of Aquatic Neutral Phytase Gene from Penicillium sp. C1 in Pichia pastoris

A new phytase gene was cloned from the genome DNA of Penicillium sp. C1 strain by degenerate primers and TAIL-PCR,and named as phyC1. The whole length of structural gene was 1 477 bp,including a 58 bp intron at the 5’ terminus. The gene encoded 472 amino acids and a termination codon,and the first 23 amino acids were signal peptides. The gene encoding the mature protein of phyC1 was efficiently expressed in Pichia pastoris GS115,and then the recombinant enzyme PhyC1 was purified to electrophoretic purity by ammonium sulfate precipitation,anion exchange column and Sephedex chromatography. The enzymatic properties showed that the optimal pH of recombinant PhyC1 was 6.5,and high stability at pH 5.0-10.0. The optimal temperature of recombinant PhyC1 was 50℃. Compared with the commercial phytases,PhyC1 showed two significant properties,such as high activity under low temperature,especially below 20℃,and over 90% relative enzymatic activity remained under neutral pH 7.0. Therefore,PhyC1 has potential application prospect in feed industry,especially in aquatic industry.