一株产植酸酶青霉的分类鉴定及其植酸酶基因克隆和在毕赤酵母中的表达

Identification of a Penicillium isolate,gene cloning of phytase and its expression in Pichia pastoris

植酸酶是催化植酸盐水解成磷酸盐的一类酶的总称,添加到猪和家禽动物的饲料中可以提高植酸磷的利用率和促进对矿质元素的吸收利用。根据形态特征和ITS分子序列,作者对该实验室筛选得到的高产植酸酶菌株青霉RCEF4908进行了鉴定,确定该菌株在系统分类上属于草酸青霉Penicillium oxalicum Currie&Thom。利用特异性引物扩增出植酸酶成熟肽基因片段,蛋白质序列含有保守区域RHGXRXP和HD,属于组氨酸酸性磷酸酶家族。通过基因操作成功实现了植酸酶在Pichia pastoris GS115的表达。转化子P49在诱导培养144 h后酶活性可以达到约8 000U·mL-1。重组植酸酶在pH 2.5～7.0之间都有活性,在pH 5.5时酶活性最高;在90℃水浴处理10、20和30 min后酶活性仍分别保留53.8%、52.5%和45.2%,具有较高的耐热性,适宜于作为饲料添加剂,有进一步开发的价值。

Phytases are a group of enzymes capable of releasing phosphate from phytate, and have been ap- plied mainly to animal （swine and poultry） diets in order to improve phytate P digestibility and mineral bioavail- ability. RCEF4908, a strain with high phytase yield, was screened from a lot of phytase-producted fungal isolates. According to morphological characters and internal transcribed spacer sequences of rDNA, and it was identified as Penicillium oxalicurn Currie ＆ Thorn. The fragment of the mature phytase was cloned directly from DNA of P. oxalicum RCEF4908 with the special primers designed from phytase gene in GenBank No. AY071824. The deduced amino acid sequence of this phytase with the conserved motifs RHGXRXP and HD belongs to a typical histidine acid phosphatases. The phytase was overexpressed in the recombinant P49, and its specific activity could reach 8 000 U.mL-1 after induced 144h at optimum pH 5.5, and showed high activity with phytic acid sodium salt at a pH range of 2.5 to 7.0. This recombinant phytase studied here can retain 53.8%, 52.5% and 45.2% of its activ- ity after denaturation at 90℃ forl 0, 20 and 30 min, respectively. The broad pH optima and high thermostability of the phytase makes it a better candidate for feed-pelleting applications.