大肠杆菌植酸酶appA基因的克隆、表达及性质分析

Cloning,Expression and Properties of Phytase appA Gene in Escherichia coli

通过维生素C-钼蓝法,从饲喂青饲料的猪的粪便中分离筛选出1株产较高活性appA植酸酶的大肠杆菌菌株;采用PCR方法从该菌株的基因组中克隆获得植酸酶appA基因,测序结果显示PCR产物全长1447 bp,该植酸酶基因编码区有1296个核苷酸,编码432个氨基酸。将该编码区克隆至pPIC9K载体,并转化入巴斯德毕赤酵母进行甲醇诱导表达,获得了约55 k Da的特征条带,表达96 h后其上清液的酶活力达到368 U/m L。经Ni亲和柱纯化后进行部分酶学性质分析,结果表明:重组appA植酸酶的最适反应温度为55℃,温度高于60℃后其酶活下降明显;其最适pH值为5.0。

Through adopting vitamin C-molybdenum blue method,we isolated and screened out an Escherichia coli strain which could produce high-activity appA phytase from the excrement of swine feeding on green fodder. The phytase appA gene was cloned from the genome of this strain by using PCR method. The sequencing results showed that the overall length of this PCR product was 1447 bp,and in the coding region of this phytase gene there were 1296 nucleotides encoding 432 amino acids. This coding region was cloned into p PIC9 K vector,and then the p PIC9 K-appA was transformed into Pichia pastoris to induce the expression of appA gene by methanol. A characteristic band of about 55 k Da was obtained,and the enzyme activity of the supernatant reached 368 U/m L after 96-h expression. The target protein was purified by Ni affinity column,and the analysis of partial enzymatic properties indicated that： the optimum reaction temperature for recombinant appA phytase was 55 ℃,and the enzyme activity decreased obviously when the temperature was higher than 60 ℃; the optimum p H-value for this enzyme was 5.0.