江西农业学报2017，Vol.29Issue(11)：1-6,16,7.DOI:10.19386/j.cnki.jxnyxb.2017.11.01
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
摘要
Abstract
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 pPIC9K vector, and then the pPIC9K-appA was transformed into Pichia pastoris to induce the ex-pression of appA gene by methanol.A characteristic band of about 55 kDa was obtained, and the enzyme activity of the superna-tant reached 368 U/mL after 96-h expression.The target protein was purified by Ni affinity column , and the analysis of partial en-zymatic properties indicated that:the optimum reaction temperature for recombinant appA phytase was 55℃, and the enzyme ac-tivity decreased obviously when the temperature was higher than 60 ℃;the optimum pH-value for this enzyme was 5.0.关键词
植酸酶/appA基因/大肠杆菌/克隆/表达/毕赤酵母Key words
Phytase/appA gene/Escherichia clo i/Cloning/Expression/Pichia pastoris分类
生物科学引用本文复制引用
冉瑞法,刘淑娟,肖圣燕,冯蔚,李镇刚..大肠杆菌植酸酶appA基因的克隆、表达及性质分析[J].江西农业学报,2017,29(11):1-6,16,7.基金项目
国家自然科学基金项目( 31360190). （ 31360190）
