新型植酸酶基因YkAPPAS在毕赤酵母中的表达及其酶学特性分析  

Expression of a novel phytase gene YkAPPAS in Pichia pastoris and its enzymological characterization

作　　者：朱恒文 田永生[2] 李大伟 于成野 陈磊[1] 彭日荷[2] 姚泉洪[2] 

机构地区：[1]上海海洋大学食品学院,上海201306 [2]上海市农业科学院生物技术研究所,上海201106 [3]未名生物医药有限公司,福建361009

出　　处：《上海农业学报》2018年第1期1-7,共7页Acta Agriculturae Shanghai

基　　金：上海市科委农业成果转化项目(143919N0300);上海市种业发展项目[沪农科种字(2016)第1-2号]

摘　　要：根据密码子的偏好性,采用PTDS方法化学合成了一个来源于Yersinia kristesenii的新型植酸酶基因(YkAPPAS),并且作为外分泌蛋白在毕赤酵母GS115中成功表达。重组植酸酶YkAPPAS的蛋白分泌量可达到106.73μg/mL。对重组植酸酶的酶学性质研究表明:该酶的比活力为2 330 U/mg;最适pH为4.5和6.0;最适温度为45℃;100℃处理5 min后其剩余活性为47.55%;90℃处理5 min后其剩余活性为56.75%;在pH 3—12时的活性均在30%以上。Li^+、EDTA对酶活有一定的激活作用;Zn^(2+)、Cu^(2+)对酶活有一定的抑制作用。重组植酸酶还具有良好的抗胃蛋白酶和胰蛋白酶水解的特性。A novel phytase gene YkAPPAS from Yersinia kristensenii was chemically synthesized by the PCRbased two-step DNA synthesis according to yeast codon preference and successfully expressed as secreted protein in Pichia pastoris GS115. The recombinant YkAPPAS protein secretion yield could reach 106. 73 μg/mL. The research of its enzymological property showed that its specific activity was 2 330 U/mg; Its optimal pH was at 4. 5 and 6. 0 and the optimal temperature was 45 ℃; Its residual activity was 47. 55% after 5-min treatment at100 ℃ and 56.75% after 5-min treatment at 90 ℃; Between pH 3—12 its activity was above 30%. To some extent its enzyme activity could be activated by Li~＋ and EDTA but inhibited by Zn^2＋ and Cu^2＋. It had a good ability to resist hydrolyzation by pepsin and trypsin.

关 键 词：植酸酶 毕赤酵母 YERSINIA kristensenii 酶学性质 

分 类 号：S816.7[农业科学—饲料科学]