文献类型：期刊文献

中文题名：应用毕氏酵母高效表达耐高温植酸酶

英文题名：High Expression of a Heat-stable Phytase inPichia pastoris

作者：彭日荷[1];郭美锦[2];等

机构：[1]上海市农业科学院生物技术研究中心、上海市农业遗传育种重点实验室，上海201106;[2]华东理工大学生物工程学院，上海200237

年份：2002

卷号：34

期号：6

起止页码：725

中文期刊名：生物化学与生物物理学报：英文版

外文期刊名：Acta Biochimica et Biophysica Sinica

收录：CSTPCD;;Scopus;北大核心:【北大核心2000】；CSCD:【CSCD2011_2012】；PubMed;

基金：上海市科委重大项目 (No .993913002 );; 上海永业农科生物工程公司资助~~

语种：中文

中文关键词：毕氏酵母;高效表达;耐高温植酸酶;热稳定植酸酶;基因合成

外文关键词：heat-stable phytase; gene synthesis; high expression;Pichia pastoris

摘要：自然界很难获得大量的耐高温植酸酶，采取连续延伸PCR方法，按照毕氏酵母的偏爱密码，合成1.3kb烟熏曲霉耐高温植酸酶基因fphy。合成基因与野生型基因核苷酸序列同源性为74％，但编码的氨基酸序列一致，将耐高温植酸酶基因fphy插入毕氏酵母高效表达载体pPIC9中，与9分泌信号肽序列融合，通过同源重组将耐高温植酸基因整合到酵母染色体中，通过SDS－PAGE检测的和表达产物的酶活性筛选，得到重组转化子，证明植酸酶获得有效分泌和高效表达，经过5L小罐高密度发酵，蛋白质表达量为5．6g/L，每毫升发酵液中植酸酶的活力单位达130000u；表达产物高温性很强，90℃处理80min后仍有40％的活性。
It is difficult to obtain naturally occurring phytase having the required thermostability for application in animal feeding. The 1.3 kb thermal stable phytase gene (fphy) ofAspergillus fumigatus was synthesized by using a successive PCR method for the optimal expression inPichia pastoris. Though the nucleotides of syntheticfphyshare only 74% homology with the natural gene, the amino acid sequences coded by both genes were identical. After being cloned into the yeast expression vector (pPIC9) and inserted into the chromosome ofPichia pastoris by homologous recombination, phytase was expressed in the yeast and secreted from the cell. The strains for phytase over-expression were selected out by SDS-PAGE and enzyme analysis. After fermentation in 5 L fermention tank and induced by 0.5% methanol for 60 h, about 5.6 g purified phytase was obtained per liter culture fluid. The activity of phytase was 130 000 u per microlitre fluid. The thermostable phytase remained 40% active after exposure at 90 ℃ for 80 min.