现代食品科技2017，Vol.33Issue(5)：77-84,108,9.DOI:10.13982/j.mfst.1673-9078.2017.5.013
小麦蛋白质二硫键异构酶的毕赤酵母表达及重组酶性质研究
Expression of Wheat Protein Disulfide Isomerase in Pichia pastoris and the Properties of the Recombinant Enzyme
摘要
Abstract
Wheat protein disulfide isomerase (wPDI) was expressed in Pichia pastoris to develop a novel and healthy enzyme as a flour improver.The recombinant plasmid,pMD19-T-wpdi,was used as a template and subcloned into a P.pastoris expression vector,pPIC9K,and then expressed in P.pastoris GS115,which was used as a eukaryotic host.After the expressed products were purified by ammonium sulfate precipitation and anion exchange chromatography,the enzymatic properties of the recombinant wPDIs from P pastoris and E.coli were compared,and their effects on the farinograph characteristics were investigated using a farinograph.The results showed that the cloned wpdi gene contained 1347 bp,encoded 449 amino acids,and had a molecular weight of about 50.2 ku.Western blot analysis showed that the recombinant wPDI was expressed in the yeast expression system.The enzymatic characteristics of the wPDI purified with anion exchange chromatography were analyzed.The results revealed that the recombinant wPDI expressed in P.pastoris showed both oxidoreductase activity from the disulfide bonds,as well as chaperone activity.The wPDI expressed in P.pastoris showed higher reductase activity than that expressed in E.coli;however,the oxidase and chaperone activity of the wPDI from P pastoris were lower than those of the wPDI from E.coli.The results of the farinograph assay showed that the recombinant wPDI expressed in P pastoris was better able to weaken the processing quality of flour than that expressed in E.coli.These results provided a basis for in-depth studies on wPDI and its application in flour products.关键词
小麦蛋白质二硫键异构酶/毕赤酵母/克隆表达/酶学性质/粉质特性Key words
wheat protein disulfide isomerase/Pichia pastoris/cloning and expression/enzymatic properties/farinograph properties引用本文复制引用
曹佩,卫娜,刘光,王敬敬,徐勇,胡松青..小麦蛋白质二硫键异构酶的毕赤酵母表达及重组酶性质研究[J].现代食品科技,2017,33(5):77-84,108,9.基金项目
国家自然科学基金项目(31471691) （31471691）
广州市科技计划项目(201604020032) （201604020032）
高等学校博士学科点专项科研基金项目(20130172110018) （20130172110018）
广东省科技计划项目(2014A010107002) （2014A010107002）
佛山市科技计划项目(2015AG10011) （2015AG10011）
