文献类型：期刊文献

中文题名：Accelerated directed evolution of dye-decolorizing peroxidase using a bacterial extracellular protein secretion system(BENNY)

作者：Abdulrahman H.A.Alessa[1];Kang Lan Tee[1];David Gonzalez-Perez[1];Hossam E.M.Omar Ali[1];Caroline A.Evans[1];Alex Trevaskis[1];Jian-He Xu[2];Tuck Seng Wong[1]

机构：[1]Department of Chemical & Biological Engineering and Advanced Biomanufacturing Centre,University of Sheffield,Sir Robert Hadfield Building,Mappin Street,Sheffield,S1 3JD,UK;[2]Laboratory of Biocatalysis and Bioprocessing,State Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,130 Meilong Road,Shanghai,200237,People’s Republic of China

年份：2019

卷号：6

期号：1

起止页码：383

中文期刊名：Bioresources and Bioprocessing

外文期刊名：生物资源与生物加工(英文)

基金：We thank the Department of Chemical and Biological Engineering,ChELSI,EPSRC(EP/E036252/1);the Open Project Funding of the State Key Laboratory of Bioreactor Engineering,The Leverhulme Trust Senior Research Fellowship(to TSW),University of Sheffield GCRF Fellowship(to KLT),BBSRC(BB/R020183/1);University of Tabuk,Singapore A*STAR and COST action(CM1303,Systems Biocatalysis)for financial support.

语种：英文

中文关键词：Directed evolution;Extracellular protein secretion;Dye-decolorizing peroxidase;Osmotically-inducible protein Y;Hydrogen peroxide tolerance

摘要：Background Dye-decolorizing peroxidases(DyPs)are haem-containing peroxidases that show great promises in industrial biocatalysis and lignocellulosic degradation.Through the use of Escherichia coli osmotically-inducible protein Y(OsmY)as a bacterial extracellular protein secretion system(BENNY),we successfully developed a streamlined directed evolution workflow to accelerate the protein engineering of DyP4 from Pleurotus ostreatus strain PC15.Result After 3 rounds of random mutagenesis with error-prone polymerase chain reaction(epPCR)and 1 round of saturation mutagenesis,we obtained 4D4 variant(I56V,K109R,N227S and N312S)that displays multiple desirable phenotypes,including higher protein yield and secretion,higher specific activity(2.7-fold improvement in k cat/K m)and higher H_(2)O_(2) tolerance(sevenfold improvement based on IC50).Conclusion To our best knowledge,this is the first report of applying OsmY to simplify the directed evolution workflow and to direct the extracellular secretion of a haem protein such as DyP4.