Polyethylene terephthalate(PET),one of the most widely used plastics in the world,causes serious environmental pollution.Recently,researchers have focused their efforts on enzymatic degradation of PET,which is an attr...Polyethylene terephthalate(PET),one of the most widely used plastics in the world,causes serious environmental pollution.Recently,researchers have focused their efforts on enzymatic degradation of PET,which is an attractive way of degrading and recycling PET.In this work,PET hydrolase Sb PETase from Schlegelella brevitalea sp.nov.was biochemically characterized,and rational design was performed based on its sequence similarity with the previ-ously reported Is PETase from Ideonella sakaiensis,resulting in a triple mutant with increased activity.Furthermore,using a sec-dependent signal peptide PeIB and colicin release protein Kil,we set up a high-efficiency secretion system of PETase in Escherichia coli BL21(DE3),enabling higher PETase secretion.Utilizing this secretion system,we established a high-throughput screening method named SecHTS(sec retion-based h igh-throughput s creening)and performed directed evolution of Is PETase and Sb PETase through DNA shuffling.Finally,we generated a mutant Is PETase S139T with increased activity from the mutant library.展开更多
In vitro characterization experiments revealed the formations of 3-(trans-2’-aminocyclopropyl)alanine((3-Acp)Ala)and 3-(trans-2’-nitrocyclopropyl)alanine((3-Ncp)Ala)are originated via two homologous proteins,BelK an...In vitro characterization experiments revealed the formations of 3-(trans-2’-aminocyclopropyl)alanine((3-Acp)Ala)and 3-(trans-2’-nitrocyclopropyl)alanine((3-Ncp)Ala)are originated via two homologous proteins,BelK and HrmI,which regioselectively catalyze the N𝜀-oxygenation of l-lysine.The two enzymes belong to the emerg-ing heme-oxygenase-like diiron oxidase and oxygenase(HDO)superfamily and the catalytic center of BelK is validated by homology modeling and site-directed mutations.Based on the in vitro characterization,the biosyn-thetic pathways of(3-Acp)Ala and(3-Ncp)Ala are proposed.展开更多
基金supported by the Qilu Youth Scholar Startup Funding of Shandong University(L.H.)National Natural Science Foundation of China(32170038)as well as the Sino-German mobility programme(M-0348).
文摘Polyethylene terephthalate(PET),one of the most widely used plastics in the world,causes serious environmental pollution.Recently,researchers have focused their efforts on enzymatic degradation of PET,which is an attractive way of degrading and recycling PET.In this work,PET hydrolase Sb PETase from Schlegelella brevitalea sp.nov.was biochemically characterized,and rational design was performed based on its sequence similarity with the previ-ously reported Is PETase from Ideonella sakaiensis,resulting in a triple mutant with increased activity.Furthermore,using a sec-dependent signal peptide PeIB and colicin release protein Kil,we set up a high-efficiency secretion system of PETase in Escherichia coli BL21(DE3),enabling higher PETase secretion.Utilizing this secretion system,we established a high-throughput screening method named SecHTS(sec retion-based h igh-throughput s creening)and performed directed evolution of Is PETase and Sb PETase through DNA shuffling.Finally,we generated a mutant Is PETase S139T with increased activity from the mutant library.
基金supported by the National Key R&D Program of China(2019YFA0905700)the National Natural Science Foundation of China(21907057,32070060)+3 种基金the Shan-dong Provincial Natural Science Foundation,China(ZR2019JQ11,ZR2019ZD18)the Natural Science Foundation of Jiangsu Province,China(BK20190201)the 111 project(B16030),the Youth Interdisci-plinary Innovative Research Group(2020QNQT009)the Future Plan for Young Scholars,and the Fundamental Research Funds(2019GN032)of Shandong University.
文摘In vitro characterization experiments revealed the formations of 3-(trans-2’-aminocyclopropyl)alanine((3-Acp)Ala)and 3-(trans-2’-nitrocyclopropyl)alanine((3-Ncp)Ala)are originated via two homologous proteins,BelK and HrmI,which regioselectively catalyze the N𝜀-oxygenation of l-lysine.The two enzymes belong to the emerg-ing heme-oxygenase-like diiron oxidase and oxygenase(HDO)superfamily and the catalytic center of BelK is validated by homology modeling and site-directed mutations.Based on the in vitro characterization,the biosyn-thetic pathways of(3-Acp)Ala and(3-Ncp)Ala are proposed.