Improved degradation of azo dyes by lignin peroxidase following mutagenesis at two sites near the catalytic pocket and the application of peroxidase-coated yeast cell walls  

作　　者：Karla IlićĐurđić Raluca Ostafe Olivera Prodanović AleksandraĐurđevićĐelmaš Nikolina Popović Rainer Fischer Stefan Schillberg Radivoje Prodanović 

机构地区：[1]University of Belgrade-Faculty of Chemistry,Studentski trg 12-16,11000 Belgrade,Serbia [2]Molecular Evolution Protein Engineering and Production facility(MEPEP),Purdue University,West Lafayette,IN 47907,USA [3]Institute of Molecular Biotechnology,RWTH Aachen University,52074 Aachen,Germany [4]Institute for Multidisciplinary Research,Kneza Višeslava 1,11030 Belgrade,Serbia [5]Departments of Biological Sciences and Chemistry,Purdue University,West Lafayette,IN 47907,USA [6]Fraunhofer Institute for Molecular Biology and Applied Ecology IME,52074 Aachen,Germany

出　　处：《Frontiers of Environmental Science & Engineering》2021年第2期33-42,共10页环境科学与工程前沿（英文）

基　　金：supported by funds from the Ministry of Education,Science and Technological Development of the Republic of Serbia via project numbers ON172049,ON173017 and III46010.

摘　　要：The enzymatic degradation of azo dyes is a promising alternative to ineffective chemical and physical remediation methods.Lignin peroxidase(LiP)from Phanerochaete chrysosporium is a hemecontaining lignin-degrading oxidoreductase that catalyzes the peroxide-dependent oxidation of diverse molecules,including industrial dyes.This enzyme is therefore ideal as a starting point for protein engineering.Accordingly,we subjected two positions(165 and 264)in the environment of the catalytic Trp171 residue to saturation mutagenesis,and the resulting library of 104 independent clones was expressed on the surface of yeast cells.This yeast display library was used for the selection of variants with the ability to break down structurally-distinct azo dyes more efficiently.We identified mutants with up to 10-fold greater affinity than wild-type LiP for three diverse azo dyes(Evans blue,amido black 10B and Guinea green)and up to 13-fold higher catalytic activity.Additionally,cell wall fragments displaying mutant LiP enzymes were prepared by toluene-induced cell lysis,achieving significant increases in both enzyme activity and stability compared to a whole-cell biocatalyst.LiPcoated cell wall fragments retained their initial dye degradation activity after 10 reaction cycles each lasting 8 h.The best-performing mutants removed up to 2.5-fold more of each dye than the wild-type LiP in multiple reaction cycles.

关 键 词：BIOREMEDIATION Enzyme immobilization Protein engineering Yeast surface display 

分 类 号：O64[理学—物理化学]