定向进化提高灰盖鬼伞过氧化物酶染织废水脱色效率

摘要

[Objective] The aim of our study is to improve Coprinus cinereus peroxidase (CIP) for textile dyes decolorization.[Methods] We synthesized CIPmt4,a CIP gene with Pichia pastori codon bias and 4 amino acid substitutions (I49S,V53A,M166F and M242I),using our gene synthesis and site-specific mutagenesis platform.Then,we carried out random mutagenesis using CIPmt4 as template,after three rounds of error-prone PCR and high-throughput screening by assaying enzymatic activity toward 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid ABTS),we obtained a mutant (CIPmt5) with improved enzymatic activity.Furthermore,we built the 3-dimensional model of CIPmt5 and wild type CIP using Swiss-model software,analyzed their thermostability using molecular dynamics simulation and investigated the decolorization ability of CIPmt5 and wild-type CIP for 7 textile dyes (Congo red,Amino black,Methyl orange,Methylene blue,Aniline blue,Bromophenol blue and Crystal violet) subsequently.[Results] Sequence analysis revealed that 5 amino acid substitutions (I49S,V53A,T121A,M166F and Y272F) were accumulated in CIPmt5.Compared to the wild-type enzyme,the specific activity of CIPmt5 toward ABTS was increased to 2.01-fold (24.44 U/mg),and its optimal temperature and pH were changed from 25 ℃ and 5.0 to 45 ℃ and 6.5 of the wild-type enzyme,respectively.The optimal decolorization pH of CIPmt5 shifted toward neutral to alkaline pH except for methylene blue.[Conelusion] The new variant obtained through directed evolution shows its potential in industrial application in textile dyes decolorization.%[目的]获得染织废水脱色能力增强的灰盖鬼伞过氧化物酶.[方法]使用基因合成及定点突变平台合成突变灰盖鬼伞过氧化物酶基因CIPmt4 (149S、V53A、M166F和M242I),并调整密码子至毕赤酵母偏好性.以CIPmt4为模板进行定向进化,经过三轮易错PCR和高通量筛选得到一个酶学性质显著改善的突变体(CIPmt5).通过3D建模和分子动力学模拟分析蛋白的结构及热稳定性,并进一步研究CIPmt5和野生型CIP对刚果红、氨基黑、甲基橙、次甲基蓝、苯胺蓝、结晶紫、溴酚蓝共7种染料的脱色能力.[结果]序列分析显示该突变体积累了149S、V53A、T121A、M166F和Y272F共5个氨基酸突变,与野生型灰盖鬼伞过氧化物酶相比,以ABTS为底物酶活性是野生型的2.01倍(24.44 U/mg),最适反应pH由5.0提高到6.5,最适反应温度由25℃提高到45℃.除次甲基蓝外对其它染料脱色的最适pH都往中、碱性方向偏移,脱色率普遍高于野生型.模型分析显示CIPmt5活性中心更开放,热稳定性增强.[结论]突变体酶CIPmt5能够更好地替代野生型灰盖鬼伞过氧化物酶应用于染织业染料脱色、化工废水和染织废水的生物修复.