Abstract:[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 °C and 5.0 to 45 °C 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. [Conclusion] The new variant obtained through directed evolution shows its potential in industrial application in textile dyes decolorization.
