ISOLATION AND IN SILICO STUDIES OF A LACCASE GENE FROM Trametes hirsuta EDN 082: STRUCTURAL AND FUNCTIONAL ANALYSIS FOR BIOREMEDIATION OF SYTHETIC DYES

Abstract

Trametes hirsuta produces multiple laccases capable of decolorizing synthetic dyes. In this study, a thermostable, high-redox-potential extracellular blue laccase gene named Thlacc1 was successfully isolated from the transcriptome of T. hirsuta EDN 082. The Thlacc1 gene was isolated using the cDNA library construct, followed by PCR using degenerate and specific primers, respectively. The Thlacc1 gene (1575 bp) encodes a 524-amino acid protein with 98.47% similarity to laccase 3 (AOX15704.1), classifying it within the minor laccase group F. Primary structure analysis on ExPASy ProtParam server predicted a molecular weight of 56.54 kDa, an isoelectric point of 4.88, a stability index of 29.13, an aliphatic index of 97.33, and a GRAVY score of -0.130. Secondary structure analysis highlighted β-strands and helices, while tertiary structure modeling on the SwissModel server presented Model 03 as the most accurate model. The enzyme contains three multicopper oxidase motifs, a signal peptide, four N-glycosylation and nine O-glycosylation sites, four copper-binding domains (L1-L4), two disulfide bonds, and a catalytic pocket (221 ų).  Molecular docking at the T1 copper site using the SwissDock server revealed potential interactions between Model 03 and synthetic dyes. DB71 and RB5 showed the highest (-8.44 kcal/mol) and lowest (-5.825 kcal/mol) binding affinities, respectively, but neither was decolorized experimentally. In contrast, recombinant Thlacc1 laccase efficiently decolorized AB113 and AB129, indicating that decolorization efficiency depends on both binding affinity and key-interacting residues. This study provides structural insight and experimental validation for the application of Thlacc1 laccase in the decolorization of synthetic dyes.