Loops constructing the substrate-binding site controlled the catalytic efficiency of Thermus thermophilus SG0.5JP17-16 laccase

Laccase (EC1.10.3.2) belongs to the family of multicopper oxidases. It oxidazes various substrates and uses the dioxygen as an electron acceptor, the only by-product is the water, thus, laccase was considered as green catalysts in the biotechnology field. In this work, the function of amino acid residues D357 and K430 in the loops 5 and 7 of Thermus thermophilus SG0.5JP17-16 laccase (lacTT) was studied. The residues D357 and K430 were mutated into methionine by site-directed mutagenesis. Kinetic assays revealed that the catalytic efficiency of D357M and K430M mutants was 1.9, 1.8 times higher than that of the wild type enzyme, respectively. Double mutant D357MK428M was also examined, the catalytic efficiency of D357MK428M decreased by 24% as compared with that of the wild type lacTT. We also examined the decolorization ability of D357M, K430M and D357MK428M for four industrial dyes, and found that D357M, K430M and D357MK428M mutants took less time than lacTT when the same degree of decolorization was achieved for three azo dyes, Congo Red, Reactive Black WNN (RBWNN), Reactive Black B (RBB) within 3 h. For the anthraquinone dye Remazol Brilliant Blue R, the decolorization ability of D357M, K430M was improved significantly. Within 24 h, as compared with that of the wild type enzyme, the decolorization efficiency of D357M and K430M mutants was enhanced by 21.3% and 21.6%, respectively, while the decolorization efficiency of D357MK428M mutant decreased by 15.2%. (c) 2022 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

Automated summary

“”In this study, the function of two amino acid residues, D357 and K430, in Thermus thermophilus SG0.5JP17-16 laccase (lacTT) were investigated. Mutations of these residues led to changes in the catalytic efficiency and decolorization ability of the enzyme, providing insights for understanding and improving the functionality of laccase.