Semi-rational engineering of microbial laccase Lac15 for enhanced activity

Abstract:

[Objective] Laccases can oxidize a wide range of compounds and have great application value in diverse industries.Lac15,a microbial laccase with considerable application potential,can be modified by protein engineering to expand the application range.[Methods] By structure-based analysis,we chose some residues supposed to contribute directly or indirectly to electron/proton transfer or substrate interaction for site-directed mutagenesis,and measured the activities of the mutants for various substrates and the enzymatic properties.[Results] Some of the mutants showed significantly enhanced activity towards particular substrates.To be specific,the catalysis efficiency of mutant D216N toward 2,2'-azino-bis (3-ethylbenzthiazoline-6-suphonic acid)(ABTS) was 1.0 fold higher,and that of mutants R178V and K433T toward dopamine was 1.2 and 11.1 folds,respectively,higher than that of Lac15.Moreover,the mutants maintained the advantages of Lac15 in application,such as the neutral optimum pH and salt activation.[Conclusion] By structure-based semi-rational engineering,the specific activities of Lac15 toward some substrates can be improved.A conserved Asp in laccases,corresponding to D216 in Lac15,can be a common engineering target for enhancing the activity of the enzymes with ABTS as substrate or mediator.