title: A semi-rational approach to obtain an ionic liquid tolerant bacterial laccase through π-type interactions.
abstract: Laccases are particularly promising enzymes for biotechnology and bioremediation purposes. They are among the most effective enzymes capable of catalyzing the degradation of phenolic compounds with poor water solubility. The technological utility of lacasses can be enhanced greatly by their use in ionic liquids rather than in conventional organic solvents or in their natural aqueous reaction media. In the current study, a laccase from Bacillus HR03 has been engineered through a semi rational method. By screening a library of 450 clones, Glu188Tyr and Glu188Phe showed a distinct improvement in thermal stability and ionic liquid tolerance. In comparison with the wild type, selected mutants exhibited higher kcat/Km against ABTS in the imidazolium based ionic liquids, (1-ethyl-3-methyl imidazolium chloride [EMIm][Cl], butyl-3-methyl imidazolium chloride [BMIm][Cl] and hexyl-3-methyl imidazolium chloride [HMIm][Cl]). Glu188Tyr had a catalytic efficiency, two times greater when compared to the wild type in [HMIm][Cl]. Far-UV circular dichroism (CD) exhibited no significant changes in the secondary structure of the mutants and wild type. Glu188Tyr revealed a more compact structure using Near-UV CD and fluorescence spectroscopy that could account for its high thermal stability. According to bioinformatic analysis, π-π and anion-π interactions played the dominant role in stabilizing both variants.
authors.affiliation: Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran.
authors.forename: Bahareh
authors.initials: B
authors.lastname: Dabirmanesh
authors.affiliation: Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran. Electronic address: khajeh@modares.ac.ir.
authors.forename: Khosro
authors.initials: K
authors.lastname: Khajeh
authors.affiliation: Department of Genetics and Molecular Biology, Medical School, Iran University of Medical Sciences, Tehran, Iran.
authors.forename: Farideh
authors.initials: F
authors.lastname: Ghazi
authors.affiliation: Department of Biophysics, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran.
authors.forename: Bijan
authors.initials: B
authors.lastname: Ranjbar
authors.affiliation: Department of Organic Colorants, Institute for Color Science and Technology, Tehran, Iran.
authors.forename: Seyed-Masoud
authors.initials: SM
authors.lastname: Etezad
mesh_terms: D001407:Bacillus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D001709:Biotechnology; D002384:Catalysis; D002942:Circular Dichroism; D004795:Enzyme Stability / Q000187:drug effects; D052578:Ionic Liquids / Q000737:chemistry* / Q000633:toxicity; D042845:Laccase / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D012995:Solubility; D012997:Solvents / Q000737:chemistry; D013696:Temperature; D014867:Water
chemical_list: D052578:Ionic Liquids; D012997:Solvents; D014867:Water; D042845:Laccase
keywords: Ionic liquids; Laccase; Semi-rational method