title: Enhancement of a bacterial laccase thermostability through directed mutagenesis of a surface loop.
abstract: Laccases (benzenediol oxygen oxidoreductases, EC 1.10.3.2) are used in many biotechnological processes, including removal of polyphenols in beverages, decolorizing and detoxifying effluents, drug analysis and bioremediation. In the present work, we have tried to increase thermal stability of laccase from Bacillus HR03 using site directed point mutations. Glu188 was substituted with 2 positive (Lys and Arg) and one hydrophobic (Ala) residues. All mutations showed improved thermal stability. Thermal activation of laccase was also increased after introducing the mutations. Remarkably, the Glu188Lys variant showed 3-fold higher thermal activation and higher T(50) (5 °C) with respect to the native enzyme. Furthermore steady-state k(cat) and K(m) values were influenced despite the distance between the mutated position and the catalytic site. In Glu188Arg mutation, the k(cat) was improved 3-fold and K(m) reduced by 25%. Interestingly, all three variants showed higher stability against urea as a chemical denaturant. Structural analyses of the native and mutated variants were carried out using fluorescence and far-UV circular dichroism.
authors: Department of Biochemistry and Biophysics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
authors: Nasrin
authors: N
authors: Mollania
authors: Khosro
authors: K
authors: Khajeh
authors: Bijan
authors: B
authors: Ranjbar
authors: Saman
authors: S
authors: Hosseinkhani
mesh_terms: D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D004789:Enzyme Activation; D004795:Enzyme Stability; D007700:Kinetics; D042845:Laccase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D014508:Urea; D042845:Laccase