title: Thermostabilization of firefly luciferase by in vivo directed evolution.
abstract: Firefly luciferase is widely used in a number of areas of biotechnology and molecular biology. However, rapid inactivation of wild-type (WT) luciferases at elevated temperatures often hampers their application. A simple non-lethal in vivo screening scheme was used to identify thermostable mutants of luciferase in Escherichia coli colonies. This scheme allowed carrying out each cycle of mutagenesis in a rapid and efficient manner. Four rounds of directed evolution were conducted on a part of the gene coding for amino acid residues 130-390 of Luciola mingrelica luciferase. The resultant mutant designated 4TS had a half-life of 10 h at 42°C, which is 65-fold higher compared with the WT luciferase. Moreover, the mutant 4TS showed a 1.9-fold increase in specific activity, 5.7-fold reduction of K(m) for ATP and a higher-temperature optimum compared with the WT enzyme. 4TS contains eight mutations, four of which are suggested to be mainly responsible for the enhancement of thermostability: R211L, A217V, E356K and S364C. Thus, directed evolution with non-lethal colony screening for in vivo bioluminescence activity proved to be an effective and efficient approach for increasing thermal stability of luciferase while retaining high catalytic activity.
authors: Division of Chemical Enzymology, Department of Chemistry, Lomonosov Moscow State University, Moscow 119991, Russia. mkoksharov@gmail.com
authors: Mikhail I
authors: MI
authors: Koksharov
authors: Natalia N
authors: NN
authors: Ugarova
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D055162:Biocatalysis; D019020:Directed Molecular Evolution / Q000379:methods*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000254:growth & development; D048888:Fireflies / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D006207:Half-Life; D006358:Hot Temperature; D007700:Kinetics; D049409:Luciferases, Firefly / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D049408:Luminescent Agents / Q000737:chemistry / Q000494:pharmacology*; D008163:Luminescent Measurements / Q000379:methods; D008969:Molecular Sequence Data; D009154:Mutation
chemical_list: D049408:Luminescent Agents; D000255:Adenosine Triphosphate; D049409:Luciferases, Firefly