title: Improving the functional expression of a Bacillus licheniformis laccase by random and site-directed mutagenesis.
abstract: BACKGROUND
  Laccases have huge potential for biotechnological applications due to their broad substrate spectrum and wide range of reactions they are able to catalyze. These include, for example, the formation and degradation of dimers, oligomers, polymers, and ring cleavage as well as oxidation of aromatic compounds. Potential applications of laccases include detoxification of industrial effluents, decolorization of textile dyes and the synthesis of natural products by, for instance, dimerization of phenolic acids. We have recently published a report on the cloning and characterization of a CotA Bacillus licheniformis laccase, an enzyme that catalyzes dimerization of phenolic acids. However, the broad application of this laccase is limited by its low expression level of 26 mg l-1 that was achieved in Escherichia coli. To counteract this shortcoming, random and site-directed mutagenesis have been combined in order to improve functional expression and activity of CotA.
  
  
  RESULTS
  A CotA double mutant, K316N/D500G, was constructed by combining random and site-directed mutagenesis. It can be functionally expressed at an 11.4-fold higher level than the wild-type enzyme. In addition, it is able to convert ferulic acid much faster than the wild-type enzyme (21% vs. 14%) and is far more efficient in decolorizing a range of industrial dyes. The investigation of the effects of the mutations K316N and D500G showed that amino acid at position 316 had a major influence on enzyme activity and position 500 had a major influence on the expression of the laccase.
  
  
  CONCLUSIONS
  The constructed double mutant K316N/D500G of the Bacillus licheniformis CotA laccase is an appropriate candidate for biotechnological applications due to its high expression level and high activity in dimerization of phenolic acids and decolorization of industrial dyes.
authors.affiliation: Institute of Technical Biochemistry, Universitaet Stuttgart, Allmandring 31, 70569 Stuttgart, Germany. itbvur@itb.uni-stuttgart.de
authors.forename: Katja
authors.initials: K
authors.lastname: Koschorreck
authors.forename: Rolf D
authors.initials: RD
authors.lastname: Schmid
authors.forename: Vlada B
authors.initials: VB
authors.lastname: Urlacher
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000378:metabolism; D004396:Coloring Agents / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D062385:Hydroxybenzoates / Q000378:metabolism; D042845:Laccase / Q000096:biosynthesis* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D004396:Coloring Agents; D062385:Hydroxybenzoates; C533003:CotA protein, Bacillus licheniformis; D042845:Laccase; C017616:phenolic acid